Cloning vectors are small DNA molecules that are used to artificially carry foreign genetic material into host cells. They contain features like an origin of replication, antibiotic resistance genes, and restriction enzyme sites. The document discusses different types of cloning vectors used for plant gene cloning, including plasmids, Ti/Ri plasmids from Agrobacterium, and plant viruses. Agrobacterium-mediated transformation uses disarmed Ti plasmids from Agrobacterium tumefaciens and involves co-cultivation of plant explants with Agrobacterium, selection of transformed cells, and regeneration of whole plants. Binary vector systems are now commonly used, involving transfer of a binary plasmid without integration into the Ti plasmid
Tumor formtion , ti ri plasmid , dna trnsfr.Sukirti Vedula
This document summarizes information about tumor formation in plants caused by Agrobacterium tumefaciens and Agrobacterium rhizogenes bacteria. It discusses how the Ti and Ri plasmids are transferred into plant cells, causing crown gall and hairy root diseases respectively. The Ti plasmid contains T-DNA which is integrated into the plant genome, inducing tumor formation and opine synthesis. DNA transfer techniques like electroporation, microprojectile bombardment, and microinjection are also summarized for introducing foreign genes into plant cells.
What are an expression vector? Detailed description of plant gene structure. Plant expression vector systems are generally consists of Ri and Ti plasmids.
The other vectors which are generally used are DNA and RNA viruses.
Genetic engineering involves directly manipulating an organism's DNA using biotechnology. The DNA of interest is isolated from a source organism and inserted into a vector, which is then introduced into a host cell. Common vectors include plasmids, bacteriophages, cosmids, phagemids, and artificial chromosomes. Artificial chromosomes, such as Bacterial Artificial Chromosomes and Yeast Artificial Chromosomes, can carry large DNA fragments of up to 300,000 base pairs, making them useful for cloning and transforming large genes. However, constructing and maintaining artificial chromosomes can be challenging due to their size and potential for rearrangements.
The document discusses Agrobacterium rhizogenes, which causes hairy root disease in plants. It contains genes on Ri plasmids, including rol genes, that are transferred to plant cells and cause root proliferation. The T-DNA from the plasmid is integrated into the plant genome. Agrobacterium attaches to wounded plant cells, vir genes are activated by compounds from the plant, and the T-DNA and virulence genes work together to transfer T-DNA to the plant cell. Hairy roots can be grown in vitro for research on plant-pathogen interactions and production of compounds.
Agrobacterium tumefaciens is a soil bacterium that causes crown gall disease in plants. It does this by transferring a segment of its tumor-inducing plasmid (Ti plasmid) called T-DNA into the plant's DNA. The T-DNA contains genes that cause uncontrolled cell growth. Researchers developed techniques using the Ti plasmid and Agrobacterium to genetically transform plants by replacing the tumor-causing genes with desired genes. This involves either a binary vector system with the T-DNA on a separate small plasmid, or co-integration of a new plasmid containing the gene of interest into the Ti plasmid. Transformed plants can be regenerated from infected plant cells or tissues.
The document discusses different expression vectors and systems used for recombinant protein expression. It describes key elements required for an expression vector including an origin of replication, selective marker, promoter, multiple cloning site, and terminator. It provides details on commonly used expression systems in E. coli such as the lac, tac, lambda PL, and T7 promoters. It also summarizes protein expression in yeast using the galactose-inducible GAL promoter system.
Haploid Production - Techniques, Application & Problem ANUGYA JAISWAL
Haploid is applied to any plant originating from a sporophyte (2n) and containing (n) number of chromosomes.
Artificial production of haploids was attempted through distant hybridization, delayed pollination, application of irradiated pollen, hormone treatment and temperature shock.
The artificial production of haploids until 1964 was attempted through:
1. Distant hybridization
2. Delayed pollination
3. Application of irradiated pollen
4. Hormone treatments
5. Temperature shocks
The development of numerous pollen plantlets in anther cultures of Datura innoxia, first reported by two Indian scientists (Guha and Maheshwari, 1964, 1966), was a major breakthrough in haploid breeding of higher plants.
The technique of haploid production through anther culture ('anther - androgenesis') has been extended successfully to numerous plant species, including many economically important plants, such as cereals and vegetable, oil and tree crops.
Tumor formtion , ti ri plasmid , dna trnsfr.Sukirti Vedula
This document summarizes information about tumor formation in plants caused by Agrobacterium tumefaciens and Agrobacterium rhizogenes bacteria. It discusses how the Ti and Ri plasmids are transferred into plant cells, causing crown gall and hairy root diseases respectively. The Ti plasmid contains T-DNA which is integrated into the plant genome, inducing tumor formation and opine synthesis. DNA transfer techniques like electroporation, microprojectile bombardment, and microinjection are also summarized for introducing foreign genes into plant cells.
What are an expression vector? Detailed description of plant gene structure. Plant expression vector systems are generally consists of Ri and Ti plasmids.
The other vectors which are generally used are DNA and RNA viruses.
Genetic engineering involves directly manipulating an organism's DNA using biotechnology. The DNA of interest is isolated from a source organism and inserted into a vector, which is then introduced into a host cell. Common vectors include plasmids, bacteriophages, cosmids, phagemids, and artificial chromosomes. Artificial chromosomes, such as Bacterial Artificial Chromosomes and Yeast Artificial Chromosomes, can carry large DNA fragments of up to 300,000 base pairs, making them useful for cloning and transforming large genes. However, constructing and maintaining artificial chromosomes can be challenging due to their size and potential for rearrangements.
The document discusses Agrobacterium rhizogenes, which causes hairy root disease in plants. It contains genes on Ri plasmids, including rol genes, that are transferred to plant cells and cause root proliferation. The T-DNA from the plasmid is integrated into the plant genome. Agrobacterium attaches to wounded plant cells, vir genes are activated by compounds from the plant, and the T-DNA and virulence genes work together to transfer T-DNA to the plant cell. Hairy roots can be grown in vitro for research on plant-pathogen interactions and production of compounds.
Agrobacterium tumefaciens is a soil bacterium that causes crown gall disease in plants. It does this by transferring a segment of its tumor-inducing plasmid (Ti plasmid) called T-DNA into the plant's DNA. The T-DNA contains genes that cause uncontrolled cell growth. Researchers developed techniques using the Ti plasmid and Agrobacterium to genetically transform plants by replacing the tumor-causing genes with desired genes. This involves either a binary vector system with the T-DNA on a separate small plasmid, or co-integration of a new plasmid containing the gene of interest into the Ti plasmid. Transformed plants can be regenerated from infected plant cells or tissues.
The document discusses different expression vectors and systems used for recombinant protein expression. It describes key elements required for an expression vector including an origin of replication, selective marker, promoter, multiple cloning site, and terminator. It provides details on commonly used expression systems in E. coli such as the lac, tac, lambda PL, and T7 promoters. It also summarizes protein expression in yeast using the galactose-inducible GAL promoter system.
Haploid Production - Techniques, Application & Problem ANUGYA JAISWAL
Haploid is applied to any plant originating from a sporophyte (2n) and containing (n) number of chromosomes.
Artificial production of haploids was attempted through distant hybridization, delayed pollination, application of irradiated pollen, hormone treatment and temperature shock.
The artificial production of haploids until 1964 was attempted through:
1. Distant hybridization
2. Delayed pollination
3. Application of irradiated pollen
4. Hormone treatments
5. Temperature shocks
The development of numerous pollen plantlets in anther cultures of Datura innoxia, first reported by two Indian scientists (Guha and Maheshwari, 1964, 1966), was a major breakthrough in haploid breeding of higher plants.
The technique of haploid production through anther culture ('anther - androgenesis') has been extended successfully to numerous plant species, including many economically important plants, such as cereals and vegetable, oil and tree crops.
The Ri plasmid is a plasmid found in Agrobacterium rhizogenes bacteria that causes hairy root disease in plants. The plasmid contains genes that allow it to integrate portions of its DNA (T-DNA regions) into the plant genome. These integrated genes cause uncontrolled root growth and the formation of hairy roots. The Ri plasmid shares similarities with the Ti plasmid found in Agrobacterium tumefaciens, including virulence genes that mediate the transfer of T-DNA into plant cells and opine synthesis genes. Integration of the T-DNA from the Ri plasmid alters plant hormone production and induces hairy root formation.
It is a part of Ti Plasmid which takes part intransfer and integration of T-DNA into plant chromosome.
The vir sequence consist of 8 operons which take part in different functions associated with virulence of Ti Plasmid. These are vir H, vir A, vir B, vir G, vir C, vir D, vir E, & vir F. ( vir H & vir F present occasionally).
Genomic DNA libraries contain representative copies of all DNA fragments in an organism's genome, including both expressed and non-expressed sequences. They are constructed by isolating genomic DNA, fragmenting it, and cloning the fragments into suitable vectors like lambda phage or BACs. cDNA libraries contain only expressed sequences, as they are constructed by isolating mRNA from tissues, reverse transcribing it to cDNA, and cloning the cDNA fragments. Both library types are useful for gene discovery, sequencing, mapping genomes, and studying regulatory sequences.
Introduction
Components of binary vector
Development of binary vector system
Properties of binary vector
Types of binary vector
Plant transformation using binary vector
Advantage of using binary vector
Conclusion
References
This document provides information on various methods of gene transfer in plants, including Agrobacterium-mediated gene transfer and direct gene transfer methods. Direct methods rely on delivering large amounts of DNA to plant cells through techniques like particle bombardment, electroporation, and microinjection. Agrobacterium-mediated gene transfer utilizes the bacterium Agrobacterium, which transfers genes into plant genomes. The document discusses several direct and Agrobacterium-mediated methods in detail and provides advantages and limitations of each approach.
The document discusses the plasmid vector pBR322, which was constructed in 1977 and is one of the most commonly used cloning vectors. It describes the origins and components of pBR322, including two antibiotic resistance genes, the origin of replication, and restriction enzyme cleavage sites. The document also summarizes the construction of several derivatives of pBR322, including pBR327, pUC18, and pBR118/119, and notes their applications and advantages over the original pBR322 vector.
This document discusses plant molecular pharming (PMP), which uses plants as bioreactors for producing recombinant pharmaceutical proteins. It covers the definition, history, strategies, host systems, production of antibiotics/enzymes/vaccines in plants, advantages/disadvantages of plant systems, and issues of transgene pollution. Key points include:
- PMP uses whole plants, plant cells or tissues to produce commercially valuable proteins like vaccines via recombinant DNA.
- Early work in 1986 produced human growth hormone in tobacco and sunflower. Commercial production of various proteins in plants has occurred.
- Strategies include transforming host plants, growing biomass, processing/purifying the product of interest.
- Plants,
Organ culture involves maintaining small fragments of whole organs or tissues in culture media while retaining their three-dimensional structure and spatial distribution of cells. There are several methods of organ culture including culturing on plasma clots, agar, liquid media, or raft methods. Organ culture has various applications and allows studying cell interactions in a way that mimics the in vivo organ. It is currently being used to develop replacement organs and tissues for applications such as growing bladders, lungs, and heart patches. While progress is being made, developing fully functional human organs remains a challenge.
A comprehensive study of shuttle vector & binary vector and its rules of in ...PRABAL SINGH
Vector: A vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host cell and into which the DNA fragment to be cloned is integrated for cloning
Construction of rDNA molecules and bacterial transformationT. Tamilselvan
Recombinant DNA technology involves introducing genes of interest into the genome of an organism using genetic engineering techniques. This is done by cutting DNA fragments using restriction enzymes, joining the fragments to a vector plasmid, and introducing the recombinant DNA or rDNA into a host cell. The host cell then replicates and produces multiple copies of the rDNA. This allows genes from one species to be introduced into another, such as joining human DNA to E. coli bacteria. The rDNA can then be used to produce beneficial proteins and genetically modified organisms.
Direct Gene Transfer method (gene gun method).ShaistaKhan60
Direct gene transfer methods rely on delivering naked DNA directly into plant cells without the use of Agrobacterium. Biolistic or gene gun particle bombardment is a direct gene transfer method where gold or tungsten particles are coated with DNA and shot into plant tissue using a gene gun. The DNA can then integrate into the plant genome. The method involves creating recombinant plasmids with the gene of interest, coating them onto microcarriers, bombarding embryogenic plant callus, selecting transformed cells, and regenerating plants. Transgenic papaya developed using the gene gun method were resistant to papaya ring spot virus.
pBR322 is a 4,361 base pair plasmid vector originally constructed in 1977 for use in cloning experiments. It contains genes conferring resistance to ampicillin and tetracycline, which allow selection of recombinant clones, as well as an E. coli origin of replication. Recombinant selection involves insertional inactivation of the tetracycline resistance gene, rendering clones sensitive to tetracycline but resistant to ampicillin. pBR322 was widely used for cloning due to its small size, two selectable markers, and ability to be amplified in host cells. However, it is limited by its mobility between cells and small carrying capacity.
1. Bovine papillomavirus (BPV) vectors utilize the circular, double-stranded DNA genome of BPV. The BPV genome contains early and late regions and can transform cells without integrating.
2. There are three main types of BPV vectors. All contain the transforming 69% fragment of BPV and bacterial sequences. One type inserts a gene of interest, another adds a stimulating gene, and the third uses the full BPV genome.
3. Transformation efficiency is highest with the full BPV genome due to an enhancer in the non-transforming region. Stimulating genes can replace this enhancer's function when parts of the genome are removed. BPV vectors provide amplified
1. There are two main methods of gene transfer - direct and indirect gene transfer. Indirect transfer uses Agrobacterium-mediated transformation while direct transfer uses physical or chemical methods.
2. Agrobacterium-mediated transformation uses Agrobacterium tumefaciens to transfer T-DNA containing the gene of interest into the plant genome. The process involves co-cultivation of plant explants with Agrobacterium followed by selection and regeneration of transgenic plants.
3. Direct physical methods include biolistic transformation, microinjection, electroporation, and macroinjection. Direct chemical methods include PEG-mediated, calcium phosphate co-precipitation, and liposome-mediated transformation
This document discusses somaclonal variation, which refers to genetic variation that arises during tissue culture or plant regeneration from cell cultures. It provides definitions and history of the term as coined by Larkin and Scowcroft in 1981. The document outlines the various causes and types of somaclonal variation including physiological, genetic, and biochemical causes. It also describes methods for generating somaclonal variation both with and without in vitro selection. Finally, it discusses applications for detecting and isolating somaclonal variants, particularly for developing disease resistance in various crop species.
Genetic manipulation of plant and animal cells have to be confirmed for further application. One such confirmatory method is the use of stains/dyes which produces fluorescence when the recombination is successful.
pET vector. Plasmid for Expression by T7 RNA Polymerase.MuhammadMujahid58
The pET vector system is a powerful tool for expressing cloned genes in E. coli. It utilizes the strong T7 promoter to drive high-level expression of the gene when induced. The T7 promoter is tightly regulated by the Lac repressor protein so expression is low without induction. This prevents toxicity from overexpression. Key features include the T7 promoter, Lac repressor binding site, ribosome binding site, and antibiotic resistance gene. Expression is induced by adding IPTG which binds Lac repressor and allows transcription by T7 RNA polymerase. This results in high protein yields while avoiding metabolic burden on the host cell.
1. Yeast plasmids like the 2 micron circle have been extensively studied and developed into yeast cloning vectors.
2. Shuttle vectors like YEp vectors contain selectable marker genes like LEU2 and bacterial plasmid origins of replication like pBR322, allowing them to replicate in both E. coli and yeast.
3. The 2 micron circle is a 6kb endogenous yeast plasmid that replicates autonomously through an ARS sequence and is maintained at 50-100 copies per cell.
This document discusses various direct or vectorless gene transfer methods for introducing foreign DNA into plant cells without the use of Agrobacterium. These include chemical methods like PEG-mediated transformation, calcium phosphate precipitation, DEAE-dextran, and liposome-mediated transformation. Physical methods include electroporation, microinjection, and gene guns. The procedures, advantages, and disadvantages of some key methods like PEG-mediated transformation, calcium phosphate precipitation, electroporation, microinjection, and fiber-mediated transformation are described in detail. Direct transformation through imbibition of dehydrated plant tissues is also mentioned.
Gene cloning allows for the creation of identical copies of genes. It involves amplifying genes using polymerase chain reaction, cutting DNA with restriction enzymes, and joining DNA fragments together with DNA ligase. Cloning vectors like plasmids and bacteriophages are used to move genes into host cells. Transformed cells are selected using antibiotic resistance or reporter genes. Cloned genes have applications in pharmaceutical production, disease diagnosis using probes or PCR, and controlling insect pests by producing bacterial pesticides, transgenic plants, or viral pesticides.
The document discusses different types of cloning vectors. It describes cloning vectors as DNA fragments capable of self-replication that can transport foreign genetic material into host cells. The main types discussed are plasmids, bacteriophages, cosmids, BACs, YACs, and retroviral vectors. Each type has distinct features like replication origin and cloning capacity that determine their suitability for different applications. Plasmids were among the earliest vectors and can clone inserts up to 10kb, while BACs and YACs can accommodate much larger DNA fragments. The key factors in choosing a vector are the size of the DNA insert and cloning efficiency required.
The Ri plasmid is a plasmid found in Agrobacterium rhizogenes bacteria that causes hairy root disease in plants. The plasmid contains genes that allow it to integrate portions of its DNA (T-DNA regions) into the plant genome. These integrated genes cause uncontrolled root growth and the formation of hairy roots. The Ri plasmid shares similarities with the Ti plasmid found in Agrobacterium tumefaciens, including virulence genes that mediate the transfer of T-DNA into plant cells and opine synthesis genes. Integration of the T-DNA from the Ri plasmid alters plant hormone production and induces hairy root formation.
It is a part of Ti Plasmid which takes part intransfer and integration of T-DNA into plant chromosome.
The vir sequence consist of 8 operons which take part in different functions associated with virulence of Ti Plasmid. These are vir H, vir A, vir B, vir G, vir C, vir D, vir E, & vir F. ( vir H & vir F present occasionally).
Genomic DNA libraries contain representative copies of all DNA fragments in an organism's genome, including both expressed and non-expressed sequences. They are constructed by isolating genomic DNA, fragmenting it, and cloning the fragments into suitable vectors like lambda phage or BACs. cDNA libraries contain only expressed sequences, as they are constructed by isolating mRNA from tissues, reverse transcribing it to cDNA, and cloning the cDNA fragments. Both library types are useful for gene discovery, sequencing, mapping genomes, and studying regulatory sequences.
Introduction
Components of binary vector
Development of binary vector system
Properties of binary vector
Types of binary vector
Plant transformation using binary vector
Advantage of using binary vector
Conclusion
References
This document provides information on various methods of gene transfer in plants, including Agrobacterium-mediated gene transfer and direct gene transfer methods. Direct methods rely on delivering large amounts of DNA to plant cells through techniques like particle bombardment, electroporation, and microinjection. Agrobacterium-mediated gene transfer utilizes the bacterium Agrobacterium, which transfers genes into plant genomes. The document discusses several direct and Agrobacterium-mediated methods in detail and provides advantages and limitations of each approach.
The document discusses the plasmid vector pBR322, which was constructed in 1977 and is one of the most commonly used cloning vectors. It describes the origins and components of pBR322, including two antibiotic resistance genes, the origin of replication, and restriction enzyme cleavage sites. The document also summarizes the construction of several derivatives of pBR322, including pBR327, pUC18, and pBR118/119, and notes their applications and advantages over the original pBR322 vector.
This document discusses plant molecular pharming (PMP), which uses plants as bioreactors for producing recombinant pharmaceutical proteins. It covers the definition, history, strategies, host systems, production of antibiotics/enzymes/vaccines in plants, advantages/disadvantages of plant systems, and issues of transgene pollution. Key points include:
- PMP uses whole plants, plant cells or tissues to produce commercially valuable proteins like vaccines via recombinant DNA.
- Early work in 1986 produced human growth hormone in tobacco and sunflower. Commercial production of various proteins in plants has occurred.
- Strategies include transforming host plants, growing biomass, processing/purifying the product of interest.
- Plants,
Organ culture involves maintaining small fragments of whole organs or tissues in culture media while retaining their three-dimensional structure and spatial distribution of cells. There are several methods of organ culture including culturing on plasma clots, agar, liquid media, or raft methods. Organ culture has various applications and allows studying cell interactions in a way that mimics the in vivo organ. It is currently being used to develop replacement organs and tissues for applications such as growing bladders, lungs, and heart patches. While progress is being made, developing fully functional human organs remains a challenge.
A comprehensive study of shuttle vector & binary vector and its rules of in ...PRABAL SINGH
Vector: A vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host cell and into which the DNA fragment to be cloned is integrated for cloning
Construction of rDNA molecules and bacterial transformationT. Tamilselvan
Recombinant DNA technology involves introducing genes of interest into the genome of an organism using genetic engineering techniques. This is done by cutting DNA fragments using restriction enzymes, joining the fragments to a vector plasmid, and introducing the recombinant DNA or rDNA into a host cell. The host cell then replicates and produces multiple copies of the rDNA. This allows genes from one species to be introduced into another, such as joining human DNA to E. coli bacteria. The rDNA can then be used to produce beneficial proteins and genetically modified organisms.
Direct Gene Transfer method (gene gun method).ShaistaKhan60
Direct gene transfer methods rely on delivering naked DNA directly into plant cells without the use of Agrobacterium. Biolistic or gene gun particle bombardment is a direct gene transfer method where gold or tungsten particles are coated with DNA and shot into plant tissue using a gene gun. The DNA can then integrate into the plant genome. The method involves creating recombinant plasmids with the gene of interest, coating them onto microcarriers, bombarding embryogenic plant callus, selecting transformed cells, and regenerating plants. Transgenic papaya developed using the gene gun method were resistant to papaya ring spot virus.
pBR322 is a 4,361 base pair plasmid vector originally constructed in 1977 for use in cloning experiments. It contains genes conferring resistance to ampicillin and tetracycline, which allow selection of recombinant clones, as well as an E. coli origin of replication. Recombinant selection involves insertional inactivation of the tetracycline resistance gene, rendering clones sensitive to tetracycline but resistant to ampicillin. pBR322 was widely used for cloning due to its small size, two selectable markers, and ability to be amplified in host cells. However, it is limited by its mobility between cells and small carrying capacity.
1. Bovine papillomavirus (BPV) vectors utilize the circular, double-stranded DNA genome of BPV. The BPV genome contains early and late regions and can transform cells without integrating.
2. There are three main types of BPV vectors. All contain the transforming 69% fragment of BPV and bacterial sequences. One type inserts a gene of interest, another adds a stimulating gene, and the third uses the full BPV genome.
3. Transformation efficiency is highest with the full BPV genome due to an enhancer in the non-transforming region. Stimulating genes can replace this enhancer's function when parts of the genome are removed. BPV vectors provide amplified
1. There are two main methods of gene transfer - direct and indirect gene transfer. Indirect transfer uses Agrobacterium-mediated transformation while direct transfer uses physical or chemical methods.
2. Agrobacterium-mediated transformation uses Agrobacterium tumefaciens to transfer T-DNA containing the gene of interest into the plant genome. The process involves co-cultivation of plant explants with Agrobacterium followed by selection and regeneration of transgenic plants.
3. Direct physical methods include biolistic transformation, microinjection, electroporation, and macroinjection. Direct chemical methods include PEG-mediated, calcium phosphate co-precipitation, and liposome-mediated transformation
This document discusses somaclonal variation, which refers to genetic variation that arises during tissue culture or plant regeneration from cell cultures. It provides definitions and history of the term as coined by Larkin and Scowcroft in 1981. The document outlines the various causes and types of somaclonal variation including physiological, genetic, and biochemical causes. It also describes methods for generating somaclonal variation both with and without in vitro selection. Finally, it discusses applications for detecting and isolating somaclonal variants, particularly for developing disease resistance in various crop species.
Genetic manipulation of plant and animal cells have to be confirmed for further application. One such confirmatory method is the use of stains/dyes which produces fluorescence when the recombination is successful.
pET vector. Plasmid for Expression by T7 RNA Polymerase.MuhammadMujahid58
The pET vector system is a powerful tool for expressing cloned genes in E. coli. It utilizes the strong T7 promoter to drive high-level expression of the gene when induced. The T7 promoter is tightly regulated by the Lac repressor protein so expression is low without induction. This prevents toxicity from overexpression. Key features include the T7 promoter, Lac repressor binding site, ribosome binding site, and antibiotic resistance gene. Expression is induced by adding IPTG which binds Lac repressor and allows transcription by T7 RNA polymerase. This results in high protein yields while avoiding metabolic burden on the host cell.
1. Yeast plasmids like the 2 micron circle have been extensively studied and developed into yeast cloning vectors.
2. Shuttle vectors like YEp vectors contain selectable marker genes like LEU2 and bacterial plasmid origins of replication like pBR322, allowing them to replicate in both E. coli and yeast.
3. The 2 micron circle is a 6kb endogenous yeast plasmid that replicates autonomously through an ARS sequence and is maintained at 50-100 copies per cell.
This document discusses various direct or vectorless gene transfer methods for introducing foreign DNA into plant cells without the use of Agrobacterium. These include chemical methods like PEG-mediated transformation, calcium phosphate precipitation, DEAE-dextran, and liposome-mediated transformation. Physical methods include electroporation, microinjection, and gene guns. The procedures, advantages, and disadvantages of some key methods like PEG-mediated transformation, calcium phosphate precipitation, electroporation, microinjection, and fiber-mediated transformation are described in detail. Direct transformation through imbibition of dehydrated plant tissues is also mentioned.
Gene cloning allows for the creation of identical copies of genes. It involves amplifying genes using polymerase chain reaction, cutting DNA with restriction enzymes, and joining DNA fragments together with DNA ligase. Cloning vectors like plasmids and bacteriophages are used to move genes into host cells. Transformed cells are selected using antibiotic resistance or reporter genes. Cloned genes have applications in pharmaceutical production, disease diagnosis using probes or PCR, and controlling insect pests by producing bacterial pesticides, transgenic plants, or viral pesticides.
The document discusses different types of cloning vectors. It describes cloning vectors as DNA fragments capable of self-replication that can transport foreign genetic material into host cells. The main types discussed are plasmids, bacteriophages, cosmids, BACs, YACs, and retroviral vectors. Each type has distinct features like replication origin and cloning capacity that determine their suitability for different applications. Plasmids were among the earliest vectors and can clone inserts up to 10kb, while BACs and YACs can accommodate much larger DNA fragments. The key factors in choosing a vector are the size of the DNA insert and cloning efficiency required.
Recombinant dna techaniques and its applicationBasharatAli103
Recombinant DNA technology involves inserting foreign DNA into a vector, such as a plasmid, and introducing it into a host cell. This allows the gene to be replicated in large quantities. Key steps include using restriction enzymes to cut the DNA into fragments, joining DNA fragments with DNA ligase, transforming host cells with the recombinant DNA, and selecting cells containing the inserted gene. Recombinant DNA technology has many applications, including producing human proteins and hormones, genetically engineering crops, and aiding forensics and disease diagnosis.
This document provides an overview of various gene transfer tools and techniques. It discusses vector-mediated methods like Agrobacterium and viral vectors as well as direct or vector-less methods such as electroporation, biolistics, microinjection, liposome mediated, and calcium phosphate mediated gene transfer. For each method, it describes the basic process and provides some key details and applications. It also notes some advantages and limitations of different techniques. The document aims to inform readers about the various options available for inserting genes into plant cells.
This document discusses various methods for improving microbial strains, including selecting naturally occurring variants, manipulating existing genetics, and introducing new genetics. It focuses on mutation and selection techniques like chemical or UV mutagenesis followed by selection on selective media. Genetic engineering techniques are also summarized, including restriction digestion, ligation into vectors, transformation, and screening of recombinants. Common vectors like pBR322, pUC18, phages like M13, and cosmids are described. The overall goal is to outline strategies for isolating industrially useful microbial mutants.
cloning and expression vector in plantsAlex Mathew
This document discusses cloning and expression vectors used in plants. It describes cloning vectors as small DNA molecules that can stably maintain foreign DNA for cloning purposes. Expression vectors are designed for protein expression and must contain strong promoters and terminators. Common vectors used in plants include plasmids, viruses, bacteriophages, and cosmids. Agrobacterium-mediated transformation is described as a method for introducing DNA into plants using Ti or Ri plasmids. Viral vectors like CaMV can also be used to directly or indirectly insert genes into plants.
Recombinant DNA (rDNA) refers to DNA created outside living cells by joining DNA from multiple sources. Common techniques for creating rDNA include restriction enzymes to cut DNA strands, ligation to join strands, and transformation or transfection to introduce rDNA into host cells. Vectors like plasmids, viruses, and artificial chromosomes are often used to replicate and express rDNA in host cells. rDNA techniques have applications in gene cloning, DNA sequencing, genetic engineering of plants and animals, and gene therapy to treat diseases.
Vector engineering involves designing expression vectors that allow for optimal transcription of heterologous genes transferred between organisms. Key components of expression vectors include an origin of replication for stability in the host, a selection marker for identifying transformed cells, and a multiple cloning site for inserting genes of interest. Vectors are classified as cloning vectors for copying DNA or expression vectors for high-level protein production. Perspectives in vector design focus on copy number control, plasmid incompatibility, stability, use of minimal and characterized parts, and selection of appropriate cloning methods. Codon optimization is also important for high expression, by introducing synonymous mutations that favor translation efficiency in the target host.
This document provides an overview of various gene transformation techniques, including both vector-mediated and direct methods. It discusses natural transformation mechanisms like conjugation and transduction, as well as artificial vector-mediated techniques like Agrobacterium-mediated transformation. Direct methods like microinjection, electroporation, particle bombardment, and chemical methods using PEG or calcium phosphate are also covered. The applications, advantages, and limitations of different techniques are summarized. Overall, the document serves as an informative introduction to the key gene transfer methods used in plant biotechnology.
This document provides an overview of various gene transformation techniques, including both vector-mediated and direct methods. It discusses natural transformation mechanisms like conjugation and transduction, as well as artificial vector-mediated techniques like Agrobacterium-mediated transformation. Direct methods like microinjection, electroporation, particle bombardment, and chemical methods using PEG or calcium phosphate are also covered. The applications, advantages, and limitations of different techniques are summarized. Overall, the document serves as an informative introduction to the key gene transfer methods used in plant biotechnology.
This document discusses cloning vectors. It begins with a brief history of cloning vectors, noting that the first designed cloning vector was the plasmid pBR322 created in 1977. It then describes the key features of cloning vectors, including an origin of replication, cloning sites, selectable markers like antibiotic resistance genes, and reporter genes. Examples of different types of cloning vectors are also provided, such as plasmids, bacteriophages, cosmids, and artificial chromosomes that can be used in prokaryotes or eukaryotes. The document concludes by differentiating between cloning vectors and expression vectors.
Recombinant DNA technology allows DNA from different species to be isolated, cut, and spliced together to form new recombinant molecules. Key tools for recombinant DNA technology include restriction enzymes, ligases, polymerases, vectors, and host cells. Recombinant DNA technology has many applications, including producing human insulin and other proteins for medical use, genetically engineering plants for crop improvement, and DNA fingerprinting for criminal investigations.
Plasmid vectors like pBR322 and pUC are commonly used cloning vectors. pBR322 was one of the first vectors created and has advantages like a small size, antibiotic resistance markers, and a high copy number. pUC vectors also have a small size and high copy number, and contain a multiple cloning site within the lacZ gene allowing visual selection of recombinants. Artificial vectors combine elements from different sources to overcome limitations of natural plasmids, and are designed for efficient cloning and expression of foreign DNA in host cells.
Biotechnology refers to the use of living organisms or their components to develop products and processes. It has applications in fields like agriculture, medicine, and industry. Modern biotechnology techniques include genetic engineering and aseptic techniques. Genetic engineering involves altering genetic material through techniques like recombinant DNA, gene transfer into host organisms, and gene cloning. It allows scientists to modify organisms for useful purposes. Restriction enzymes, vectors, DNA polymerase and ligase are important tools used in genetic engineering and recombinant DNA technology.
Vectors can perform their functions in two main ways: transcription and expression. There are several types of vectors, including plasmids, which are small, self-replicating DNA molecules commonly used in molecular cloning. Plasmids are important tools in genetic engineering as they can be easily manipulated and transformed into bacteria to generate multiple copies of recombinant DNA. Common plasmid vectors include transcription vectors, which amplify DNA sequences without expressing proteins, and expression vectors, which are used to express foreign genes in cells.
This document discusses various techniques in microbial genetics including transformation, transduction, conjugation, plasmids, and transposons. Transformation involves the uptake of genetic material like DNA by bacterial cells. Transduction occurs when viruses called bacteriophages transfer genetic material between bacteria. Conjugation is the transfer of genetic material like plasmids through direct contact between bacteria. Plasmids are small circular DNA molecules that are distinct from chromosomal DNA and often provide genetic advantages to bacteria. Transposons are genetic elements that can move to different locations in a genome and contribute to the spread of traits like antibiotic resistance.
recominant DNA technology and its applicationRimshaRizwan4
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1. VECTORS USED FOR
GENE CLONING IN
PLANTS
Submitted By :
Arunodaya Maji
CARA-2018-110
Batch - D
Submitted To :
Dr. G. Uma Devi Ma’am
College of Agriculture, Rajendranagar
Course No. – PATH – 271
Course Title – Principles of Plant Pathology
Course Credits – 2(2+0)
Assignment - 3
2. Content
1. What is cloning vector ?
2. Why cloning vector ?
3. History
4. What determines choice of vector
5. Features of a cloning vector
6. Types of cloning vector
7. Agrobacterium Mediated Cloning Vectors
Ti Plasmid
Ri Plasmid
8. Attempts to use Plant Viruses as Cloning Vectors
9. Conclusion
10. References
3. Cloning Vectors
The molecular analysis of DNA has been made possible by the cloning of DNA.
The two molecules that are required for cloning are the DNA to be cloned and a
cloning vector.
A cloning vector is a small piece of DNA taken from a virus, a plasmid or the
cell of a higher organism, that can be stably maintained in an organism and
into which a foreign DNA fragment can be inserted for cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to the size and type of DNA to be cloned.
The vector therefore contains features that allow for the convenient insertion or
removal of DNA fragment in or out of the vector, for example by treating the
vector and the foreign DNA with a restriction enzyme and then ligating the
fragments together.
After a DNA fragment has been cloned into a cloning vector, it may be further
subcloned into another vector designed for more specific use.
4. Vector
In molecular cloning, a vector is a DNA molecule used as a vehicle to
artificially carry foreign genetic material into another cell, where it can be
replicated and/or expressed.
A vector containing foreign DNA is termed recombinant DNA.
5. Why Cloning Vectors
Cloning vector is used as a vehicle to artificially carry foreign genetic
material into another cell, where it can be replicated and expressed.
It is used to amplify a single molecule of DNA into many copes.
Cloning vectors are DNA molecules that are used to "transport" cloned
sequences between biological hosts and the test tube.
Without Cloning Vector, Molecular Gene Cloning is totally impossible.
6. History
Scientists (Herbert Boyer, Keiichi Itakura and Arthur Riggs) working in Boyer’s lab (University of
California) recognized a general cloning vector with unique restriction sites for cloning in foreign
DNA and the expression of antibiotic resistance genes for selection of transformed bacteria.
Rodriguez Raymon along with Paco Bolivar constructed the vector “pBR 322” in the year
1977
This vector was small, ~4 kb in size, and had two antibiotic resistance genes for selection.
Arthur Riggs Herbert BoyerRodriguez Raymon
7.
8. Features of A Cloning Vector
All commonly used cloning vectors have some essential features:
Origin of replication (ori):
This makes autonomous replication in vector.
ori is a specific sequence of nucleotide from where
replication starts.
When foreign DNA is linked to the sequence along with vector
replication, foreign (desirable) DNA also starts replicating within
host cell.
Cloning Site:
Cloning site is a place where the vector DNA can be digested and
desired DNA can be inserted by the same restriction enzyme.
It is a point of entry or analysis for genetic engineering work.
Recently recombinant plasmids contain a multiple cloning site
(MCS) which have many (up to ~20) restriction sites.
9. Selectable Marker
Selectable marker is a gene that confers resistance to particular antibiotics or selective
agent that would normally kill the host cell or prevent its growth.
A cloning vector contains a selectable marker, which confer on the host cell an ability to
survive and proliferate in a selective growth medium containing the particular antibiotics.
Reporter Gene or MarkerGene
Reporter genes are used in cloning vectors to facilitate the screening of successful clones by
using features of these genes that allow successful clone to be easily identified.
Such feature present in cloning vectors is used in blue- white selection.
10. Replicate autonomously.
Restriction sites.
Self replication, multiple copies.
Replication origin site.
Small size.(Larger plasmids are more difficult to characterize by restriction
mapping and replicate to lower copy numbers)
Low molecular weight.
No pathogenicity
Easily isolated & purified.
Easily isolated into host cell.
Control elements – promoter, operator, ribosome binding site.
13. Types Of Cloning Vectors
S. No. Vectors Targeted Host
1 Plasmid Bacteria, Streptomyces
2 Ti & Ri Plasmid Transformation of cloned genes
in Higher Plants
3 Bacteriophages/Phagemids/Phasmids Bacteria
4 Cosmid (Hybrid Vector) Bacteria
5 Fosmid E. coli
6 Bacterial Artificial Chromosome (BAC) Bacteria
7 Yeast Artificial Chromosome (YAC) Yeast
8 Human Artificial Chrosome (HAC) Human Cells
9 Mammalian Artificial Chromosome (MAC) Mammalian Cells
10 Shuttle Vectors E. coli, Yeast
11 Retroviral Vectors Human and Animal Cells
14. Agrobacterium Mediated Transformation
The important requirements for Agrobacterium- mediated gene
transfer in higher plants are as follows:-
The plant explants must produce acetosyringone for activation of
Vir genes.
The induced Agrobacterium should have access to cells that are
competent for transformation.
Explants include cotyledon, leaf, thin tissue layer, peduncle,
hypocotyls, stem, microspores
18. Agrobacterium tumifaciens
Agrobacterium tumefaciens—nature’s smallest genetic
engineer
A. tumefaciens causes CROWN GALL DISEASE in many
species of dicotyledonous plants.
Causes a cancerous proliferation of the stem tissue in the
region of the crown.
A. tumifaciens is a Gram –ve soil bacterium
Infects plants through breaks or wounds.
Tumor formation is the result of integration of T-DNA
(Transfer DNA) in plant genome.
21. Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(3), 121-129
Cellular Processes of Agrobacterium Host Infection
22. In general, the transformation procedure is as follows:
The recombinant small replicon is transferred via bacterial conjugation or direct transfer to A. tumefaciens harboring a
helper Ti plasmid, the plant cells are co-cultivated with the Agrobacterium, to allow transfer of recombinant T-DNAin to the
plant genome, and transformed plant cells are selected under appropriate conditions.
1. Binary vector system involves only the transfer of a binary plasmid to Agrobacterium without any integration.
2. This is in contrast to co-integrate vector system wherein the intermediate vector is transferred and integrated with
disarmed Ti plasmid.
3. Due to convenience, binary vectors are more frequently used than co-integrate vectors.
Compared with co-integrated vectors, binary vectors present some advantages:
No recombination process takes place between the molecules involved.
Instead of a very large, recombinant, disarmed Ti plasmid, small vector s are used, which increases transfer
efficiency from E. coli to Agrobacterium.
This vector system is most widely used nowadays.
Different types of binary vectors have been devised to suit different needs in a plant transformation process.
25. Transformation Protocols
Transformation was performed using minor modifications of given protocols, using
I. Leaf disk
II. Scutellum-derived callus, or
III. Floral dip methods, respectively.
AGROBACTERIUM-MEDIATED PLANT TRANSFORMATIONS HAVE THE FOLLOWING BASIC
PROTOCOL :-
Development of Agrobacterium carrying the cointegrate or binary vector with the
desired gene.
Identification of a suitable explant e.g. cells, protoplasts, tissues, calluses, organs.
Co-culture of explants with Agrobacterium.
Killing of Agrobacterium with a suitable antibiotic without harming the plant tissue.
Selection of transformed plant cells.
Regeneration of whole plants.
26. Example-scutellum-derived callus method
Transformation Protocol for Rice – Abbreviated
Seed plating on 2N6 – dark
↓ 4 weeks Subculture onto 2N6 – dark
↓ 4 – 10 days
Co cultivation onto 2N6-AS – dark
↓ 3-7 days
Selection on 2N6-TCH – dark
↓ 4 weeks, subculture onto 2N6-TCH every 2 weeks Transfer proliferating calli onto 2N6-TCH-dark
↓ 2 weeks
Regeneration onto RGH6-dark
↓ 7 days Transfer to light
↓ 4-6 weeks Plantlets onto ½ MSH - light
↓
Transfer plants to the glasshouse
27. Seed Material: Oryza sativa L. ssp japonica cvs. Millin or Nipponbare.
Steps-
1. Callus Induction
2. Callus Subculture
3. Bacteria Preparation
4. Transformation
5. Callus washing
6. Selection
7. Regeneration
8. Plantlet Formation
To ensure that the gene transfer did not result from contamination with Agrobacterium cells, controls including species specific PCR,
selective plating, and use of atagged binary vector were implemented.
Thus, diverse plant associated bacteria, when harbouring a disarmed Ti plasmid and binary vector(or presumably a cointegrate or whole Ti
plasmid), are readily able to transfer TDNA to plants. The Ti plasmid is self transmissable, perhaps in dicating the existence of a ubiquitous
natural mechanism effecting horizontal gene transfer from bacteria to plants.
28. Procedure for Plant Transformation
IMAGE: Mol bio of the cell by Albert (pg no:599)
29. Regeneration, Selection And Detection
Regeneration: for shoot organogenesis, cytokinin (lower amounts of auxin) are required
Selection: two antibiotics are required
• An antibiotic to kill the Agrobacterium, while not affecting theplant's cell growth and
division
• a second antibiotic allows growth of transformed shoots (w/selectable
marker) but inhibits growth of untransformed plantcells.
Detection of the "trait" gene
PCR methods can detect the presence of the "trait" DNA
protein detection methods are used where a gene product is producedthat defines the trait
verification of the incorporation of the trait gene into the plant's chromosome:
• by Southern hybridization
• by demonstrating transfer of the trait to the originaltransformant's progeny.
30. Scientists can insert any gene they want into the plasmid in place of thetumor
causing genes and subsequently into the plant cell genome.
By varying experimental materials, culture conditions, bacterial strains, etc.
scientists have successfully used A. tumefaciens Gene Transfer to produce BT Corn.
This method of gene transfer enables large DNA strands to be transferred into the
plant cell without risk of rearrangement whereas other methods like the Gene Gun
have trouble doing this .
The vast majority of approved genetically engineered agriculture has been
transformed by means of Agrobacterium tumefaciens Mediated Gene Transfer.
Original problems existed in that Agrobacterium tumefaciens only affects
dicotyledonous plants.
Monocotyledon plants are not very susceptible to the bacterial infection.
Benefits and Problems with Agrobacteria
31. Ti Plasmid
The Ti plasmid (approx.size 200 kb each) exist as independent replicating
circular DNA molecules within the Agrobacterium cells.
The T-DNA is variable in length in the range of 12 to 24 kb.
The Ti plasmid is lost when Agrobacterium is grown above 28 °C.
The plasmid has 196 genes that code for 195 proteins. There is one structural
RNA. The plasmid is 206,479 nucleotides long, the GC content is 56% and
81% of the material is coding genes. There are no pseudogenes.
The modification of this plasmid is very important in the creation of
transgenic plants.
Genes in the virulence region are grouped into the operons virABCDEFG,
which code for the enzymes responsible for mediating conjugative transfer of
T-DNA to plant cells.
32. Though Ti plasmids are effective natural vectors they had certain limitations.
The phytohormone produced by transformed cells growing in culture
prevents their regeneration into mature plants. Hence auxins and cytokinin
genes must be removed from the Ti –plasmid derived cloning vector.
The opine synthesis gene must be removed as it m ay divert plant resources
into opine production in transgenic plant.
Generally, Ti- plasmids are large in size (200-800kb) For effective cloning,
large segments of DNA that are not essential for cloning has to be removed.
As Ti plasmid does not replicate in E.coli Ti-plasmid based vectors require an
ori that can be used in E.coli.
33. Binary vector
Plasmid DNA
VIR genes
Bacterial
ChromosomeBacterial ORI
t-DNA
Ampicillin
resistance
Construction of vector
with disired genes
34. To overcome these constraints, Ti plasmid based vectors were organized with the
following components:
A selectable marker gene that confers resistance to transformed plant cells. As these marker genes
are prokaryotic origin, it is necessary to put them under the eukaryotic control (plant) of post
transcriptional regul ation signals, including promoter and a termination- poly adenylation
sequence, to ensure that it is efficiently expressed in transformed plant cells.
An origin of replication that allows the plasmid to replicate in E.coli.
The right border sequence of the T-DNA which is necessary for T- DNA integration into plant cell
DNA.
A polylinker (MCS) to facilitate the insertion of cloned gene into the region between T-DNA
border sequences.
37. 1. T-DNA Region :This region has the genes for the Biosynthesis
of Auxin (aux), Cytokinin (cyt), and Opine (ocs)
T-DNA Border : A set of 24 bp sequence present on either side of T-DNA
2. Virulence Region : The genes responsible for the transfer of T-
DNA into the host plant are located outside the T-
DNA ant the region referred to as vir or virulence
region
3. Opine Catabolism region : Uptake and Metabolism of Opine
Organization of Ti-Plasmid
A B C D E G
39. virA- Transports acetosyringone into bacterium, activates virG post-
translationally (by phosphorylation)
virG - Promotes transcription of other vir genes
virD2- Endonuclease/integrase that cuts T-DNA at the borders but only on one
strand.
virE2 - Can form channels in membranes
virE1 - Chaperone for virE2
virD2 & virE2 also have NLSs, gets T-DNA to the nucleus of plant cell
virB - Operon of 11 proteins, gets T-DNA through bacterial membranes
Function of Vir genes
40. Opines
Derivatives of amino acids synthesized by T-DNA.
Ti plasmids can be classified according to the opines produced :
1. Nopaline plasmids
2. Octopine plasmids
3. Agropine plasmids
Nopaline plasmids : carry gene for synthesizing nopaline in the plant
and for utilization (catabolism) in the bacteria.
Octopine plasmids : carry genes to synthesize octopine in the plant
and catabolism in the bacteria.
Agropine plasmids : carry genes for agropine synthesis and
catabolism.
42. 1. Signal Induction : Wounded Plant cells release certain phenolic compounds which
are recognized as signals by agrobacterium.
Wound Phenolic compounds Signal
2. Attachment : The Agrobacterium attaches to palnt cells through polysaccharides, particularly via
cellulosefibers
Plant CellBacteria
Vir G VirB,C,D,E
3. Production of Virulance Proteins :
Signal Vir A
Vir A
43. 4. Production of T-DNA Strand : The right and left border of T-DNA are recognized by
D1/D2 proteins and these proteins involved in the
production of ss-DNA.
5. Transfer of T-DNA out of Agrobacterium : Vir B form transport apparatus and ss-T-
DNA in association with vir D2 exported from the
bacterial cell.
6. Transfer of T-DNA into plant cells and its Integration : In plant cell ss T-DNA get
covered with vir E2 for its protection. Vir D2 and E2 interact
with variety of plants proteins which influences T-DNA transport
and its integration into plant genome. (illegitimate
Recombination)
Transcription Translation Crown GallIntegration
44. B
B
A
G
D
2
D
2
D1/D2
D1/D2
B C D E
E2
SS-T DNA
Wounded Plant cell
E2
T
i
1
2
3 4
5 6
Transcription
Translation
Production of
auxin, cytokinin
and opine
Autostimulation of
Cell division
Bacteria
45. Making of Co-Integrate Vectors
In this strategy, both the T-DNA with our gene of interest and vir region are present in the
same vector used for transformation.
At first; an intermediate vector is made using E.coli plasmid + vir region + T-DNA borders +
origin of replication+pBR 322 sequences.
Second vector is a disarmed pTi vector = gene of interest+ some markers+pBR322 sequences.
Both intermediate vector and disarmed pTi has some sequences in common (pBR322
sequences).
Therefore by homologous recombination, co-integration of two plasmids will take place within
Agrobacterium.
Now we have a co-integrate vector that has both T-DNA with our gene of interest with in the T-
DNA borders and vir region. This complete vector is used for transformation eg:pGV2260.
There are two types of Ti plasmid vectors are used for genetic transformation of plant
they are cointegrate vector and binary vector.
47. Advantages of Co-integrate Vector
Target genes can be easily cloned.
The plasmid is relatively small with a number of restriction
sites.
Intermediate plasmid is convenientiy cloned in E.coli and
transferred to Agrobacterium.
48. Binary vector strategy: Two vector strategy
Here two vectors are used. This vector was devised based on the knowledge that vir region need not be in the
same plasmid along with T-DNA for T DNA transfer.
Binary vector consists of a pair of plasmids
1) A disarmed Ti plsmid: This plasmid has T-DNA with gene of interest + ori for both E.coli and
Agrobacterium. Also called as mini-Ti or micro Ti plasmid eg: Bin 19
2) Helper Ti plasmid has virulence region that mediates transfer of T-DNA in micro Ti plasmid to the
plant.
49. The binary vector system consist of an Agrobacterium strain along with a disarmed Ti plasmid called vir helper plasmid
(the entire T-DNA region including borders deleted while vir gene is retained). It may be noted that both of them are not
physically linked (or integrated). A binary vector with T-DNA can replicate in E.coli and Agrobacterium.
The binary vector has following components-:
1. Left and right borders that delimit the T-DNA region.
2. A plant transformation marker (PTM) e.g. npt2 that confers kanamycin resistance in plant transformed cells.
3. A multiple cloning site (MCS) for introducing target/foreign genes.
4. A bacterial resistance marker e.g. tetracycline resistance gene for selecting binary vector colonies in E.coli and
Agrobacterium.
5. Ori T sequence for conjugal mobilization of the binary vector from E.coli to Agrobacterium.
6. A broad host- range origin of replication such as RK2 that allows the replication of binary vector in Agrobacterium.
The target (foreign) gene of interest is inserted into the multiple cloning site of the binary vector.
In this way, the target gene is placed between the right and left border repeats and cloned in E.coli.
By a mating process, the binary vector is mobilised from E.coli to Agrobacterium.
Now, the virulence gene proteins of T-DNA of the vector into plant cells.
The binary vector system involves only the transfer of a binary plasmid to Agrobacterium without any
integration.
This is in contrast to cointegrate vector system wherein the intermediate vector is transferred and integrated
with disarmed Ti plasmid.
Due to convenience, binary vectors are more frequently used than cointegrate vectors.
50. Limitations of Ti – Plasmid
LARGE SIZE.
TUMOR INDUCTION PROPERTY.
ABSENCE OF UNIQUE RESTRICTION SITES.
51.
52. Ri Plasmid
The virulence plasmid of A. rhizogenes is commonly known as Ri- Plasmid (pRi).
Agrobacterium rhizogene is a soil borne , gram negative bacterium.
All strains of A. rhizogenes are known to produce agrocinopine.
Causes hairy root disease, i.e., massive proliferation of a highly branched root system.
This is used for obtaining large amounts of protein from genes cloned in plants
Ri plasmids are large (200 to greater than 800 kb) .
Contain one or two regions of T-DNA and a vir (virulence) region, all of which are necessary
for hairy root formation.
The Ri-plasmids are grouped into two main classes according to the opines synthesized by
hairy roots.
First, agropine-type strains induce roots to synthesise agropine, mannopine and the related
acids.
Second, mannopine-type strains induce roots to produce mannopine and the corresponding
acids.
55. D
D
irect gene transfer by precipitation of
NA onto the surfaces of protoplasts.
Direct gene transfer.
56. Limitations of cloning with Agrobacterium plasmids
Extensively used in dicots, but much more difficult to obtain the same
results with monocots.
But yet eventually artificial techniques for achieving T-DNA transfer
in monocots were devised.
Ease of recovery of plants varies with species.
Hence gene gun technique is being used
57. ATTEMPTS TO USE PLANT VIRUSES AS CLONING VECTORS
The potential of plant viruses as cloning vectors has been explored for several years, but without great success.
One problem here is that the vast majority of plant viruses have genomes not of DNA but of RNA. RNA viruses
are less useful as potential cloning vectors because manipulations with RNA are more difficult to carry out. Only
two classes of DNA virus are known to infect higher plants – the caulimoviruses and geminiviruses – and
neither is ideally suited to gene cloning.
Caulimovirus vectors
Although one of the first successful plant genetic engineering experiments, performed back in 1984, used a
caulimovirus vector to clone a new gene into turnip plants, two general difficulties with these viruses have
limited their usefulness.
59. The first problem is that the total size of a caulimovirus genome is, like that of λ, constrained by the need
to package it into its protein coat.
Even after the deletion of non-essential sections of the virus genome the capacity for carrying inserted DNA
is still very limited.
Recent studies have shown that it might be possible to circumvent this problem by adopting a helper virus
strategy, similar to that used with phagemids .
In this strategy, the cloning vector is a cauliflower mosaic virus (CaMV) genome that lacks several of the
essential genes, which means that it can carry a large DNA insert but cannot, by itself, direct infection.
60. Plants are inoculated with the vector DNA along with a normal CaMV genome.
The normal viral genome then provides the genes needed for the cloning vector to be packaged into virus
proteins and spread through the plant.
Although this approach has considerable potential it does not solve the second problem, which is the
extremely narrow host range of caulimoviruses.
This restricts cloning experiments to just a few plants, mainly brassicas such as turnips, cabbages and
cauliflowers.
Caulimoviruses have, however, been important in genetic engineering as the source of highly active
promoters that function in all plants and that are used to obtain expression of genes introduced by Ti plasmid
cloning or direct gene transfer.
61. Geminivirus Vectors
The geminiviruses are particularly interesting because their natural hosts include plants such as maize and
wheat, and they could therefore serve as potential vectors for these and other monocots.
But geminiviruses have presented their own set of difficulties, one problem being that during the infection
cycle the genomes of some geminiviruses undergo rearrangements and deletions, which would scramble up
any additional DNA that has been inserted – an obvious disadvantage for a cloning vector.
Investigations performed over the years have addressed these problems and geminiviruses are now
beginning to find some specialist applications in plant gene cloning.
One such application is virus-induced gene silencing (VIGS), a technique used to investigate the functions
of individual plant genes.
62.
63. The use of a geminivirus vector to silence a
plant gene via virus-induced gene silencing.
• This method exploits one of the natural defence
mechanisms that plants use to protect themselves
against viral attack.
• This method, termed RNA silencing, results in the
degradation of viral mRNAs.
• If one of the viral RNAs is transcribed from a cloned
gene contained within a geminivirus genome, then not
only are the viral transcripts degraded but also the
cellular mRNAs derived from the plant’s copy of the
gene . Consequently, the plant gene becomes silenced
and the effect of its inactivation on the phenotype of the
plant can be studied.
64. Genome maps of (a) African cassava mosaic virus (ACMV)
and (b) maize streak virus (MSV), representative of the
majority of dicot- and monocot-infecting geminiviruses,
respectively. Coding regions are referred to as virion-sense
(V) and complementary-sense (C) depending on their
orientation relative to the virion ssDNA. ACMV AVI and MSV
V2 encode coat proteins. ACMV AC1 and the MSV C1-C2
fusion protein are essential for viral-DNA replication, while
ACMV BVl and BCI, and MSV Vl participate in virus spread.
ACMV AC2 trans-activates coat-protein expression and AC3
is required for efficient viral-DNA replication. The functions
of ACMV AV2 and AC4 are unknown. Intergenic sequences
conserved between ACMV DNA A and DNA B (dark shading)
contain c/s-acting elements that participate in DNA replication
and the control of bidirectional gene expression. Positions of
the large intergenic region (LIR) and small intergenic region
(SIR) of MSV are indicated. Sequences that are dispensible
for viral-DNA replication are light-shaded; the ACMV
sequences are also dispensible for systemic infection of
Nicotiana benthamiana.
65. References
1. www.asgct.org › General Public › Educational Resources
2. www.link.springer.com
3. www.bios.net
4. Wikipedia
5. http://nptel.ac.in/courses/102103016/module3/lec23/2.html
6. Plant biotechnology – the genetic manipulations of plants 2nd edition by Adrian Slater, Nigel W.Scott & Mark R. Fowler
7. Principles of gene Manipulations & Genomics – Primrose & Twyman 7th edition
8. Joseph Sambrook, David Russell. "Chapter 1". Molecular Cloning – A Laboratory Manual 1 (3rd ed.)
9. https://www.ndsu.edu/pubweb/~mcclean/plsc431/cloning/clone3.htm
10. http://www.chemistrylearning.com/cloning-vector/
Editor's Notes
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