Promoters in Plant Genetic Eng

1. WELCOME
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2. BOT.515 : Plant Genetic Engineering
Presented by-
Group:4
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3. Topic 4
Promoters used in plant genetic engineering

4. Fig : Central Dogma of Life
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5. Transcription
Transcription is the first step in gene expression.It involves copying a gene’s
DNA sequence to make an RNA molecule .
Transcription is performed by enzymes called RNA polymerases,which link
nucleotides to form an RNA strand (using a DNA strand as a template)
Three steps of transcription-
1.Initiation
2.Elongation
3.Termination
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7. Promoter
In genetics,a promoter is a region of DNA that leads to initiation of
transcription of a particular gene.
An obvious requirement for any genes that are to be expressed as transgenes
in plants is that they are expressed correctly.
The major determinant of gene expression (level,location,time) is the region
upstream of the coding region,termed Promoter.
Promoters can be about 100-1000 base pairs long.
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9. Location of promoters
A promoter is a region of DNA that initiates transcriotion of a particular
gene.
Promoters are located near the transcription start sites of genes
,upstream on the DNA (towards the 5̍ region of the sense strand)
The promoter region controls when and where the RNA polymerase will
attach to DNA, so transcription can commence.
CONTINUE…..
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10. DNA sequences called response elements are located within promoter region, and
they provide a stable binding site for RNA polymerase and transcription fact.
In order to produce a transgenic plant ,an isolated promoter is inserted into a vector
and linked to a heterologous DNA sequence.
The presence or absence of protein in promoter can affect the strength of
promoter,such type of promoter are called regulated promoter.
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12. TATA Box
 The promoter region in general has a conserved sequence (T/A) or (A/T) at about -
25 to -30 base pairs (bp) of the transcription initiation point which is termed as TATA
box and elements near the promoters which are located approximately 100 (CCAAT)
and 200 base pair (GC box) above the start point of transcription.
It is a type of promoter sequence ,which specifies to other molecules where
transcription brgins.
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13. AGGA Box
Another sequence of DNA occur at -40 to -180 upstream of capsite

14. Portions of promoter
There are three main portions that make up a promoter-
1.Core promoter
2.Proximal promoter
3.Distal promoter

15. 1.Core promoter :The minimal portion of the promoter required to properly initiate
transcription.
2.Proximal promoter : The proximal sequence upstream(it is specific transcription
factor binding sites) of the gene that tends to contain primary regulatory elements.
3.Distal promoter :The distal sequence upstream of the gene that may contain
additional regulatory elements, often with a weeker influence than the proximal
promoter.
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17. Prokaryotic promoter
In prokaryotes,the promoter consists of two short sequences at -10 and -35
positions upstream from the transcription start site.
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18. Eukaryotic promoter
Eukaryotic promoters are extremely diverse and are difficult to characterize.
They typically lie upstrem of the gene and have regulatory elements several
kilobites away from the transcriptional start site.
Many eukaryotic promoters,contain a TATA box (sequence TATAAAA) , which in
turn binds a TATA binding protein which assists in the formation of the RNA
polymerase transcriptional complex.
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19. The TATA box typically lies very close to the transcriptional start site (often
within 50 bases).
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20. Functions of promoter
1.A region of DNA where transcription of a gene is initiated.
2.A vital component of expression vectors because they control the binding of RNA
polymerase to DNA.
3.RNA polymerase transcribes DNA to mRNA which is ultimately translated into a
functional protein.
4.The elements contained in the promoter sequences usually determine the correct
starting point of transcription.
5.Promoters are a key tool in biotechnological processes to ensure that expression of
a gene of interest is effective and regulated.
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21. Types of promoter
 Promoters used in biotechnology are of different types according to the intended
type of control of gene expression . They can be generally divided into four types .
such as-
1.constitutive promoter
2.Inducible promoter
3.Tissue specific promoter
4.synthetic promoter
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22. 1.Constitutive promoter
An unregulated promoter that allows for continual transcription of its associated gene
is called constitutive promoter.
It includes the expression of the downstream located coding region in all tissues
irrespective of environmental or developmental factors.
Its expression is normally not conditioned by endogenous factors.
These are usually active across species and even in kingdom.
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23.  Constitutive promoter are three types based on origin.These are-
 Viral origin
 Bacterial origin
 Plant origin
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24.  Viral origin
Promoters that originate from viruses are called viral origin promoter.some viral origin
promoter are-
i. CaMV 35s promoter (cauliflower mosaic virus)
ii. CsVMV promoter (cassava vein mosaic virus)
iii. 34s promoter from figwort mosaic virus (FMV)
iv. Sugarcane bacilliform badnavirus (ScBV) promoter
v. Carnation etched ring virus promoter
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25.  CaMV 35s promoter
 35s and 19s promoters of cauliflower mosaic virus are commonly used .
 35s promoter is a very strong constitutive promoter,causing high levels of gene
expression in dicot plants.
 It is less effective in monocot,especially in cereals.
 The differences in behaviour are due to differences in quality or quantity of
regulatory factors.
 Minimal promoter does not drive the expression of a gene by itself.
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26.  CsVMV (cassava vein mosaic virus) promoter
 Because of its constitutive properties,the CsVMV promoter can be used in plant
biotechnology as an alternative to the wider used 35s CaMV promoter.
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27. Bacterial origin
Promoters that originate from bacteria are known as bacterial origin promoter.some
bacterial origin promoter are-
1.octopine synthase (ocs) promoter
2.nopaline synthase (nos) promoter
3.mannopine synthase (mas) promoter
nos gene and ocs gene can be mentioned which respectively encoded the nopaline
synthase and octopine synthase both of Agrobacterium tumefaciens.
nos ,ocs are compact promoters less than 400 bp long.
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28. Promoters from the nopaline synthase(nos),octopine synthase(ocs),and mannopine
synthase(mas)genes have been isolated and inserted into transformation vectors
upstream of foreign genes to control the expression of those genes.
nos is the weakest promoter in the group while ocs and mas are stronger in the older
given.
The nos promoter is highest in older leaves,stem, and flower of tobacco .
Used mainly for transformation of dicotyledonous (dicot) plants.
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30.  Plant origin
In the case of plants,the most widely used constitutive promoters are the promoter
of the gene encoding ubiquitin of maize (ubi-1) and the actin gene from rice (Act-1)
The promoter of the rice Act-1 gene has been used as a strong constitutive promoter
drives the expression of gene of interest in monocots.
Plant promoters that are activated specifically when and where needed are ideal for
genetic engineering applications .
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Merits of constitutive promoters
1.They can be used to drive scorable/selectable reporter gene ,which are critical for
molecular biology studies,and for the development of transgenic plants.
2.Their use will be essential in the case of such proteins that are required in all
tissues and during all stages of plant development finally.
3.High level of production of proteins used to select transgenic cells or plants.
4.High level of expression of reporter proteins or scorable markers,allowing easy
detection or quantification.

32. 5.High level of production of a transcription factor that is part of a regulatory
transcription system.
6.Production of compounds that requires ubiquitious activity in the plant.
7.Production of compounds that are required during all stages of plant
development.
8.Specifically activated constitutive promoters are ideal for genetic engineering
applications.
9.Maize ubiquitin gene (ubi-1)promoter functioned in both monocot and dicot
plants.
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33. Limitations of constitutive promoter
The use of constitutive promoters causes unnecessary gene expression increasing
the possibility of interference with other routes of plant development .
 Solanum tuberosum contain the Atcbf gene regulated by the constitutive promoter
, the leaves showed reduced size, retarded flowering and reduction and lack of tuber
production.
Bacterial constitutive promoter affected bt hormones and wounding.
It cannot be controlled when needed.
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34. 2. Inducible promoter
Promoters that are activated in response to a specific physical factor e.g., light ,
temperature, heat, cold, wound etc or specific chemical compound are called
inducible promoters .
These promoters especially chemical inducible promoters provide fine control on
the regulation of gene expression.
Inducible promoters are a very powerful tool in genetic engineering because the
expression of genes linked to them can be turned on or of at certain stage of
development of an organism or in a particular tissue.
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35. A number of native plant gene promoters are sttimulus responsive ,e.g., Adhl
promoter (responds to anaerobic condition ),cab promoter and rbcs promoter (respond to
light), etc .But chemical inducible promoters are synthesized from promoter sequences
of different organisms.
Inducible promoters can be grouped into two categories . These are-
1.chemically regulated inducible promoter.
2.physically regulated inducible promoter.
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36. 1.Chemically regulated inducible promoter
Promoters whose transcriptional activity is regulated by the presence or absence of
alcohol,tetracycline,steroids,metal and other compounds.
It is important that chemically regulated promoters should be derived from such
organisms that are distantly related to plants as possible,e.g., from bacteria like
Escherichia coli ,yeast Drosophila ,and mammals.
there are virtually hundreds of inducible promoters that vary according to the
organism source and cells or tissues where they regulate gene expression.
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37. Alcohol regulated: It placed under the control of a strong constitutive promoter
such as CaMV 35s,and a modified alcA (alcohol dehydrogenase 1-Adh 1,encoded by
the alcA gene)promoter linked to a gene of interest or target gene.
Tetracycline regulated: A CaMV 35s promoter is modified by introducing a
tet-operator sequence upstream and downstream of the TATA box.
Pathogenesis related (PR)-proteins are a heterogenous group of proteins
induced in plants by pathogen infection and exogenous chemicals.
Chemicals such as salicylic acid,ethylene,thiamine have been identified as inducer
of PR protein.
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38. 2.Physically regulated inducible promoter
Promoters induced by environmental factors such as water or salt
stress,temperature,illumination and wounding have potential for use in the
development of plants resistant to various stress conditions.
These promoters contain regulatory elements that respond to such environmental
stimuli.
expression can be enhanced by the application of higher or lower temperature as
compared to the normal temperature conditions.
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39. Temperature regulated promoter : promoter linked to the chloramphenicol
acetyl transferase (CAT) coding sequence and construct introduced in tobacco and
thermo regulated expression of CAT activity examined in leaf extracts.
Light regulated promoter :In plants , light regulated promoters are critical in
regulating plant growth and development through the modulation of expression of
light responsive genes.
Light responsive elements from genes such as –small subunit of ribulose-1,5-
bisphosphate carboxylase-oxygenase (rbcS) gene ,chlorophyl a/b binding protein,and
the chalcone synthase have been widely studied.
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Merits of inducible promoter
1.It can easily be maintained.
2.It can easily be manipulated.
3.It can be used to drive the expression of transgenes of promote bacterial
disease resistance.
Limitations of inducible promoter
 Endogenous inducible promoters are leaky.

41. 3. Tissue specific promoter
 These promoters are capable of selectively expressing heterologous DNA
sequences in certain plant tissues. eg., anther specific promoter for induction of male
sterility, LEA protein promoters for gene expression during embryonic development.
 Fruit-specific promoter –
promoter region from the ethylene regulated genes E4 and E8 and from the fruit-
specific polygalacturonase gene have been used to direct fruit specific expression of a
heterologous DNA sequence in
transgenic tomato plants.
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42.  Root specific promoters-
 These promoters have been of particular use in engineering resistance to nematodes
and improving plant tolerance to environmentally stressful conditions such as water,
salt and heavy metals.
 An attempt to engineer resistance to the parasite in transgenic tomato plants, the
root specific promoters (tob) was used to direct the expression of the sarcotoxin IA .
• Result: sarcotoxin IA gene was selectively toxic to the parasite and non-toxic to the
plant.
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43.  Seed Specific Promoters-
 The majority of available seed specific promoters originate from seed storage
proteins (SSPs) such as rice glutelin & globulin, soybean lectin & β-phaseolin ,
Brassica napin , Maize zein .
 The genes that encode for the prolamin storage proteins are an ideal source for the
isolation of seed specific promoters as these proteins are exclusively synthesized in the
endosperm & are expressed at high levels during seed development in most cereals.
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44. Merits of Tissue specific promoters
Tissue-specific promoters are indispensable in such cases where it is desired to
limit the expression of trans gene to a specific tissue
Limitations of tissue specific promoter
Tissue-specific promoters of interest in genetic engineering strategies include those
associated with photosynthesis where expression is restricted to "green" tissues, and is
considerably lower or absent in non-photosynthetic tissues like roots, mature flowers
(excluding sepals) and mature fruit/nuts.
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