chloroplast genome and their role in modern biotechnology

1. WelcomeWelcome

2. Dr. BalasaheB sawant KonKan
Krishi viDyapeeth,Dapoli
College of Agriculture, Dapoli

3. Topic:- Chloroplast genome and it’s importance.
Course No. :- MBB-501
Course Title:- Principles of Biotechnology.

4. Presented By:- Mr. Umesh Ramdas Phondekar.
Reg. No. :-ADPM/18/2608
M.Sc.(Agri.)
DEPARTMENT:-PLANT PATHOLOGY

5. - organelle found in plant cells and eukaryotic
algae
- The main role of chloroplasts is to
conduct photosynthesis, where the photosynthetic
pigment chlorophyll captures
the energy from sunlight and converts it and stores it
in the energy-storage
molecules ATP and NADPH while
freeing oxygen from water
- Photosynthesis.
IntroductionIntroduction:-:-
Chloroplast:

6. Abstract:
Chloroplasts play a crucial role in sustaining life on earth.
The availability of over 800 sequenced chloroplast genomes from a
variety of land plants has enhanced our understanding of chloroplast
biology, intracellular gene transfer, conservation, diversity, and the
genetic basis by which chloroplast transgenes can be engineered to
enhance plant agronomic traits or to produce high-value agricultural or
biomedical products.
In this review, we discuss the impact of chloroplast genome
sequences on understanding the origins of economically important
cultivated species and changes that have taken place during
domestication.

9. Chloroplasts visible in the cells of Bryum capillare, a
type of moss

10. - Chloroplast DNA (cpDNA) is also known as plastid
DNA (ptDNA).
- Circular double stranded DNA molecule
- Chloroplast genome size ranges 120-217kb with
majority of plants fall into 120-160kb. (Pelargonium
has a chloroplast genome size 217kb)
- contain about 100 genes to synthesize proteins
- cpDNA regions includes Large Single-Copy (LSC)
& Small Single-Copy (SSC) regions, and Inverted
Repeats (IRA & IRB).
- Conifers and a group of legumes lack Inverted
Repeats.
Chloroplast genomeChloroplast genome

11. IRA
IRB
LSC
SSC

12. - Complete chloroplast DNA sequences of four land
plants (Nicotiana tabacum, Marchantia polymorpha,
Oryza sativa and Epifagus virginiana) were available
for comparative study on structure and gene content
of chloroplast genomes in 1980s.
- At present, the number of complete chloroplast
genome sequences is 122 (from 114 different
organisms).
eg. Arabidopsis thaliana, Coffea arabica, Eucalyptus
globulus, Glycine max, Gossypium hirsutum,
Helianthus annuus, Lycopersicon esculentum,
Nymphaea alba, Phaseolus vulgaris, Pinus koraiensis,
Piper cenocladum, Solanum tuberosum, Triticum
aestivum, Vitis vinifera, Zea mays etc.
Chloroplast genomeChloroplast genome

13. - cpDNA is a relatively abundant component of
plant total DNA, thus facilitating extraction
and analysis.
- Conservative rate of nucleotide substitution
enables to resolve plant phylogenetic
relationships at deep levels of evolution.
eg. familial level; mono- & dicotyledonous;
- Chloroplast protein-coding genes evolve at a
rate that is on average fivefold slower than
plant nuclear genes.
Characteristics of Chloroplast GenomeCharacteristics of Chloroplast Genome

14. - Strictly maternally inherited in most
angiosperms while in conifers, inheritance
is paternal.
- Chloroplast DNA is passed on from one
generation to the next with only an
occasional mutation altering the molecule;
sexual recombination does not occur.

15. - cpDNA regions can be amplified by means
of PCR.
- The resulted PCR products may be
subjected to RFLP or DNA sequencing.
- Common cpDNA regions used in
systematic study:
rbcL (1400bp), trnL-trnF (250-800bp), atpB-
rbcL (1000bp), trnL intron (300bp), matK
(2600bp), trnT-trnL (400-800bp), 16S (1400bp),
rpoC (3600bp) etc.
Molecular Systematics on cpDNAMolecular Systematics on cpDNA

16. IRA
IRB
LSC
SSC
rbcL
matK
atpB-rbcL
trnL intron
trnL-trnF
trnT-trnL
rpoC
16S

17. - Restriction site mapping of
the entire chloroplast
genome. (involve the
isolation of chloroplast
DNA from the total DNA)
Molecular Systematics on cpDNAMolecular Systematics on cpDNA
The whole chloroplast genomes of
different Brassica species were
digested with SacI

18. - Singular structural rearrangements (e.g.
inversions and intron losses).
- Loss of intron of rpl2 gene was found in
species of order Caryophyllales (cacti,
amaranths, carnations, carnivorous plants).
Molecular Systematics on cpDNAMolecular Systematics on cpDNA

19. - On of the most comprehensive phylogenetic
study of cpDNA rearrangement involved a
22kb inversion found to be shared by 57
genera representing all tribes of the family
Asteraceae (sunflowers), a large plant family
with 20,000 species and 1100 genera.
- 50kb inversion brought psbA closer to rbcL in
legumes.
- 25kb inversion brought atpA closer to rbcL in
wheat.
Molecular Systematics on cpDNAMolecular Systematics on cpDNA

20. H. nervosa
H. dyeri
H. dryobalanoides
H. beccariana
H. pierrei
H. latifolia
H. mengerawan
H. myrtifolia
H. ferruginea
H. sangal
H. nutans
H. odorata
H. helferi
H. apiculata
H. wightiana
Neobalanocarpus heimii
69
84
89
100
75
5192
Subsection
Hopea
Subsection
Dryobalanoides
Subsection
Sphaerocarpae
Subsection
PierreaHopea clade
Dryobalanoides
clade
Outgroup
72-bp deletion
in the trnL-trnF
H
o
p
e
a
D
r
y
o
b
a
l
a
n
o
i
d
e
s
Phylogeny based on the trnL-trnF, trnT-trnL and atpB-rbcL sequences.
Upuna borneensis
H. subalata
72
96
H. bilitonensis
Tree length = 143
CI = 0.8811
RI = 0.8651
H. pubescens

21. Chloroplast genome for biotechnology
Conferring stress tolerance
n the past decade, chloroplast genetic engineering has focused
primarily on the overexpression of target genes with the potential
to enhance biotic stress tolerance, which is very important for
plant protection and yield enhancement. Yield loss due to insect
pests can be very serious in many countries. In addition to cotton
bollworm resistance conferred by hyper-expression of Bt protein
in chloroplasts [137], there are many other striking recent
examples of improved biotic stress tolerance.

22. Retrocyclin-101 and Protegrin-1 protect against Erwinia soft rot and
tobacco mosaic virus (TMV), which result in yield loss in several
cultivated crops [140]. Whitefly and aphid resistance has been
accomplished by expressing β-glucosidase [141], which releases
insecticidal sugar esters from hormone conjugates. Multiple
resistances against aphids, whiteflies, lepidopteran insects, and
bacterial and viral pathogens were achieved by expressing
the Pinellia ternata agglutinin (PTA) gene in the chloroplast genome
[142]. More than 40 transgenes have been stably integrated into and
expressed within the chloroplast genome, conferring important
agronomic traits, including insect resistance in edible crops cabbage
(Brassica oleracea) [143], soybean [144, 145], and eggplant
(Solanum melongena) [146].

23. Definition: A chloroplast
genome is a circular, double-
stranded DNA molecule located
in stroma of chloroplast.
Chloroplast genomes are highly
conserved among plant species.
There is more than one copy of
genome in each chloroplast. The
exact number varies during
development, but mesophyll
cells in young leaves contain
about 100 copies of genome.

24. Origin: All plastids studied to date contain their own
DNA, which is actually a reduced "genome" derived from
a cyanobacterial ancestor that was captured early in the
evolution of the eukaryotic cell. The genomes of the
organelles have maintained many of the features of their
prokaryote ancestors. They are circular, like prokaryotes,
and do not form complex structures like chromatin of
nuclear genome. Some of the genes are organized as
operons, with common regulatory element or promoter.

25. Function: Most of the chloroplast genome are involved in either protei
synthesis, or photosynthesis (note that most of the genes encoding
proteins participating in photosynthesis are located in nucleus). Many o
genes are in clusters, allowing expression in form of large polycistronic
mRNA transcripts, which are processed to oligo- or monocistronic
mRNAs.

26. Example: Let's have a look at chloroplast genome of maize for
instance.

27. The chloroplast genome of
maize (Zea mays) is 140,387
base pairs. It contains a pair if
inverted repeats, IRa and IRb,
which are separated by small
single-copy region (SSC of
12,536 bp) and large single-
copy region (LSC of 82,355 bp)
. Genes drawn outside the circle
are transcribed clockwise.

28. Applications in Genetic engineering: Chloroplasts are
maternally inherited. This fact is exploited for clean
gene technology . There is no danger to any gene
transfer to related weedy species through pollen. It also
ensures very large gene copy number. One of the major
additional advantage is absence of reports of gene
silencing with chloroplast transformation. These facts
combined with ability of chloroplast to correctly fold
and crosslink transformed proteins, means that there is
tremendous potential in chloroplat transformation for
very high level gene expression and synthesis of active
proteins.

29. Chloroplast genomes are typically 100–200 kbp in size, and include a set
of genes for proteins essential to photosynthesis. Other genes present in
the ancestral symbiont have been lost or relocated to the nucleus.
Chloroplasts originated in the ancestor of plants and red and green algae
by endosymbiotic acquisition of a cyanobacterium, and then spread to
many other eukaryotic lineages.
In many organisms, gene transfer from chloroplast to nucleus can still
take place, at an unexpectedly high frequency.
Many nonphotosynthetic organisms retain remnant chloroplasts, with
genomes, reflecting the fact that photosynthesis is not the only
biochemical process that takes place in the chloroplast.
In many organisms, gene transfer from chloroplast to nucleus can still
take place, at an unexpectedly high frequency.
Establishment of the chloroplast has resulted in the development of
nuclear encoded RNA binding protein families and, in land plants,‐ ‐
additional RNA polymerases that play a central role in chloroplast gene
expression.