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Identification and characterization of drought stress responsive genes from Pennisetum glaucum (L.) R. Br.

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Title Identification and characterization of drought stress responsive genes from Pennisetum glaucum (L.) R. Br.
 
Creator RAJENDRA PRASAD MEENA
 
Contributor Jasdeep Chatrath Padaria
 
Subject null
 
Description t-9777
Global warming is a matter of concern worldwide as it poses increasing risks to crop
growth and yield via biotic and abiotic stresses. Abiotic stresses such as drought,
salinity, cold and heat stress have detrimental role in agricultural production and
productivity. Presently, over 60 percent of yield losses in all the major crops are due
to various abiotic stresses. Identification and isolation of genes from the various
sources which can survive in extreme climatic conditions is one of the promising
approaches to develop stress tolerant crops. Pearl millet [Pennisetum glaucum (L.) R.
Br.] is one such crops which has a natural ability to withstand high levels of abiotic
stresses. It is a reservoir of many crucial genes and alleles, which can be utilized for
developing stress tolerant crops. Therefore, the present study was undertaken to
identify genes for drought tolerance from P. glaucum cv. 841B and further validate
these genes in the model plant Arabidopsis thaliana under drought stress conditions.
For this purpose, two genotypes P. glaucum cv. 841B and P. glaucum cv. 411B were
analyzed for their drought tolerance ability. Drought stress was given by water
withdrawal to both genotypes at flowering stage for different time intervals (10, 15,
20, 25 days). Various physiological and biochemical parameters such as relative water
content (RWC), cell membrane injury (CMI), malondialdehyde level (MDA),
chlorophyll content and proline content were analyzed in both the genotypes, which
showed that genotype P. glaucum cv. 841B displayed higher tolerance in
comparison to P. glaucum cv. 411B. Further, suppression subtractive hybridization
(SSH) library was generated in P. glaucum cv. 841B at flowering stage to identify
genes for drought tolerance. The forward SSH libraries were constructed from leaf
tissues of plants at different time interval of drought stress and drought responsive
genes were identified through SSH library. A total of 889 high quality drought stress
responsive expressed sequence tags (ESTs) were identified. These ESTs were
assembled into 346 unigene sequences with 60 contigs and 286 singleton sequences.
Gene Ontology search through Blast2GO analysis classified the unigenes on the basis
of molecular function, biological processes and cellular process. Most of the unigene
sequences showed homology to genes directly involved in abiotic stress responses.
Differential expression of 23 selected genes was validated by quantitative RT-PCR at
different time intervals (10, 15, 20, 25 days) of drought stress in both the genotypes P.
glaucum cv. 841B and P. glaucum cv. 411B. Most of the genes showed higher
expression levels in P. glaucum cv. 841B as compared to P. glaucum cv. 411B. Based
on expression analysis, three genes CaM, ASR1, DREB2A were selected for by 5' and
3' RACE. The genes PgASR1 and PgDREB2A were cloned in binary vector under
CaMV35 constitutive promoter and transformed in Agrobacterium tumefaciens.
Functional validation of PgASR1 and PgDREB2A, transformation was done in
Arabidopsis thaliana using floral dip method. The putative transgenic plants were
screened on kanamycin selection medium and presence of transgenes was confirmed
through PCR. Twenty days old T2 transgenic seedling were grown on MS media
supplemented with Mannitol 100 mM, Mannitol 150 mM; ABA 3 μM, ABA 5 μM;
NaCl 100 mM, or NaCl 150 mM as well as under cold (4°C) and heat (37°C) stress
along with wild type plants. ASR1 and DREB2A transgenic seedlings under stress
conditions showed better performance as compared to wild type seedling in terms of
root length and shoot length. Leaves were more greenish and bigger in size in
transgenic in comparison to wild type plants. These transgenic plants for PgASR1 and
PgDREB2A were subjected to water withdrawal stress for 15 days. It was observed
that RWC, chlorophyll, proline content were higher, while MDA and CMI were lower
in transgenic as compared to wild type plants. Overexpression of PgASR1 and
PgDREB2A genes in transgenic plants was confirmed by qRT-PCR. Thus, the present
study provides an insight into the drought responsive genes from pearl millet and
showed that ASR1 and DREB2A provided better tolerance to drought stress in
Arabidopsis. Further, these genes could be deployed for drought tolerance in other
crops.
 
Date 2018-10-09T04:50:07Z
2018-10-09T04:50:07Z
2017
 
Type Thesis
 
Identifier DIVISION OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY NATIONAL RESEARCH CENTRE ON PLANT BIOTECHNOLOGY ICAR-INDIAN AGRICULTURAL RESEARCH INSTITUTE NEW DELHI
http://krishikosh.egranth.ac.in/handle/1/5810075019
 
Language en_US
 
Format application/pdf