Isolationand charatarization of stress responsive genes from stress-

Views:
 
     
 

Presentation Description

By: Prakash Venkateshgowda Department of crop Physiology University of Agricultural Sciences GKVK, Bnagalore-65 OBJECTIVES:  Identification of tolerant species and developing stress protocols for stress transcriptome profiling.  Construction of subtracted drought stress responsive cDNA library and screening of stress responsive clones by reverse northern expression for sequencing.  e-Northern analysis of upstream regulatory genes against Arabidopsis microarray data of whole genome under drought, salt and cold stress conditions.  Validation of putative sigma factor for stress responsiveness and through Virus Induced Gene Silencing approach. Stress responsive genes from tolerant species may provide better protection of cellular structures, these novel potential candidate genes will improve tolerance of susceptible species

Comments

Presentation Transcript

PowerPoint Presentation:

Drought is one of the most serious world-wide problems for agriculture. Four-tenths of the world's agricultural land lies in arid or semi-arid regions. Other agricultural regions have consistently low rain-fall and rely on irrigation to maintain yields In both circumstances, crop plants which can make the most efficient use of water and maintain acceptable yields will be at an advantage. DROUGHT

PowerPoint Presentation:

Genetic and physiological mechanisms that control stress tolerance Potential physiological, biochemical and architectural modifications that will allow crops to escape, avoid – Stress avoidance A complex trait, is the result of the co-ordination of physiological and biochemical alterations at the cellular and molecular level - Stress tolerance

PowerPoint Presentation:

Resurrection Plant ( Selaginella lepidophylla ) Finger millet Groundnut Outsourcing for stress genes Stress tolerant crops

PowerPoint Presentation:

Resurrection plants:- Unique stress adaptive mechanisms due to specific candidate genes associated with stress tolerance Diminished growth rates and perennial nature Crop Plants:- Some of our recent work and publications supports the fact that : “Stress responsive genes from tolerant species may provide better protection of cellular structures, these novel potential candidate genes will improve tolerance of susceptible species”

PowerPoint Presentation:

In this background: Work was carried out, to clone and characterize stress genes from Finger millet for better understanding of stress tolerance. Those candidate genes may play role in stress tolerance, so as to alter the response of plant to environmental constraints. “ Isolation and transcription profiling of finger millet drought stress responsive genes and functional characterization through VIGS approach”

PowerPoint Presentation:

OBJECTIVES: Identification of tolerant species and developing stress protocols for stress transcriptome profiling. Construction of subtracted drought stress responsive cDNA library and screening of stress responsive clones by reverse northern expression for sequencing. e-Northern analysis of upstream regulatory genes against Arabidopsis microarray data of whole genome under drought, salt and cold stress conditions. Validation of putative sigma factor for stress responsiveness and through Virus Induced Gene Silencing approach.

PowerPoint Presentation:

Identification of tolerant species and developing stress protocols for stress transcriptome profiling. 1 A) Identification of tolerant species: it is clear that significant variation exists among the species and Finger millet was more tolerant than Rice, Cowpea and Sunflower.

PowerPoint Presentation:

100%FC 20%FC 80%FC 60%FC 40%FC B) Developing stress protocol for stress transcriptome expression: We have assessed the leaf, Membrane integrity- EC - 17% leakage – less under severe stress Osmotic potential- MPa - reduced from –0.82 to –1.62 MPa under severe stress Relative water content - % - 51.74% in severe stressed plants- but sustained growth These results indicates, the stress induction procedures is efficient to achieve the main goal of developing stress protocol for optimum expression of stress responsive genes.

PowerPoint Presentation:

Extent of Stress induction by expression of NCED (9-cis-epoxycarotenoid dioxygenase) C 20% 40% 60% Total RNA Actin Ahd N CED With increase in severity of stress, there was increase in expression of NCED. With maximum expression under severe stress. Inference: The results suggested that tolerant crop (finger millet), in spite of reduced RWC, there is relatively less membrane leakage, Correspondingly increased leaf area during stress recovery indicating finger millet is more tolerant than Rice followed by Cowpea and Sunflower

PowerPoint Presentation:

RNA isolated from leaves of severely stressed (20%FC) and a control (100% FC) plants was used to carry out subtractive hybridization. Adapter ligation and Modified Smart Kit protocol. Construction of subtracted drought stress responsive cDNA library and screening of stress responsive clones by reverse northern expression for sequencing. 2 Stress adaptation is mainly due to differential expression of SRG. The stress cDNA library was constructed by following …

PowerPoint Presentation:

27 Recombinant clones - Dot blot expression studies Ec DS clones probed with cDNA prepared from RNA of drought stressed plant leaves (maintained at 20%FC) Ec DS clones probed with cDNA prepared from RNA of well irrigated plant (maintained at 100%FC) Stress Control Amongst the 27 clones tested, Only 4 clones were moderately expressed under control and 8 clones were highly expressed under 20% FC. Later all 27 clones were sequenced and annotated against Nucleotide EST database in NCBI - Resulted in 13 clones - Table

PowerPoint Presentation:

Modified Smart Kit : We have screened around 500 recombinants with insert size > 400bp Resulted in about 280 clones. Dot blot expression studies: cDNA prepared from RNA of pooled drought stressed plant leaves maintained at 20%, 40% and 60%FC and probed cDNA prepared from RNA of control plant leaves maintained at 100%FC and probed

PowerPoint Presentation:

Dot blot analysis -/+ = No expression ++/+++ = moderate Expression +++/++++ = High Expression Not Expressed moderately Expressed Highly Expressed Dot blot analysis depicting the expression of Ec DR clones in control and stressed plants. Most of the clones (71 %) were not expressed under control conditions, but 57 % were moderately expressed and 31 % were highly expressed under stress condition.

PowerPoint Presentation:

All the clones with insert size more than 400 bp whether or not expressed in dot blot were sequenced, later analyzed by annotation Homology analysis:

PowerPoint Presentation:

The maximum number of clones under cellular communication, signal transduction and transcription -accounts to 22%, Cell rescue, defense and DNA processing proteins accounts to 14%. The genes functioning under energy and metabolism together accounts to 17% Only 4% of the genes falling under development and cellular organization. 17% of genes were unclassified category – expressed, but hypothetical, having homology to DNA and protein sequences- Novel SRG,s

PowerPoint Presentation:

List of accession numbers of Finger millet subtracted drought stress cDNA clones in EST data base, obtained by Modified Smart kit protocol and Adapter ligation methods . DV549313 – DV549319, DV565207 – DV565213 DV635137 – DV635149, CV478067 – CV478069 EB390428 – EB390462, EB637072 – EB637139 EB643372 - EB643429, EB643629 - EB643657 EB739719 - EB739738

PowerPoint Presentation:

Adapter ligation method: Stress responsive nature of the clones by dot blot studies revealed that nearly 50% of the clones were found to be stress responsive Upregulation under stress was detected for ATP dependent DNA helicase , amino oxidase , putative sigma facter2 suggesting possible role of these genes under stress The number of clones obtained was very less because of redundancy- Many PCR cycles, Only severely stressed RNA was used, Due to redundancy cloning efficiency is less Modified smart Kit protocol: The library constructed by modified SMART kit protocol was efficient. Expression analysis by dot blot revealed that most of the clones are stress responsive Inference:

PowerPoint Presentation:

e-Northern analysis of upstream regulatory genes against Arabidopsis microarray data of whole genome under drought, salt and cold stress conditions. 3 Various transcriptional regulatory mechanisms function under different stress signal transduction pathways Variety of genes induced by various abiotic stresses, function not only in stress tolerance but also in regulation of gene expression and signal transduction in stress responses - TF’s Characterization and analysis of these stress-inducible transcription factors should provide more information on the complex regulatory gene networks that are involved in different stresses

PowerPoint Presentation:

So only 43 upstream regulatory genes were selected for e-northern analysis against Arabidopsis microarray data base at www.bbc.botany.utoranto.ca . Relative expression of Ec DR upstream regulatory genes in Arabidopsis under (A) Drought, (B) Salt and (c) Cold We confirmed that a total of 25 genes were stress inducible, and among them, 6,10 and 9 genes were drought , salt and cold inducible respectively It was noticed that certain degree of similarity between drought and salt stress regulated transcripts expression, but dissimilarity in expression under drought and cold stress regulated transcripts was observed

PowerPoint Presentation:

One specific clone Ec DR37 (Proline-rich extensin like protein) was highly over expressed in both drought and salt but moderate expression in cold. The genes induced by drought stress in various species are also induced by salt and low temperature leading to adaptation, this suggests the existence of similar mechanisms in response to stress.

PowerPoint Presentation:

Validation of putative sigma factor for stress responsiveness and through Virus Induced Gene Silencing approach. 4 Plastid encoded RNA poly. called PEP, Present in chloroplast which has one of the core sub unit, nuclear encoded - Sigma Factor PEP is responsible for transcription of photosynthetic genes in plastids. Specific Sigma factors are responsible for activation under abiotic stress conditions. Activation of BLRP under abiotic stress conditions through the function of specific Sigma factors. ( plant cell physiol. 45(4): 357-368, 2004 ) BLRP activation drives the expression of PSII genes

PowerPoint Presentation:

C 20% 40% 60% Ec SIG2 Total RNA A) Stress responsive nature of Ec SIG2 RT-PCR in Finger millet C 20% 40% 60% Ec SIG2 Total RNA Northern analysis in Groundnut Fold change over control Quantitative RT-PCR in Finger millet

PowerPoint Presentation:

m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PCR amplification of putative sigma factor ( EC SIG2) in different monocot and dicot crop species. m=1 kb marker, Monocots: 1= Finger millet,2= Rice,3= Wheat,4= maize,5= Oat,6= Bajra,7= Barn yard millet, Dicots: 8= Soybean9= Red gram10= Horse gram11= Field bean12= Bengal gram13= Cowpea and 14= Beans

PowerPoint Presentation:

Zm SIG2A Ec SIG2 Os SIG2A At SIGB Zm SIG2A Ec SIG2 Os SIG2A At SIGB Zm SIG2A Ec SIG2 Os SIG2A At SIGB Alignment of the deduced amino acid sequence of Ec SIG2 with different putative sigma factor2 sequences using ClustalW a multiple alignment programme. 93% Per cent identity of Ec SIG2- a putative sigma factor observed with sigma factor2A of maize (Zea maize) and it also has complete 3’ end. The deduced amino acid sequence of Ec SIG2, 132 stretch of amino acid also showed 66% and 53% identity with Rice and Arabidopsis SIG2 sequences with 3’ complete sequence

PowerPoint Presentation:

Since Sig2-1 mutant showed pale green phenotype and reduced chlorophyll content ( Nucleic Acids Research, 2003 ) Pale green phenotype can be seen morphologically by silencing the Sigma factor endogenously in the plant system. B) Functional validation of Ec SIG2 through VIGS approach Understanding of functional relevance of SRG will provide the basis for engineering the strategies for stress tolerance

PowerPoint Presentation:

LB RB 2X 35S 134 K 194 K mP 16 K RZ NOS pTRV1 A EcoR1, Xba1 , Stu1, Nco1, BamH1 , Kpn1, Sac1, mtu1, Xho1, Srf1, Sma1 2X 35S CP MCS RZ NOS LB RB pTRV2 B pTRV2:: Ec SIG2 2X 35S CP Ec SIG2 RZ NOS LB RB C Diagrammatic representation of pTRV vectors, (A) pTRV1: 2X35S = duplicated CaMV promoter, open reading frame correspond to 134 and 194 kDa replicase, movement protein (mP) and 16 kDa cystein rich protein, RZ= self cleaving ribozyme, NOS= nopaline synthase transcriptional terminator, RB and LB refers to right and left borders of T-DNA. (B) pTRV2: open reading frame corresponds to (CP), MCS= multiple cloning site. (C) Ec SIG2 gene placed in between XbaI and BamHI site of pTRV2 MCS resulting pTRV2:: Ec SIG2 driven by 2X35S promoter. Cloning of EC SIG2 into pTRV2 vector

PowerPoint Presentation:

m 1 2 3 4 850 bp Confirmation of cloning Colony PCR analysis of recombinant clones obtained from bacterial transformation of pTRV2:: Ec SIG2 into agobacterium strain GV2260. m 1 2 424 bp Restriction digestion analysis of pTRV2:: Ec SIG2 plasmid DNA upon digestion with XbaI and BamHI restriction enzymes. P E Dot blot of the positive colonies of pTRV2:: Ec SIG2. All above results indicating that Ec SIG2 was cloned into pTRV2

PowerPoint Presentation:

M 1 2 3 424 bp Amplification of putative Sigma factor ( Ec SIG2) from Finger millet and Nicotiana benthamiana genomic DNA. M=Marker, 1&2 = Finger millet genomic DNA, 3= N.benthamiana genomic DNA Ec SIG2 homologues in Nicotiana benthamiana

PowerPoint Presentation:

Control Mock Sigma Factor Sigma Factor M C M I 824 bp 400 bp RT-PCR analysis showing the confirmation of systemic virus spread in fresh leaves Infectivity of pTRV2:: Ec SIG2 agrobacterium cultures leaf infiltrated in Nicotiana banthamiana. 10 days of post inoculation

PowerPoint Presentation:

M C I Phenotypic symptoms of Ec SIG2 silencing in Nicotiana banthamiana showing pale green on freshly formed leaves. The plants were photographed after 14 days of inoculation showing pale green color in upper fresh leaves. M 1 2 3 4 5 6 RT-PCR expression analysis showing reduced transcript levels in leaves of Nicotiana benthamiana infected with pTRV2 :: Ec SIG2 inserts M=marker, 1=RNA from control plants, 2= RNA from vector plants and 3 -6 = RNA from pTRV2:: Ec SIG2 infiltrated plants.

PowerPoint Presentation:

M C pTRV2:: Ec SIG2 Virus-induced gene silencing of Ec SIG2 in Nicotiana benthamiana showing pale green on freshly formed leaves on whole plant. After 24 days post inoculation plants showing pale green leaves were photographed 1 2 3 4 5 6 Ec SIG2 Total RNA Northern expression analysis showing reduced transcript levels in Nicotiana benthamiana infected with pTRV vectors carrying Ec SIG2 inserts. RNA was extracted from plants of Nicotiana benthamiana after 14 days post 1= RNA of control plants, 2 -5 = RNA of pTRV2:: Ec SIG2 infiltrated plants and 6= RNA of vector control plants.

PowerPoint Presentation:

Measurement of photosynthetic gas exchange parameters There was significant reduction in photosynthesis in Ec SIG2 silenced plants when compared to control. The total chlorophyll content as assessed by SPAD meter reading also showed reduction compared to control Subsequently photosynthetic efficiency using A/gs analysis showed reduction in photosynthetic efficiency in silenced plants compared to mock and control,

PowerPoint Presentation:

Response of silenced plants to salt stress A B Relative levels of chlorophyll content of Ec SIG2 silenced plants of Nicotiana benthamiana subjected to different concentrations of NaCl stress. The reduction was linearly correlated with the increasing salt concentration. Results showed reduced Chla and Chlb in silenced plants compared to control.

PowerPoint Presentation:

OUT COME OF THE STUDY A stress protocol for maximum expression of stress responsive genes have been developed. The stress cDNA library was diverse, with maximum number of clones under cellular communication and signal transduction and transcription altogether accounts to 22%, cell rescue, defense and DNA processing proteins accounts to 14%. The genes functioning under energy and metabolism together accounts to 17% and only 4% of the genes falling under development and cellular organization. The unknown function category includes genes with unknown function in plants but they are expressed, hypothetical having homology to DNA or protein sequences are grouped into Unclassified which accounts to 17%.

PowerPoint Presentation:

Based on e-northern analysis we show that only a few Arabidopsis homologues showed higher expression either in cold, salinity of moisture stress suggesting that stress specific genes expressed in finger millet were different from those of Arabidopsis stress genes reported. We demonstrated in this study that VIGS approach could be a potential option to validate the stress responsive genes. For the first time we show that Ec SIG2 ( Ec DS 75) a putative sigma factor2 involved with a bacteria type multisubunit RNA polymerase (PEP), is upregulated under stress. In VIGS silenced plants with reduced transcript levels of Ec SIG2 substantially affected photosynthetic characters inferring its role in regulating the expression of plastid encoded photosynthetic genes. Ec SIG2 was identified as sigma factor2 as a core component of plastid encoded RNA polymerase. Ec SIG2 showed 93% homology with dicot crop species maize sigma factor2. Sigma factors are key regulatory factors in transcription of photosynthetic genes.

PowerPoint Presentation:

First best poster presentation award, for “Molecular and functional characterization of putative sigma factor in finger millet for desiccation response” in 2005 Deliverables “Isolation and Functional Characterization of Stress Responsive Genes from Stress Adapted Species Finger millet” --- Presented in International seminar on January.2006. Osmania University Hyderabad. Isolation and functional characterization of putative sigma factor in Finger millet for desiccation response. Manuscript in preparation…. Manuscript in preparation Finger millet EST sequences deposited in NCBI database

PowerPoint Presentation:

Acknowledgements Chairman and members of ADC Department of Crop Physiology AP Netherlands Biotechnology programme

authorStream Live Help