Expression analysis of stress responsive pathway genes linked to drought hardiness in an adapted crop, finger millet (Eleusine coracana)

Drought is one of the major stresses limiting plant growth and productivity. Drought tolerance is regulated by multiple plant traits and examining the tolerance mechanisms from adapted species would assist in identification of novel pathways and superior genes. Since cellular tolerance is one of the major traits in drought acclimation we made in this study, an attempt to prospect candidate genes associated with the trait in drought hardy crop plant, finger millet (Eleusine coracana (L.) Gaertn). A novel gravimetric approach was employed to simulate field level drought stress for examining stress responsiveness of a few selected genes implicated in different stress response pathways. Gene expression was studied initially by e-northern analysis, and subsequently in leaf tissues experiencing different levels of drought stress by semi-quantitative and quantitative RT-PCR. A few stress responsive genes identified include metallothionein, farnesylated protein ATFP6, protein phosphatase 2A, RISBZ4 and farnesyl pyrophosphate synthase which probably have crucial roles in imparting hardiness to finger millet. Taken together the results suggest that multiple cellular tolerance pathways operate in a coordinated manner in drought tolerant crops.     

Generation and analysis of drought stressed subtracted expressed sequence tags from safflower (Carthamus tinctorius L.)

Drought is the most crucial environmental factor that limits productivity of many crop plants. Exploring novel genes and gene combinations is of primary importance in plant drought tolerance research. Stress tolerant genotypes/species are known to express novel stress responsive genes with unique functional significance. Hence, identification and characterization of stress responsive genes from these tolerant species might be a reliable option to engineer the drought tolerance. Safflower has been found to be a relatively drought tolerant crop and thus, it has been the choice of study to characterize the genes expressed under drought stress. In the present study, we have evaluated differential drought tolerance of two cultivars of safflower namely, A1 and Nira using selective physiological marker traits and we have identified cultivar A1 as relatively drought tolerant. To identify the drought responsive genes, we have constructed a stress subtracted cDNA library from cultivar A1 following subtractive hybridization. Analysis of?~1,300 cDNA clones resulted in the identification of 667 unique drought responsive ESTs. Protein homology search revealed that 521 (78?%) out of 667 ESTs showed significant similarity to known sequences in the database and majority of them previously identified as drought stress-related genes and were found to be involved in a variety of cellular functions ranging from stress perception to cellular protection. Remaining 146 (22?%) ESTs were not homologous to known sequences in the database and therefore, they were considered to be unique and novel drought responsive genes of safflower. Since safflower is a stress-adapted oil-seed crop this observation has great relevance. In addition, to validate the differential expression of the identified genes, expression profiles of selected clones were analyzed using dot blot (reverse northern), and northern blot analysis. We showed that these clones were differentially expressed under different abiotic stress conditions. The implications of the analyzed genes in abiotic stress tolerance are discussed in our study.     

Identification and annotation of abiotic stress responsive candidate genes in peanut ESTs

Peanut (Arachis hypogaea L.) ranks fifth among the world oil crops and is widely grown in India and neighbouring countries. Due to its large and unknown genome size, studies on genomics and genetic modification of peanut are still scanty as compared to other model crops like Arabidopsis, rice, cotton and soybean. Because of its favourable cultivation in semi-arid regions, study on abiotic stress responsive genes and its regulation in peanut is very much important. Therefore, we aim to identify and annotate the abiotic stress responsive candidate genes in peanut ESTs. Expression data of drought stress responsive corresponding genes and EST sequences were screened from dot blot experiments shown as heat maps and supplementary tables, respectively as reported by Govind et al. (2009). Some of the screened genes having no information about their ESTs in above mentioned supplementary tables were retrieved from NCBI. A phylogenetic analysis was performed to find a group of utmost similar ESTs for each selected gene. Individual EST of the said group were further searched in peanut ESTs (1,78,490 whole EST sequences) using stand alone BLAST. For the prediction as well as annotation of abiotic stress responsive selected genes, various tools (like Vec-Screen, Repeat Masker, EST-Trimmer, DNA Baser, WISE2 and I-TASSER) were used. Here we report the predicted result of Contigs, domain as well as 3D structure for HSP 17.3KDa protein, DnaJ protein and Type 2 Metallothionein protein.     

Identification and characterization of phospholipase D and its association with drought susceptibilities in peanut (Arachis hypogaea)

Preharvest aflatoxin contamination has been identified by the peanut industry as a serious issue in food safety and human health because of the carcinogenic toxicity. Drought stress is the most important environmental factor exacerbating Aspergillus infection and aflatoxin contamination in peanut. The development of drought-tolerant peanut cultivars could reduce aflatoxin contamination and would represent a major advance in the peanut industry. In this study, we identified a novel PLD gene in peanut (Arachis hypogaea), encoding a putative phospholipase D (PLD, EC 3.1.4.4). The completed cDNA sequence was obtained by using the consensus-degenerated hybrid oligonucleotide primer strategy. The deduced amino acid sequence shows high identity with known PLDs, and has similar conserved domains. The PLD gene expression under drought stress has been studied using four peanut lines: Tifton 8 and A13 (both drought tolerant) and Georgia Green (moderate) and PI 196754 (drought sensitive). Northern analysis showed that PLD gene expression was induced faster by drought stress in the drought-sensitive lines than the drought tolerance lines. Southern analysis showed that cultivated peanut has multiple copies (3 to 5 copies) of the PLD gene. These results suggest that peanut PLD may be involved in drought sensitivity and tolerance responses. Peanut PLD gene expression may be useful as a tool in germplasm screening for drought tolerance. The nucleotide sequence, reported in this paper, have been submitted to GenBank under accession number AY274834.     

毛尖紫萼藓干旱胁迫cDNA文库的构建

干旱胁迫是影响植物生长发育的主要环境因素,严重影响农作物的产量。解决这个问题的有效途径是培育和利用优良的抗旱品种。应用比较功能基因组学方法筛选抗旱相关基因,并通过基因工程培育抗旱品种已成为植物遗传资源与品种改良研究的重要内容。毛尖紫萼藓（Grimmia pilifera）是典型旱生藓类,生长在向阳的裸岩上,具有很强的抗旱能力,是很好的抗旱基因资源。本研究采用SMART技术构建毛尖紫萼藓干旱cDNA文库,文库滴度为2.8×10⁵ pfu·mL^-1,重组率为91.7%,插入片段大小为500~2 000 bp,平均为800 bp。通过测序我们获得了1 045条ESTs,其中高质量的996条,通过拼接获得875个Unigenes,为进一步筛选抗旱相关基因奠定了基础。