Functional Analysis Of GhEXLB2 In Response To Drought Stress In Cotton

Cotton is an important economic and oil crop. It is also the primary source of textile material which has relative stress resistance. However, as environmental problems become more pronounced, soil salinization, and water shortage gradually become the restriction factors for cotton production and quality. Therefore, it is of great significance for agricultural production to cultivate drought-resistant cotton. Expansins are a large family of extracellular proteins in plant cell wall. The expansin superfamily of plant proteins is made up of four families, designated α-expansin(EXPA), β-expansin(EXPB), expansin-like A(EXLA), and expansin-like B(EXLB). Recent years have witnessed a rise in studies on involvement of expansin in plant in response to stress conditions, but its mechanism is still unclear. In the present study, an expansin-like gene Gh EXLB2, derived from a suppression subtractive hybridization library of cotton protoplast regeneration cell wall was characterized. The main results are as follows:Evolutionary tree analysis showed that GhEXLB2 was most homologous with TcEXLB1 with a similarity of 92%, and it had two typical domains of expansin proteins. Promoter cis-element predicting analysis revealed that many stress response elments, wound induction elments, dehydration and hormone response elements were found in the promoter region of GhEXLB2. Expression profiling analysis indicated that the expression of GhEXLB2 was induced by PEG, salt and ABA treatments.From our earlier research, we obtained two overexpression transgenic lines and four RNAi transgenic lines of GhEXLB2 in cotton. And homozygous lines were generated by selfing for several generations. These lines were then treated by drought stress factors on different growth stage. After PEG6000 treatment on germination stage, the length of main root and hypocotyl of overexpression lines was significantly longer than YZ1 and RNAi lines, and the H2O2 contents in overexpression lines were significantly lower than in YZ1 and RNAi lines. PEG6000 treatment at seedling stage resulted in lighter damage on overexpression lines than on YZ1 and RNAi lines, with low H2O2 and MDA content in overexpression lines. There was no remarkable difference between drought treatments on all lines at adult-plant stage. However, overexpression lines recovered the growth more quickly than YZ1 and RNAi lines after rehydration. These results showed that overexpression of GhEXLB2 enhanced the drought resistance of cotton.RNA-Seq profilings were conducted using the first leaf on the top of plants from adult-stage plants under drought and normal conditions(relative soil water content about 16%-19%). Contrastive analysis between each comparative group showed that there were 316 differential expression genes(DEGs) present. GO clustering analysis of these DEGs showed that they were mainly enriched in microtubules and cytoskeleton by means of annotation on ‘biological process’ and ‘molecular function’ level. Hierarchical clustering analysis of all tubulin genes expression in drought treatment group showed that most tubulin genes were up regulated in overexpression line, but down regulated in RNAi line, indicating that overexpression of GhEXLB2 in cotton could improve the transcription of most tubulin genes when plants suffered from drought stress. RT-PCR analysis of several genes related to drought resistance selected from differential expression genes was carried out. The results were consistent with RNA-seq analysis, especially the transcriptional level of α-tubulin gene(Gh_D02G1393), ACO(Gh_A07G0773) and ERF transcription factor(Gh_D07G0442). They didn’t show visible difference in the plants under normal conditions, but their transcriptional levels in overexpression line were significantly higher than YZ1 and RNAi lines under drought stress. Meanwhile, the protein of GhEXLB2 was used as bait to screen a yeast-2-hybrid library derived from cotton treated with different abiotic stress factors, and 19 positive clones that may interact with GhEXLB2 were obtained. Finally, point-to-point hybridization revealed that 7 proteins, including serine/threonine-protein kinase(Gh_D06G225), WD repeat-containing protein(Gh_A01G0493), polyubiquitin(Gh_D01G0586), nuclear transcription factor Y subunit B(Gh_D13G1387), ribose-5-phosphate isomerase(Gh_D07G2027), ubiquitin-conjugating enzyme E2(Gh_D11G0132), glutaredoxin(Gh_A10G0874), might interacte with GhEXLB2. Among them, genes encoding WD repeat-containing protein, polyubiquitin, ribose-5-phosphate isomerase, glutaredoxin were differentially expressed in RNA-Seq profiling.