Cloning and Characterization of Stress-tolerant Genes from Cotton and Analysis of Differential Expression of Proteins in Cotton under Cold-stress
|School||Central China Normal University|
|Keywords||cotton (Gossypium hirsutum ) AQP Di19 protein stress plant gene engineering transgenic proteomic two-dimensional gel electrophoresis (2-DE)|
Drought, high salt and low temperature are major environmental factors that influence the growth and output of plant. These factors even damage plant irreversibly and lead to plant death. It is an important field of modern agriculture to study the mechanism of stress-tolerance, obtain related genes in plant and cultivate tress-tolerant crops through molecular biology, molecular genetics methods and so on. In this study, 3 stress-related genes were isolated from cotton. Then the expression and the preliminary function of the genes were analyzed. Meanwhile the different expression proteins of cotton under cold stress were studied by 2-DE. The results obtained are as follows:1. One aquaporin gene was isolated from more 30 putative stress-related genes of cotton and was designated GhTIP1. It contains 251amino acids and the MIP family signature sequence HVNPAVTFG At the amino acid level, the GhTIP1 shares high identity with other plant TIPs. For example, 90% identity to bobTIP26-1 (Brassica oleracea var. botrytis TIP) and 88% identity to Atγ-TIP (Arabidopsis thalianaγ-TIP). TIP belongs to tonoplast membrane intrinsic protein and plays a key role in water transportation. RT-PCR analysis showed that accumulations of GhTIP1 mRNA were abundant in roots and lower levels in other tissues, which was development-regulated in roots. Furthermore, GhTIP1 was regulated in cotton under drought, salt and cold stress. Further experiments showed that the pattern of GhTIP1 expression was different under different stresses and in different tissues. Especially, GhTIP1 transcript was increased in cotyledon, however, decreased in roots under cold treated, suggesting that the GhTIP1 expression in different tissues play an important role in different signal transduction pathways. Over-expression of GhTIP1 gene in yeast cells can improve yeast survival in cold stress but not affected in the osmotic stress. These indicated that GhTIP1 might be a functional gene in the cold stress tolerance of cotton. Expression vector of GhTIP1 was constructed and transformed into N. tabacum. The studies of salt and cold stress to the transgenic plants showed that salt sensitivity and cold tolerance was raised in transgenetic tobacco, compared with control. The drought stress of the transgenic plants is underway.2. By the method as above described, two GhDi19-Like genes (GhDi19-1, GhDi19-2) were obtained. They, consisting of 219 and 218 amino acid residues, respectively, share high identity with other known Di19 proteins. The expression of GhDi19-2 was higher than GhDi19-1 in cotton tissues and GhDi19-2 was predominant in fiber cells. Both of them were induced by drought and high salt treatment, but not influenced by cold treated. These results showed that the two genes expression patterns were similar. Northern-blot analysis indicated that the expression of GhDi19-1 was specifically induced in cotyledons but not in roots under NaCl treatment. In order to study the difference in 5’ regultation sequence of the two genes, the promoters were obtained using Genome walker PCR method and then were fused to a GUS reporter gene. Those make a contribution for the next study about the different activity and different regulation pattern of the two promoters under stress.3. The proteins of cotton (Gossypium hirsutum) were extracted by TCA- acetone method and the technique of two-dimensional electrophoresis (2-DE) in cotton proteomics was improved. Some satisfied pictures were obtained through optimizing many key procedures, such as sample preparation, sample dissolution, IPG stripe, IEF electrophoresis, SDS-PAGE. Then proteins from cold treated cotton cotyledon for 12 hours were extracted and separated by 2-DE. Then the expression pattern of protein from cotton under cold treatment and control was studied. A large quantity of protein spots appeared in pictures. PDQuest software was used to analyze the pictures. Compared with the control, there were 50 distinct points in treated sample, which 28 proteins were up-regulated in stress and 22 proteins were down-regulated. Suggesting these proteins were related to cold stress.