Study On The Role Of GhKNL1 And GhMYBL1 Transcription Factors In Fiber Development Of Cotton (Gossypium Hirsutum)

Cotton as the world’s most important natural fibre and oil crops, has a wild production areas, and it distributes from tropical to subtropical regions. Cotton has very high economic value, for example it can be used in textile, food, paper making, etc. Cotton fiber is the outer epidermis cell of highly elongated and thickened. The content of cellulose in mature cotton is as high as 95%. cotton fiber provides an ideal model system for the study of cell elongation, cellulose synthesis and cell wall synthesis. the development stages of Cotton fiber can be divided into four stages:initiation, elongation, secondary wall thickening and maturity. Initiation stage affects the density of cotton fiber, elongation stage affects the length of cotton fiber, and the secondary wall thickening stage affects the quality of cotton fiber. We focused on the study of secondary wall thickening stage, our study will provide theoretical foundation for improving the quality and yield of the cotton fiber.This paper continued to look insight into the the roles of GhKNL1 and GhMYBLl that play during the development of cotton fiber. The main results are as following:1.GhKNLl transcription factor plays a role of negative regulation in the process of cotton fiber developmentIn order to further study the function of GhKNL1 gene during cotton fiber development, we constructed the GhKNL1 RNAi vector and transfered it into the cotton explants. We spended a lot of time on conducting the tissue culture and regeneration, Finally we got the transgenic cotton plants. Through the phenotype analysis of the T1 generation of transgenic cotton plants, we found that comparing to the wild type cotton, the fiber of transgenic cotton is significant longer, the short fiber is more intensive, and the size of the seeds is a little bit bigger. It means that GhKNL1 gene is a transcription inhibitor which negatively regulate the development of cotton fiber.2.GhKNL1 regulated the expression level of the genes that related to the development of secondary cell wall in cotton fiberwe used the RT PCR to analysis the changes of the genes which related to fiber development in the transgenic plants,. The results show that the expression of 1,3-β-G gene increased slightly, the expression of XTH was down-regulated, and most of the genes that related to the synthetic of secondary cell wall were up-regulated significantly. These results suggested that GhKNL1 regulate the development of cotton fiber.3.The construction of GhMYBL1 vectors and the genetic transformation of cottonIn the previous work,we Cloned and identified a cotton gene-GhMYBL1, and analyzed the functions of the gene in arabidopsis thaliana at the same time. In order to further study the role of GhMYBLl in the development of cotton fiber, we constructed the RNAi vector and 3 kinds of over expression vectors, all of them were conducted the genetic transformation of cotton. Now, we have already got the GhMYBL1 RNAi transgenic cotton plants. Among the 40 cotton plants of TO generation,there are 25 positive plants. And we found that the transgenic cottons of T1 generation showed a special phenotype:compared to the wild type cotton, the leaves of transgenic cottons is down-curve significantly. The cotton materials of other vectors were at the stage of non-embryogenic callus and we will get the embryogenic callus soon.4. GhMYBL1 protein influences the promoter activity of CelAl and IRX12To further study how the GhMYBLl protein works, and what kind of cell type it will regulate, we proceeded the cross-fertilize experiment.PC1301-CelAlp::GUS(CelAl Promoter fused with GUS gene) and PC1301-IRX12p::GUS (IRX12 Promotrr fused with GUS gene) transgenic arabidopsis thaliana cross with the GhMYBL1 overexpression transgenic plants respectively.Experimental results showed the GUS signal was much stronger after the cross-fertilize of CelAlpro, and GUS signal was turned down after the crossing of IRX12pro. So we proposed the suppose that the GhMYBLl protein can influence the activity of CelAl promoter and IRX12 promoter.5. GhMYBLl directly binds to the SMRE site of CelA1 promoter in vitro.In order to study whether GhMYBL1 protein can regulate the expression of CelAl through recognizing the SMRE site in CelAl promoter, we experimented with EMSA. we induced and purified the GhMYBLl protein, and then we designed the bio-probe based on the SMRE site sequence in CelAl promoter. And the EMS A experiment showed that GhMYBLl can directly bind to the SMRE site of CelAl promoter in vitro. The results indicated that the GhMYBLl may regulate the development of cellulose synthetic directly and play a role in the secondary cell wall synthesis.