| Cotton is one of the main important economic crop, widely grown in the world.Cotton fiber is an important raw material source for textile industry, chemical industry and some strategic materials. In the process of the development of cotton fiber,there are a large number of genes involved in regulation, but also by the regulation of plant hormones, such as BR. Therefore, the research of the cotton fiber development genes function, further improves the quality of the cotton fiber, has an important significance for national economy and people’s livelihood.By yeast two hybrid, we take the cotton 14-3-3e as a bait protein, screening cDNA library of cotton fibers, so get a GhMYBL2 protein with the cotton 14-3-3e interaction,and its function in the fiber development are studied. the results are as follows:1. Sequence analysis of GhMYBL2 and construction of expression vectorThe gene of coding GhMYBL2 protein has an open reading frame of 1086bp, and the length of the gene (cDNA) is 1217bp. comparing the protein sequence in NCBI website, It has two MYB structural domain. Based on expression in cotton GhMYBL2 analysis, the expresson of GhMYBL2 is the highest in 9d of cotton fiber. In order to further analyzing the biological function of this protein, so we constructed 35S-GhMYBL2 and TUA9-GhMYBL2 overexpression vectors to transform into cotton and Arabidopsis thaliana, and constructed the 35S-GhMYBL2, TUA9-GhMYBL2 and RDL1-GhMYBL2 RNAi vectors to transform into cotton.2.Trichome analysis of GhMYBL2 transgenic arabidopsisStudies have suggested the fiber development in cotton and the trichome development in Arabidopsis thaliana are similar, so the 35S-GhMYBL2 overexpression vector was transformed into Arabidopsis’ thaliana, to analyze of the phenotypes of trichome. we found that the trichomes of transgenic Arabidopsis have more 2 branches than wild type. gll mutants have no trichome, so the 35S-GhMYBL2 overexpression vector was transformed into gll mutants. We found the T1 and T2 generation of transgenic Arabidopsis still have no trichome, and have trichome in some strains of T3 generation of transgenic arabidopsis, but the number is still less than wild type,indicating that GhMYBL2 is able to partially restore the gll mutant phenotype. BRI is the key receptor in the BR signal path. bril mutants is small, so we expect to the 35S-GhMYBL2 overexpression vector was transformed into bril mutants, to analyze the phenotype. The experiment is underway.3. Phenotypic Analysis of GhMYBL2 transgenic CottonThe 35S-GhMYBL2 and TUA9-GhMYBL2 overexpression vectors were transformed into cotton, at the same time, the 35S-GhMYBL2, TUA9-GhMYBL2 and RDLl-GhMYBL2 RNAi vectors were transformed into cotton. A large number of TO transgenic cotton plants were obtained. At present, TUA9-GhMYBL2 overexpression and RNAi transgenic cotton seeds (T1 and T2 generations) have been planted in field and greenhouse to further analyze the phenotypic and genetic stability of transgenic plants.The T2 generation of transgenic cotton seeds are planted in greenhouse, and we made the positive appraisal to it for further analysis. Cultivating ovule after 12 days in vitro, we found the growth condition of RNAi strains fiber is better than the wild type,and overexpression strains and wild type has not obvious difference. After treatment with BL, the fiber length of wild type and transgenic cotton increased, but the length of overexpression strains increased more obviously. Taking the ovule of 1 day after flowering to slice observation, we also found that the fiber raised of RNAi transgenic strains on the surface of the ovule is significantly faster than the wild type, whereas overexpression strains are not obvious. A comparison of the size of the peach after 9 days of flowering revealed that the RNAi transgenic strains were larger than the wild type.Analysis of mature cotton fiber length, the result showed that RNAi transgenic cotton fiber was longer than wild type, and overexpression of transgenic cotton fiber was shorter than that of wild type. It was speculated that GhMYBL2 might be a negative regulator in cotton fiber development. In addition, we found a cotton gene GhEXP1, which down-regulated the expression in overexpression transgenic cotton, whereas the expression level in RNAi transgenic cotton was up-regulated.5.Polyclonal antibody preparation of GhMYBL2 and related experimentsWe constructed the pET32a-GhMYBL2 vector, and it was transformed into E.coli BL21 to protein-induced expression and purification. After purifying a large number of target protein,we began to immune to a negative rabbit,immuning three times in a row,so we obtained GhMYBL2 polyclonal antibody. when the dilution ratio of polyclonal antibody is in 1:4000～1:6000 by Western blot experiments,we can obtain a good experiment effect. We extracted various tissue proteins of wild type cotton, found the specific band of GhMYBL2 protein were detected in cotton fiber samples after 10 days of flowering, which indicated that the protein might be involved in the development of cotton fiber elongation. |
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