The Structure And Function Analysis Of The Gene Cluster Involved In Cotton Reproduction Development

Cotton is a kind of strategic crop, it is always a focus that how to improve cotton yield and quality. The development of cotton reproductive organ directly affect the economic value of cotton, so studying cotton reproductive development in-depth is a very important breakout for breeding high yield good quality cotton. Base on the research ago, we know that arf1 and nodulin-like genes is very close to each other in genome, and they both express in cotton reproductive organ specificly, so we deduce that there is a gene cluster correlative with cotton reproductive development around arf1 and nodulin-like genes. Aiming at this problem, we carry on a series of researches, the target is understanding the mechanism of cotton reproductive development deeply, providing theory basis and physical foundation for breeding high yield good quality cotton.First of all, we designed three pairs PCR primers base on arf1 and nodulin-like genes, and use these primers to screen cotton 18R bacterial artificial chromosome library by PCR pool screen. At last, we got two BAC clones include this two genes, G2-J-15 and G128-K-15. Restriction endonuclease analysis shows that the inserting segments of these BAC clones are 38kb and 90kb respectively, and we finished the sequencing of G2-J-15 BAC clone.The sequencing and bioinformatics analysis of G2-J-15 show that there are seven genes in the inserting segment, average 5kb lies a gene, we can conclude this gene cluster locates in a gene rich area. Except for arf1 and nodulin-like genes, by gene structure prediction and BLAST, we found four gene never be cloned in cotton, include GhGolS(galactinol synthase gene), GhAPm(clathrin adaptor complexes medium subunit family gene), GhPsbP(photosystem II reaction center PsbP family gene) and GhNeuF (neutral beta-fructofuranosidase gene), and a new gene it never be cloned in any species. By a series of bioinformatics analysis, we had understood their gene structure and protein character, furthermore, we got some information about their protein structure and function. By these bioinformatics analysis, we found a very interesting phenomenon that the arrangement and direction of GhGolS, GhAPm and GhNeuF in cotton genome is the same with their homologous genes in Agrobacterium tumefaciens, even there are two genes between GhAPm with GhNeuF, and also there are two genes between their homologous genes in Agrobacterium tumefaciens. The conservative structure of this gene cluster maybe indicate the function of this gene cluster is conservative too, and the gene cluster is necessary for cotton development.We studied the spatio-temporal expression pattern of these gene by real-time PCR, the result indicates that except for GhPsbP expresses in leaf specifically, the other four genes all express in cotton reproductive tissues specifically: GhGolS expresses in anther and 35dpa fiber specifically, GhAPm is predominant in 30dpa fiber, the expression of unknown gene is highest in anther, and the expression of GhNeuF is highest in 25dpa fiber. Contacting the spatio-temporal expression pattern of arf1 and nodulin-like gene, we had proved our supposition is correct, this gene cluster is correlative with cotton reproductive development.We had gotten the cDNA of GhGolS and GhAPm by RT-PCR, the length are 1816bp and 1590bp respectively, the gene structure prediction of GhGolS is not exactly, the gene structure prediction software treated a 90bp exon as intron, however, the prediction result is the same with cDNA approximately, on the other hand, the gene structure prediction result of GhAPm is the same with cDNA exactly.We have constructed the overexpression vectors of GhGolS and unknown gene, the RNAi vectors of silencing cotton GhGolS and unknown gene, the RNAi vectors of silencing Agrobacterium tumefaciens AtGolS2 and neutral beta-fructofuranosidase gene, and the RNAi vector of silencing Agrobacterium tumefaciens AtGolS2, clathrin adaptor complexes medium subunit family gene and neutral beta-fructofuranosidase gene together. Transferred these vectors to cotton and Agrobacterium tumefaciens respectively, and got the Agrobacterium tumefaciens T1 seeds, and the cotton T0 seeds is being screened. All of these will help us to understand the function of this gene cluster in cotton reproductive development.This paper found a gene cluster is correlative with cotton reproductive development, this gene cluster includes seven gene, and this gene is close to each other, we carry on a series of research, all of these is the foundation for studying cotton reproductive development deeply, and maybe will providing new theory and technique for breeding excellent cotton varieties.