T-DNA Tagging Male Sterile Mutant Identification And Related Genomic Sequence Cloning In Upland Cotton (Gossypium Hirsutum L.)

At present, Arabidopsis, rice, corn plant genome sequencing has been completed, this huge genetic information linked to gene function has become the focus of biology scientific research. There are many kinds of strategy in plant gene cloning and functional analysis,one of which is using kinds of mutant. Since that spontaneous mutant is in very low frequency and difficult to find out, it becomes the main method using physical, chemical and biological technology approach to induce mutant and Agrobacterium-mediated T-DNA tagging is the most effective and most widely used means. Notable success has been achieved on elucidation the physiological, developmental problems of dicotyledon through studying with important traits in Arabidopsis mutant. Genes cloned by T-DNA tagging account for 40 percent in Arabidopsis. With studying on the function of these genes we can improve crop yield, quality and resistibility.Cotton (Gossypium spp.) is the world's most important cash crop, improving cotton production and the quality of fiber has an important economic benefits. It becomes the main means to improve the lint yield and the quality of fiber by using heterosis that had proved to be existed within different cotton varieties. However, most of the current hybrid varieties of large-scale cultivation are still artificial pollination. And high cost of the cotton hybrid seed production has become the major limiting factor. Several types of cotton male sterile had been identified during the past years, but they are hardly to be used in hybrid cotton seeds production. Therefore, cloning male fertility-related genes and used to create the ideal male sterile line is a new way of improving the cotton yield and quality by cotton heterosis application.In this present paper, a male sterile mutant was got by Agrobacterium tumefaciens-mediated T-DNA insertion. Male sterility phenotype was caused by single copy of the T-DNA insertion which is proved by Southern hybridization together with PCR analysis. To investigate the phenotype of the T-DNA insertion male sterile mutant, the process of development of male gametes of the mutant was observed by paraffin sections. It was found that there was no abnormality in the gamategenesis process from pollen mother cells to tetrad when compared the mutant with the wild type plant. The remarkable abnormality was found in the late stage of the gamategenesis in which the four small spores did not separate after released from callose.In order to study the molecular basis of the male sterile mutant, TAIL-PCR, inverse PCR and genomic library screening were carried out to clone the genomic sequence that flanked the T-DNA and a total of 2240 bp genomic sequence was obtained. To see if there was any genomic structure change resulted from the T-DNA insertion, the insertion site sequence and the original genomic sequence (pre-insertion site sequence) were compared by sequence alignment. It was found that the T-DNA insertion led to a 115 bp deletion and the partial sequence recombination at the right border side of the T-DNA. Bioinformatics analysis on the cloned original sequence (2240bp) in cotton genome showed that a sequence of 125 bp upstream the T-DNA right border is homologous with 71% to the Arabidopsis snoRNA and a 444 bp sequence downstream the T-DNA left border is homologous with 94% to an upland cotton mRNA. The function of sequence is needed to be studied further.