Genetic Analysis And Cloning Of Functional Genes Of Male Gamete Sterile Mutant In Rice

Rice is one of the most important crops in the world, and it is the model crop for the research of molecular biology. In recent years, transgenic rice have been widely study and application. However, lots of transgenic rice couldn't used in breeding for the problem which develoment of gametophyte due to the genetic instability of transgene. So, screening and research of the rice male gamete sterile mutant and cloning of functional genes is not only has important theoretical meaning in the development of rice pollen and biological study but also has important practical meaning in transgenic rice breesing.In this paper, we screened 216 mutant lines from transgenic rice lines. Genetic analysis of T-DNA transmission and the male sterile candidate mutants were analyzed by I2-KI staining of mature pollen grains, and in which the cry1Ac gene expression were tested by ELISA. A male sterile mutant pool, which caused by T-DNA insertion, was obtained. The male sterile mutants' flanking sequences were obtained by TAIL-PCR, and two candidate genes were cloned and construct expression vectors. Transgenic rice plants of the constructs were produced. The main results can be described as follows.(1) We screened more than 6000 transgenic rice (Oryza sativa L. ssp. indica) lines carrying different genes by antibiotic selection and found 216 mutant lines in which hygromycin resistance and sensitivity segregation ratios showed 1:1 by x~2-test. Genetic analysis of T-DNA transmission was conducted through self-pollination, testcross and/or reciprocal cross with wild type. The result found 57 mutant lines, in which T-DNA transmission through the female gametes, but not male gametes, were male gamete sterile candidate mutants, while 39 lines were female gamete sterile candidate mutants for T-DNA transmission through the male gametes, not female gametes.The male sterile candidate mutants were analyzed by I2-KI staining of mature pollen grains, and in which the cryIAc gene expression were tested by ELISA in continuous two years. A male sterile mutant pool, which included 38 male sterile mutants caused by T-DNA insertion, was obtained.(2) Of which 22 male sterile mutants' flanking sequences were obtained by TAIL-PCR and were analyzed using BLAST tool and annotation information in GenBank. We found only 15 different insertion sites in the 22 mutants, and T-DNA of several mutants were integrated into the same site. It indicated the possible existence of insertion 'hot spots'. Most of the insertion sites were in genic regions or genic regulation regions.(3) Two candidate genes, a WRKY transcription factor and a hypothetical protein, were selected to construct fusion vectors combined promoter of the candidate genes with gus gene, while over-expression vector was generated with cDNA of the hypothetical protein gene. Transgenic rice plants of the constructs were produced and further analysis will be conducted.