A Study On Cloning And Functional Complementation Of ABCG15in Rice (Oryza Sativa L.)

Rice male sterility is a widespread phenomenon in plants, receives much concerned because of the successful application of hybrid rice.Breeders get sterile materials through various methods, study the mechanism of infertility, to get control of male sterile trait genes, by cloning of the gene, transforming and combining ability of use to get wide CMS, used for cross breeding, is of great value.Rice male sterility genetic mechanism is complex, anther development, the synthesis of substance metabolism are involved. This study used to Sichuan H2S recessive sterile material of fertility control it by cloning the gene on the chromosome of the sixth, a45kb band,between markers Inde140520and RM20366. Analysis of candidate genes for7of the band’s final preliminary locking it for the number of LOC-Os06g40550candidate genes, and their function is involved in the transport of fatty acids, gene named ABCG15.The experiment succeeded in building the complementarity of rice ABCG15gene expression vector pOsAct2-ABCG15, recessive nuclear male sterile mutant of H2S from Sichuan material for the receptor, using Agrobacterium tumefaciens-mediated genetic transformation of valuable transgenic plants. Get complementary materials laid the Foundation for in-depth study of rice ABCG15gene. Experimental study on the main results are as follows:1Sichuan recessive genic male-sterile mutant material for observation and identification of H2S, its anther small, colorless flood, other floral organs are normal, after iodine dye performance of pollen sterility.2using scanning electron microscopy (SEM) and semi-thin sectioning technique to observe Sichuan recessive nuclear male sterile mutant of H2S anther separation of materials and their wild-type material for cytological observation of epidermal cells. It turns out, from small spores begin to anther mature wild type epidermal cells in anthers of material gradually appeared on the surface roughness of the reticular formation, and mutant materials is basically the whole process was smooth. Biopsy results, abnormalities of the small spores after tetrad, tapetum degradation delay, ultimately resulting in no pollen sterility.3according to the prediction of ABCG15on rice TIGR gene sequences of CDS designed primers, PCR reaction, respectively, final amplification full length cDNA fragment was about2100bp.4clones are ABCG15on the gene expression of full length cDNA fragment attached to carrier pOsAct2-1-nos, expression vector pOsAct-ABCG15are complementary.5Transfer the complementary expression vector of pOsAct-ABCG15imported by using Agrobacterium-mediated genetic transformation of recessive genic male-sterile mutant of H2S to Sichuan material for spike induction of embryogenic callus, through tissue culture, resistance selection, differentiation and rooting and plant regeneration.6Using PCR amplification of the ABCG15gene to identify regeneration of transgenic plants is confirmed by full-length cDNA ABCG15gene fragment properly inserted into a receptor in the rice genome, and successfully got5positive transgenic rice strains.7Potassium iodide dyes for the fertility of ABCG15gene transgenic plants have positive identification, iodine dye showed ABCG15gene transgenic plants’s fertility is restored. Behind the access to complementary rice ABCG15gene transgenic plants ABCG15gene functions provide the assurance of further research.