Selection And Clone Of Candidate Resistance Genes From Wild Rice

3 wild rice(Dong-xiang, Xiao-li, Cha-ling) and part of transformation-competent large fragment library of O.officinalis were used in this research which was about selection and clone on resistance candidate gene, the results are as follows:1 .The agrose gel electrophoresis result of Cha-ling, Xiao-li, Dong-xiang total DNA indicate that the way of CTAB to distill wild rice total DNA to be feasible, and the quality of total DNA can meet the gene transformation and clone of the wild rice and other molecular biology analysis requests; the difference of molecular weight of 3 wild rice total DNA is not significant, about 23 KB;2. 9 primers were selected by PCR on Dong-xiang, Xiao-li and Cha-ling 3 wild rice total DNA, the results indicated the PCR effect of the 6th (WR-1, WF-1) and 10th (S2,AS3) primer were the best, main behave the clearest banding and the least non-specificity band. So the 6th primer were used to PCR, recovery, clone, identify, sequence and analyze the total DNA of Cha-ling wild rice, by this way, 4 kinds of materials owned different resistant candidate genes were obtained; the 10th primer were used to PCR, recovery, clone the total DNA of Dong-xiang wild rice, 34 white recombine clone were obtained also;3.Througt the process of PCR with primer WF-1 and WR-l,the Cha-ling wild rice total DNA, the dosage of degenerate primer need some times more than common primer to achieve better purpose. 5 kinds of different ladder: 1 μL (12.5μmol),2μL (25μmol), 4μL (50 μmol), 8μL (100μmol),16μL (200 μmol) of degenerate primer dosage were compared, the result showed that the 16μL primer was the best, which have clearest banding and the least non-specificity band can meet the degenerate primers compound requirement;4.The resistance candidate gene were selected by 117 board of transformation-competent large fragment BIBAC2 library of O.officinalis using primer WF-1 and WR-1, we find out 5 clones(117M18, 117M19, 117M22, 117N2, 117N5) as resistance candidate gene, recovery, connection and sequencing analysis the two clones(117M18, 117N5) from them, we knew the two sequences have homology as high as 99 %, and their homology is as high 98% as that category(CL17) of 4 kinds of Cha-ling wild rice resistant candidate gene, it has very high...