The study of flanking sequences generate methods and the insertion sites of rice T-DNA mutant lines

• Main Authors: chêng yao chung, 鄭堯中
• Other Authors: fan tsung ch'ên
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/89861213854565266510

碩士 === 亞洲大學 === 生活應用科學學系碩士班 === 95 === The researches engaged in the functional genomic of rice at present mainly used T-DNA as tag to understand the profits of rice gene and there function. In addition, for the purpose of reverse genetic, the research from gene sequence to the phenotype by T-DNA insertion the inseration sites flanking sequence is very importment.Which can let us understands the connection between phenotypes and their gene sequences of those mutane lines. The database of T-DNA insertion sites (Flanking sequence tag, FST) which can accelerate the research of the rice functional genomic, become an importment issue. In this research, we intend by develop a newly PCR method which was named as Hooker adaptor PCR to solute this problem. Totally 1301 lines were planted in the quarantine green house in Taiwan Agricultural Research Institute in this research. Among them 319 lines have obvious appearances different in phenotype. The CTAB method was used to extract plant DNA, after the genomic DNA was extract, the restriction enzyme and ligase was used, on the T-DNA insertion sites flanking sequence was generated by PCR method. The results shows, totally 885 bands was generated, among them 139 bands by Inverse PCR (IPCR),382 bands by adaptor ligation PCR(AL-PCR),and 364 bands by hooker adaptor PCR (HA-PCR). Those FST generates by IPCR, was showed by rice T-DNA mutant lines M48821-4 and M66317 as examples. After compare with TRIM, the results showed that the T-DNA was inserted in the Chr 4 and Chr 9 of rice respectively. We take M27669-8 as an example for those T-DNA inserted mutant lines their FST were generated by AL-PCR.The flanking sequences of RB and LB compared with TRIM BLAST, the result showed the RB and LB sequence were perfect inserted in the orderly closed location,which is located in rice Chr 8. As for Hooker adaptor PCR (HA-PCR) not only generated 364 bands but also succeed in solving the problem of FST which can not generated by IPCR or AD-PCR. We demonstrated by M60023 and M 60035 (2 copy) flanking sequences after blast in TRIM. The results show ed that M60023 have an insert in Chr 8. M60035 have two different T-DNA inserted which located at Chr 6 and Chr 9.From the results of 461 lines of TN67 T-DNA insertion mutants, the distribution of the T-DNA is mainly in the Chr 1, Chr 2, Chr 3 and the Chr 6. The Chr 9 and Chr 12 have the lease distribution.After analysis the cost and successed ratio, it showed that the success rate of IPCR is 17%,it take 3 days to get a line’s FST. Success rate for AL-PCR is 21%,it takes 3 days to get a line’s FST.The successful rate for HA-PCR was 56% it take one day to get a line’s FST.The result shows the HA-PCR not only easy to operate, lest cost and lease time consumption compare to the others. The HA-PCR is very efficient and novel technology of perfect at the present stage. We believe this method can be thought as the best method for generated T-DNA insertion sites flanking sequence of rice.