Cloning And Function Analysis Of Nsa CMS Candidate Restorer Gene In Brassica Napus

Rape is the main edible vegetable oil source, the use of cytoplasmic male sterility is the importantway to improve the yield, but there are some problems in actual application of infertility, such ascytoplasmic single of sterile lines, sterility is not stable. Nsa CMS is a new cytoplasmic male sterilitysystem produced in the process of exploring new sterile cytoplasmic, and it’s different with Pol CMS、Ogu CMS、nap CMS. Nsa CMS comes from somatic hybridization of S. arvensis var. Yeyou18and B.napus var. Zhongshuang4. The cloning of restorer gene has been the hot spot of infertility systemresearch. So far some restorer gene of plant were cloned and identified. Research shows that maybemore than one restorer genes in one palnt, and there are some homologous genes aroud the restorer gene.This study use Nsa CMS and its restorer lines as the main material, finding the homologous gene ofPPR618, which is cloned in our lab before. Determine the candidate restorer genes through the analysisof sequences and construct expression vector, then transferred into rape through the agrobacterium.Finding the restorer gene may as the basis of research that cytoplasmic male sterility mechanism offertility restoration, the interaction of cytoplasm and nuclear, and breeding excellent restorer lines. Themain results are as follows:1. A candidate restorer gene named PPR618of Nsa CMS was cloned based on the homologuesequencing strategy previously. In this study, based on sequence information of PPR618,22homologous sequences were identified in Nsa CMS male sterile line, Nsa CMS restorer lines, theoriginal fusion parents which gave rise to Nsa CMS and several other B. napus lines as well as one B.oleracea and one B. rapa accessions. Sequence analysis showed that there are two PPR618homologuesin each of the fusion parental lines, S. arvensis var. Yeyou18and B. napus var. Zhongshuang4, whereasthere were three, one, one, and one PPR618homologues in the four restorers, Hui1, Hui2, Hui3andHui4, respectively.2. The identity of the homologous genes in restorers was above93%to those in Yeyou18, but lessthan80%to those in Zhongshuang4, implicating the candidate restorer genes of Nsa CMS originatedfrom S. arvensis.3. After mitochondrial positoning signal analysis and compassion analysis using database, thecandidate restorer genes that PPRR1-1、PPRR1-2、PPRR1-3in restorer1and PPRR2in restorer2wereidentified. The homologous genes from restorers was also found to have relations with the restoringgenes for CMS system of radish and petunia.4. Semi-quantitative RT-PCR results showed that the candidate restoring gene expressed in alltested organs. The gene expression gradually increased along with the developmental process fromvegetative to reproductive stages, peaked in the bud of1.5–2.5mm in diameter, and slumped in rootsand stems. In contrast, the highest expression of homologous gene in male sterile line was detected instems.5. The positive and antisense expression vector of PPRR1-1and PPRR2were successfully constructed through double enzyme digestion with Sac Ⅰa nd Kpn Ⅰthen connection into PBI121.6. The expression vectors were transfered into competent cells of agrobacterium use thefreeze-thaw method, and the agrobacterium bacteria that resist to kanamycin and rifampicin wasobtained.7. The restorer genes PPRR1-1and PPRR2were transfered into Nsa male sterile lines and theirantisense genes into Nsa CMS restorer lines with the hypocotyls as receptor through agrobacteriummediated method. After culture, we obtained5resistant transgenic plants of PPRR1-1gene,23resistanttransgenic plants of antisense PPRR1-1gene,64resistant transgenic plants of PPRR2gene,33resistanttransgenic plants of antisense PPRR2gene.