The Cloning Of The Gene In Brassica Napus L. That May Interact With ATP 6 And The Constructing Of Plant Expressing Vector

Cytoplasmic male sterility and nucleus controlled fertility restoration are wide--spread plant reproductive features. Cytoplasmic male sterility not only offers goodmaterials for studying the interactions between nuclear and cytoplasmic genes, butalso plays an important role in utilizing heterosis in crops. So, the researches thatstudy the genes which have correlations with the cytoplasmic male sterility and thecreation of the plant male sterility line by engineered method has great value in appl--ying CMS in the production of hybrid seed.Brassica napus is widely grown as the oilseed crop of rape or canola, its hete--rosis are evident. So it has great value in applying Brassica CMS in the productionof hybrid rapeseed. Now, studies of CMS in Brassica napus are mainly focused onthree male sterile cytoplasms: ogu, nap, and pol. Both because the "Polima" or polcytoplasm confers a relatively temperature stable male sterility and because of theavailability of restorer genotypes this system appears to be the most advantageous forhybrid rapeseed production.The cytoplasmic male sterile (CMS) line of Brassica napus, pol confers arelatively temperature stable male sterility and because of the availability of restorergenotypes, it is considered to have great research utility for hybrid rapeseed production. Investigation of its molecular mechanism has demonstrated that ATP6,subunit of the ATP synthase, is related with CMS.The atp 6 gene that correlated with Brassica napus pol CMS was used as thebait protein, by using yeast two-hybrid system, we have screened the Brassica napus.cDNA library, identified the proteins interacting with ATP 6.One of the positive clones has been studying in this experiment. The result ofsequencing showed that the sequence obtained by yeast two-hybrid system is thegene 3'end sequence, not the gene complete sequence. Based on the knownfragment,rapid amplication of cDNA ends(RACE)was applied to amplify its 5'end.Sequencing and Gene bank blasting demonstrated it is still a fragment. Based on thesequence obtained by RACE, we designed primers, cloned the unknown sequence byChromosome Walking technology. The original pieces obtained from the yeasttwo-hybrid,RACE and genome walker were joined. According the assemblysequence we designed the primers: 359-Up-primer. 5'-ATG ATG GAT ACT TTCCCT CCT TCG-3'; 359-Down-primer. 5'-TCA CCA GAA GGG TAA CAA AACACA AG-3'. the DNA and cDNA were used as the templates separately for the PCR,the fragment were amplified and ligated vector pMD 18-T. Sequence analysis showedthat, the cDNA sequence is 1011bp, the DNA sequence is 1172bp and the introns are161bp,it has two sequences, one is 79bp and the other is 82 bp. Blast researchshowed that putative conserved domain (Gly-co_hydro₁7) has been detected.It washomologus with a gene encoding an unknown protein from Arabidopsis thaliana atthe ratio of 88%, next it had 84% identities with Putative glucanendo-1, 3-beta-glucosidase 1 precursor from Arabidopsis thaliana. Multiplebiosoftwares are used for bioinformatics analysis. The comparisons indicate that thetarget have the following characters: (1) contains no signal peptide sequence; (2) ithas two membrane-spanning domains.Because the gene sequence has not been reported, and the its function inBrassica napus is unknown. So we digested the pMD18-T-gene plasmid byPstâ… /BamHâ… , then the digested gene fragment was ligated to pSK vector which hadbeen digested by Pstâ… /BamHâ… . The recombinant plasmid was digested by EcoR â…¤/BamHâ… or EcoRâ…¤/Xbaâ… seperately. As follows, the digested gene fragmentinserted into the plant express vector-pCAMBIA 2301G. At present we have insertedthe gene to plant express vector-pCAMBIA 2301G and construct recombinantplasmid. We are ready to transform the Brassica.napus by Agrobacterium tooverexpress the target gene or inhibit it. By analyzing the change of fertility to studythe gene function in Brassica. napus cytoplasmic male sterility.