Clonning and Functional Analysis of WRI1Gene from Brassica Napus L.
|School||Hunan Agricultural University|
|Keywords||Brassica napus L. WRI1clone Arapdopsis thaliana genetictransformation yeast expression|
Oilseed rape is the most important oilseed crop planted widely in China, as well as a major source of edible vegetable oil for human. WRI1encoding WRINKLED1transcription factor is involved in the regulation of Glycolysis and fatty acid biosynthesis in plants. Six AtWRI1-Like WRI1cDNAs were isolated from Brassica napus Xiangyou NO.15by RT-PCR and named BnsWRI1-1,BnaWRI1-2,BnaWRI1-3BnaWRI1-4,BnaWRI1-5,BnaWRI1-6respectively. Amino acid sequence analysis shows that BnaWRI1-6could not encode full-length protein. Alignment of the coding regions of BnaWRI1-1, BnaWRI1-2, BnaWRI1-3, BnaWRI1-4and BnaWRI1-5detected40single nucleotide polymorphic sites. The nucleotide transition from A to G at positon892in the coding sequence of BnaWRI1-1, BnaWRI1-2, BnaWRIl-3and at position880in the coding sequence of BnaWRI1-4, BnaWRI1-5led to a specific site of restricted cleavage. Digestion profile of the WRI1CDS from Brassica napus, Brassica rapa and Brassica oleracea produced with Bel I confirmed that the AtWRI1-Like WRI1cDNAs of and Brassica oleracea origin were recognised and digested, while that of Brassica rapa origin could not. The results indicated that it was possible to distinguish between the AtWRI1-Like WRI1in A and C genome of Brassica napus by Bel I cleavage.Two different sequences were used to construct seed-specific expression vector. The recombinant pMD18-T Simple vector which contain WRI1cDNA sequence and The recombinant pGEM-T Easy vector which contain Napin promoter sequence were digested by BamH I/Sac Ⅰ and Hind Ⅲ/BamH I respectively.Target fragments were ligated into the corresponding sites of the pBI121which named PNW2and PNW4respectively. Recombinant seed-specific expression vectors were identified by restriction enzyme and PCR. PNW2and PNW4were transfered into LBA4404with heat shock method. Genetic transformation of Arapdopsis thaliana was performed by floral dip method. The To seeds were screened on medium with50mg/L kanamycin and the anti-kanamycin plants were inditified by PCR. BnaWRI1-3、BnaWRI1-5and BnaWRI1-6were ligated to the vector pYES2.0named PYESW3、PYESW5、PYESW6, which transformed to yeast NFNY.The results of SDS-PAGE electrophoresis shows that BnaWRI1-3、BnaWRI1-5could express in yeast, and BnaWRI1-6could not. Evidence for the BnaWRI1s mRNA abundance was obtained by semi-quantitative RT-PCR. The expression of BnaWRI1s gene was tissue-specific with the highest abundance of mRNA in reproductive organs such as flowers and young embryos, and BnaWRI1s mRNA was also detected in stems,leafs,pericarps and roots.