巴西橡胶树乳管高效表达焦磷酸酶基因的双元表达载体的构建

以巴西橡胶树叶片为材料提取DNA，用特异引物经PCR扩增获得了378 bp的REF启动子片段，该片段序列与NCBI报道的REF序列间的同源性分别是98.68 %和99.47 %。以胶乳为材料提取总RNA，用特异引物经RT-PCR方法获得了2310 bp的V-PPase基因片段，该片段序列与NCBI报道的V-PPase基因序列一致。通过酶切和连接，将克隆的两个片段重组到植物表达载体pCAMBIA1301, pCAMBIA2301和pCAMBIA3301中，构建了分别含有潮霉素磷酸转移酶基因（hpt Ⅱ）、新霉素磷酸转移酶基因（npt Ⅱ）和抗除草剂基因（bar）的三种橡胶树乳管高效表达V-PPase基因的载体pC1301RV、pC2301RV和pC3301RV。然后用冻融法将其导入根癌农杆菌EHA105，为进一步转化橡胶树奠定基础。

Construction of binary vectors expressing V-PPase gene efficiently in laticifer of rubber tree (Hevea brasiliensis Muell. Arg. )

Genomic DNA was extracted from leaf, and total RNA was isolated from latex of rubber tree. REF promoter fragment of 378 bp and the V-PPase fragment of 2310 bp was cloned by PCR using specific primers. Sequence analysis showed that, the cloned V-PPase gene fragment was identical with that logged on GenBank, and the cloned REF promoter fragment shared high identities with four submitted REF promoter sequences in NCBI at a rate of 98.68 % and 99.47 %, respectively. Then the two fragments was cloned into the plant expression vector pCAMBIA1301, pCAMBIA2301 and pCAMBIA3301 by restriction enzyme digestion and ligation to get the recombinant expression vector pC1301RV, pC2301RV and pC3301RV contained hygromycin phosphotransferase Ⅱ(hpt Ⅱ) gene , neomycin phosphotransferaseⅡ(npt Ⅱ) gene and bialaphos resistance(bar) gene, respectively, which might express V-PPase gene efficiently in laticifer of rubber tree. The expression vectors were transferred into Agrobacterium tumefaciens strain EHA105 by freeze-thaw method. This work set a good foundation for further genetic transformation of rubber tree.