OsMAPK7基因的克隆、序列分析及其植物表达载体的构建

Cloning and sequencing of OsMAPK7 gene and construction of its plant expression vector

以低温处理的耐盐水稻辽盐241植株叶片总RNA为模板,用OsCDPK7基因特异引物通过RT-PCR扩增出1 700 bp的片段,并将该片段克隆至pUC18上。序列分析结果表明,该序列与GenBank上的OsCDPK7基因序列相比,缺失了CDS序列中157￣234处的78个核苷酸,其编码的蛋白序列缺失了53￣78的26个氨基酸。但是缺失部分位于丝氨酸/苏氨酸蛋白激酶活性中心和钙结合基序等结构域之外,因此预计该基因产物应具备钙依赖的蛋白激酶活性。进一步将OsCDPK7基因分别克隆至植物表达载体卡盒pBE12和pB29A上,构建了分别由E12启动子、rd29A启动子调控的OsCDPK7基因植物表达载体pBC7E12和pBC729A。通过冻融法将重组质粒导入根癌农杆菌LBA4404中,为农杆菌介导法转化植物,分析OsCDPK7基因的功能奠定了基础。

A 1 700 bp DNA fragment amplified by RT-PCR use OsCDPK7 gene special primer from liaoyan241 leaf treated by 200 mmol. L^-1 NaCl was cloned into pUC 18. The result of sequencing showed that this fragment 78 nucleotides from 157-234 in CDS compare with that of OsCDPK7 gene in GenBank and lack of 26 amino acids from 53-78 in protein sequence. Further more, OsCDPK7 gene was cloned into plant express vector cassette pBE12 and pB29A. Thus two plant express vectors, pBC7E12 and pBC729A were constructed, in which OsCDPK7 gene was regulated by E12 promoter and rd29A promoter respectively. Then the recombination plasmids were introduced into Agrobacterium LBA4404 by freezing -melting transformation method. This work provides a foundation for transferring OsCDPK7 gene into plant by Agrobacterium-mediated method and its function analysis.