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水稻钙依赖型蛋白激酶(CDPKs)是一个响应逆境胁迫并在植物发育过程中起重要作用的蛋白激酶。我们采用RT-PCR从水稻品种日本晴(Oryza sativa L.cv.Nipponbare)中克隆了OsCPK9基因靠近翻译起始位点下游的一段280 bp的特异基因片段。将该片段以正、反向分别连接到来源于小麦TAK14基因的548 bp内含子片段(NCBI accession number:AF325198)两侧,从而获得pSK-OsCPK9-RNAi的中间表达载体。进而将其克隆到植物双元表达载体pCAMBIA1301中,构建shRNAi表达载体pCAMBIA-OsCPK9-RNAi。经酶切和测序鉴定正确后,利用农杆菌介导转化水稻。通过抗性筛选和标记基因hyg和gus进行PCR鉴定,筛选到20株转基因水稻植株,实时荧光定量PCR分析显示,部分转基因植株OsCPK9的表达受到显著抑制。
Rice calcium-dependent protein kinases (CDPKs) are protein kinases that respond to stress and play an important role in plant development. We cloned a 280 bp specific fragment of the OsCPK9 gene downstream of the translational start site from the rice variety Oryza sativa L. cv. Nipponbare using RT-PCR. The fragment was ligated to the 548 bp intron fragment (NCBI accession number: AF325198) derived from wheat TAK14 gene in forward and reverse directions to obtain the intermediate expression vector pSK-OsCPK9-RNAi. Then, it was cloned into plant binary expression vector pCAMBIA1301 to construct shRNAi expression vector pCAMBIA-OsCPK9-RNAi. After digestion and sequencing, the rice was transformed by Agrobacterium tumefaciens. Twenty transgenic rice plants were screened by resistance screening and hyg and gus marker genes. Real-time PCR analysis showed that the expression of OsCPK9 in some transgenic plants was significantly inhibited.