水稻钙依赖型蛋白激酶0sCPK9基因RNAi表达载体的构建及遗传转化

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280bp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548bp内含子片段（NCBIaccessionnumber：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．05CPK9一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。

Construction and Genetic Transformation of RNA Interference Vectors for Os CPK9 Gene in Rice

Rice calcium-dependent protein kinase （CDPKs） plays important roles in the growth and developmental processes and in response to bio- and abiotic stresses. A 280 bp gene specific fragment of OsCPK9 was amplified using RT-PCR method from rice variety Nipponbare and this fragment was linked both in sense and antisense directions to the ends of 548 bp intron fragment of wheat TA K14 gene as junction fragment. The resulting construct was then cloned into plant binary expression vector pCAMBIA1301 to form shRNAi expression vector pCAMBIA- OsCPK9-RNAi. After confirmation by enzyme digestion and sequencing analysis, this pCAMBIA-Os CPK9-RNAi was transferred into rice by agrobacterium-mediated transformation method. A total of 20 regenerated transgenic lines were screened through PCR identification of hyg and gus genes. Realtime quantitative PCR analysis showed that Os CPK9 expression in several transgenic lines was successfully suppressed.