水稻钙依赖型蛋白激酶OsCPK9基因启动子的克隆及在烟草中的瞬时表达分析

Cloning and transient expression analysis of OsCPK9 gene promoter in rice( Oryza sativa L. )

植物钙依赖型蛋白激酶(CDPK)调控钙信号途径下游组分,与植物的生长发育及各种逆境生理过程密切相关。通过对本课题组克隆的水稻OsCPK9基因的cDNA序列与NCBI中的水稻基因组数据库进行比对、定位,结合生物信息学的方法,预测到基因上游的一段启动子序列。进而利用PCR的方法从水稻‘日本晴’(Oryza sativa L.cv.Nipponbare)基因组DNA中克隆到了水稻OsCPK9基因5’端上游约2 kb的DNA序列,命名为POsCPK9。PLANTCARE在线分析表明,POsCPK9序列除包含植物启动子所必备的基本元件如TATAbox和CAAT-box外,还含有多个与逆境和信号物质相关的顺式表达元件。将克隆到的POsCPK9取代pBI121中的CaMV 35S启动子,构建成POsCPK9与GUS的融合表达载体POsCPK9-GUS;通过农杆菌介导的方法在烟草的根、茎、叶中进行瞬时表达。结果显示,该启动子驱动的GUS基因在烟草的根、茎、叶中都有不同程度的表达。说明OsCPK9基因上游2 kb具有启动子活性。

Calcium-dependent protein kinases （CDPKs） regulate the downstream components in calcium signaling pathways and play important roles during the growth and development of plant and in response to various biotic and abiotic stresses.Based on the cDNA sequence of OsCPK9, a CDPK gene in rice, a 2 kb putative promoter sequence was located in rice genome by searching NCBI database , and was amplified by PCR using genomic DNA of rice （Oryza sativa L.cv.Nipponbare） as template and specific primers designed according to the predicted sequence . The cloned OsCPK9 promoter was 2 114 bp in length,and designated P OsCPK9 .PLANTCARE online software analysis indicated that P OsCPK9 contains some cis-acting expression elements related to stress signals , except basic elements＆amp;nbsp;TATA-box and CAAT-box.A POsCPK9 and GUS fusion expression vector P OsCPK9-GUS was constructed by replacing CaMV 35S promoter of pBI121 with the POsCPK9 .This vector was transiently expressed in the tobacco plant through Agrobacterium-mediated transformation method .The histochemical GUS staining and activity analysis showed that GUS could be expressed in roots, stems and leaves of tobacco plant with different levels , which suggested the upstream 2 kb sequence of OsCPK9 gene has promoter function.