大豆rbcS基因启动子的克隆及在转基因烟草中的功能缺失分析
Cloning of rbcS gene promoter from Glycine max and dysfunctional analysis in transgenic tobacco

【目的】进一步验证栽培大豆rbcS基因启动子功能，为植物基因工程相关研究提供启动子资源。【方法】从栽培大豆中克隆了rbcS基因5′端上游1 538 bp的DNA序列，根据光诱导表达调控元件及顺式作用功能元件所在的位置，设计含1 089，712和190 bp 3个5′ 端缺失体，并将rbcS基因启动子（1 538 bp）及3个5′ 端系列缺失体序列分别与gus基因融合，构建植物表达载体并命名为pGmrbcS、pA、pB和pC，用农杆菌介导法转化烟草，通过gus基因活性变化检测不同缺失体的表达特性。【结果】克隆了1 538 bp的大豆rbcS基因启动子序列及1 089，712和190 bp 的5′ 端缺失体启动子片段。GUS 活性检测表明，在pGmrbcS转基因烟草的叶中GUS活性最高，且与pCAMBIA1301转基因烟草叶片中CaMV35S启动子驱使gus基因的表达量相当，而茎、根中GUS活性较低。GUS定量分析表明，T1代转基因烟草光下培养时，3种缺失体启动子叶片中的GUS表达活性均与CaMV35S 启动子相当，其中pB活性最高，是CaMV35S启动子活性的1.4 倍,pA、pC活性低于pB，分别为pB活性的84%和71%；含pA 的转基因烟草于黑暗中萌发的幼苗叶片gus基因不表达，光下萌发幼苗叶片有gus基因表达，而含有 pB、pC的转基因烟草，在黑暗和光下萌发的幼苗叶片中均有gus基因表达，且表达量以pB最高。【结论】含有长度为1 089 bp（pA）光诱导元件的rbcS基因缺失体启动子具有光诱导和组织特异表达特性，长度为712 bp （pB）的缺失体启动子的启动活性最高。
【Objective】This study aimed to verify the function of rbcS gene promoter of soybean and provide resource for plant genetic engineering.【Method】Sequence of a 5′-upstream DNA segment (1 538 bp) of rbcS gene from soybean was cloned.Based on location of light-inducible regulatory element and cis-acting functional element,three 5′-end series deletions with lengths of 1 089,712 and 190 bp were constructed and fused with gus gene in addition to rbcS promoter to form expression vectors of pA,pB,pC and pGmrbcS,respectively.All vectors were transformed into tobacco with the Agrobacterium mediated method and expression characters of different deletions were detected through the change of gus gene activity.【Result】Promoter sequence (1 538 bp) of rbcS gene from soybean and three 5′-end deletion promoters with lengths of 1 089,712 and 190 bp were cloned.GUS activity detection shows that the highest GUS activity was detected in transgenic tobacco leaves with pGmrbcS,similar to that of gus gene driven by CaMV35S promoter in transgenic tobacco leaves of pCAMBIA1301.Weak GUS activity was detected in stem and root.GUS quantitative analysis shows that the expression activities of three deletion promoters in leaves were similar to that of CaMV35S promoter when T1 progeny seeds of transgenic tobacco were cultured with light.pB had the highest activity,which was 1.4 times of that of CaMV35S.The activities of pA and pC were 84% and 71% of that of pB.Expression of gus was detected in the leaves of transgenic tobacco seedling with pA cultured with light while it was not in the dark.Expression of gus was detected in the leaves of transgenic tobacco seedling with pB and pC cultured in both dark and light and the expression in pB was highest.【Conclusion】The rbcS gene deletion promoter with length of 1 089 bp containing light-inducible element (pA) was light inducible and expressed