水稻淀粉分支酶基因(RBE4)作为转基因水稻基体标准物质的内标准基因的研究  

Studies on Rice Starch Branching Enzyme (RBE4) as Endogenous Reference Gene for the Matrix Reference Material of Transgenic Rice(Oryza sativa L.)

作　　者：余笑波[1] 崔海峰[1] 俞晓平[1] 隋志伟[2] 李亮[2] 王晶[2] 叶子弘[1] 

机构地区：[1]中国计量学院,杭州310018 [2]中国计量科学研究院,北京100013

出　　处：《农业生物技术学报》2012年第11期1234-1243,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.31000357);浙江省自然科学基金(No.Y3090173)

摘　　要：内标准基因(endogenous reference gene)是指具有物种专一性、不显示等位基因变化、拷贝数恒定的保守DNA序列,对基因定量分析时具有重要意义。本研究选取了5个水稻内标准基因:根部表达的水稻基因(rice root-specific,GOS9)、磷脂酶D基因(phospholipase D,PLD)、蔗糖磷酸合成酶基因(sucrose phosphate synthase,SPS)、水稻淀粉分支酶基因(rice starch branching enzyme,RBE4)、泛素蛋白基因(ubiquitin5,UBQ5)和2个外源基因:苏云金芽孢杆菌Cry1Ab杀虫晶体蛋白基因Cry1Ab和cry1Ab/cry1Ac融合杀虫晶体蛋白基因(Cry1Ac/Cry1Ab),分别以转基因水稻(Oryza sativa L.)克螟稻2号和TT51-1为模板,比较各自的外源基因(Cry1Ab=KMD2和Cry1Ac/Cry1Ab=TT51-1)与5个内源基因的PCR产物量,筛选出和外源基因PCR产物量最接近的内标准基因为RBE4。在此基础上,数字PCR不依赖于已知浓度的标准曲线来定值,可以避免标准样品的标准曲线和样品目的基因的扩增曲线在扩增效率上的不一致等因素所带来的误差,定值结果更加准确、可靠,采用数字PCR分析外源基因拷贝数与内标准基因RBE4拷贝数的比值,荧光定量PCR方法分析内标准基因RBE4和外源基因的定量检测稳定性、灵敏度等。结果表明,外源基因拷贝数与内标准基因RBE4拷贝数的比值在转基因水稻克螟稻2号和TT51-1上都较接近于1∶1,分别为115.9%和105.3%。RBE4的荧光定量PCR体系重复性、定量检测稳定性和灵敏度好,符合作为转基因水稻基体标准物质定量分析的内标准基因的要求,RBE4定量体系在TT51-1和克螟稻2号的最小检出限(LOD)分别为5～11copies/μL和3～12copies/μL,最小定量限(LOQ)分别为11～22和12～24copies/μL。RBE4是转基因水稻标准物质研制和产品定量检测的适宜内标准基因。研究结果为转基因作物标准物质研制和定量检测的内标准基因选取提供一定的参考。Endogenous reference genes （ERGs） refer to a class of genes characterized by species specificity, low copies and allelic conservation. The specific PCR amplification of ERGs has important meaning for quantitatively detecting the foreign gene of transgenic crops. In this paper, atter looking for present related article, 5 related endogenous reference genes （the rice root-specific gene （GOS9）, the phospholipase D gene （PLD）, the sucrose phosphate synthase gene （SPS）, the rice starch branching enzyme gene （RBE4） and the Ubiquitin 5 gene （UBQ5）） and 2 exogenous reference genes （CrylAb and CrylAc/CrylAb） were selected. Transgenic rice KMD2 and TT51-1 were selected as template. By comparing the products of PCR of 5 related endogenous reference genes with the products of PCR of exogenous genes, the endogenous reference gene which PCR products are most close to exogenous gene PCR products was picked out. On the basis, more research on the selected endogenous reference gene was analyzed. Limitations in Real-time PCR applications to relative quantification of number of DNA targets had led to new developments such as the digital PCR （d-PCR） which allows accurate measurement of DNA copies without the need for a reference calibrator, so the ratio of copy number of KMD2/RBE4 and TT51-1/RBE4 were analyzed by digital PCR, Real-time PCR analysis on the amplification efficiency of two transgenic rice, the stability of PCR amplification and detection sensitivity. The results showed that compared to other endogenous reference genes, the amplification products of RBE4 was the most close to the amplification products of two exogenous genes, the ratio of copy number of two transgenic rice were close to 1：1, 115.9% and 105.3%, respectively. The repeatability and stability of PCR system of RBE4 were also satisfied with the endogenous genes for quantify matrix reference materials of transgenic rice. In the case of the RBE4 TaqMan assay for two transgenic rice KMD2 and TT51-1, the limit of detection �

关 键 词：转基因水稻 内标准基因 定量检测 基体标准物质 

分 类 号：S511[农业科学—作物学]