采用优化的数字PCR方法分析转基因小麦外源基因拷贝数  

Analysis of Foreign Gene Copy Number in Transgenic Wheat by Optimized Digital PCR

作　　者：琚鹏举 宁蕾[1] 葛林豪 许成杰 史华伟[1] 梁凯歌 马亮 刘陶然 陈明[2] 孙黛珍[1] JU PengJu;NING Lei;GE LinHao;XU ChengJie;SHI HuaWei;LIANG KaiGe;MA Liang;LIU TaoRan;CHEN Ming;SUN DaiZhen(College of Agriculture,Shanxi Agricultural University,Taigu 030800,Shanxi;Institute of Crop Science,Chinese Academy of Agricultural Sciences,Beijing 100081;Minzu University of China,Beijing 100081;Shijiazhuang Academy of Agricultural and Forestry Sciences,Shijiazhuang 050047)

机构地区：[1]山西农业大学农学院,山西太谷030800 [2]中国农业科学院作物科学研究所,北京100081 [3]中央民族大学,北京100081 [4]石家庄市农林科学研究院,石家庄050047

出　　处：《中国农业科学》2020年第10期1931-1939,共9页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2016YFD0101802);谷子氮素高效利用重要基因克隆及价值评估(2018ZX08009-17B)。

摘　　要：[目的]探索基于数字PCR的小麦基因拷贝数分析技术,提高目标基因拷贝数的分析通量,促进小麦基因组研究及基因工程研究.[方法]采用中国农业科学院作物科学研究所小麦抗逆分子育种课题组创制的高抗小麦黄花叶病转nib8小麦为试验材料,使用小麦D基因组中的单拷贝纯合内源基因PINb-D1b(籽粒硬度基因)为内参基因,根据转化的抗病基因nib8序列设计4对特异性引物及相应探针,通过试验确定探针和引物的最优浓度,优化体系的退火温度,寻找最合适的模板浓度.然后用数字PCR检测转nib8小麦中外源基因的拷贝数;同时用Real-time PCR和Southern blot 2种不同方法分析的拷贝数结果,验证上述采用数字PCR方法分析拷贝数的准确性;以PINb-D1b内参基因检测Wx012(蜡质基因)和SSII(淀粉合成基因)2个内参基因的拷贝数,以Wx012和SSII为内参基因检测转nib8小麦中外源基因的拷贝数,验证以PINb-D1b内参基因的检测结果准确性;在nib8的不同区段设计特异性引物来检测转nib8株系拷贝数分析结果是否有差异.最终,建立基于数字PCR技术的高通量检测小麦目标基因拷贝数的分析方法.[结果]通过试验最终确定了基于数字PCR方法的小麦目标基因拷贝数的检测体系.试验确定引物和探针的最佳终浓度分别为500和250 nmol·L-1,确定体系反应最佳退火温度为59℃,最佳DNA模板量为40 ng.以PINb-D1b作为内参基因检测转nib8小麦中nib8的拷贝数,拷贝数检测结果显示第12、16、17、23、29、30株系中nib8拷贝数分别为7个、1个、1个、1个、1个、7个;同时通过比较发现此结果与Real-time PCR以及Southern blot结果一致;在转基因株系中,以PINb-D1b为内参基因,对Wx012和SSII 2个基因进行拷贝数进行分析,同时发现采用这3种内参基因分析转nib8小麦中nib8基因拷贝数的结果一致,说明这3个内参基因都适合用于数字PCR方法;在nib8上游、中游和下游不同�【Objective】In order to explore the analysis technology of wheat gene copy number based on digital PCR,and improve the analysis efficiency of target gene copy number,and promote the research of wheat genome and gene engineering.【Method】Using the transgenic wheat with high resistance to wheat yellow mosaic disease transformed by nib8 gene created by the wheat stress resistance molecular breeding group in the Crop Science Institute of Chinese Academy of Agricultural Sciences as the experimental material,and used the single copy homozygous endogenous gene PIN-D1b(grain hardness gene)in wheat D genome as the internal reference gene,and four pairs of specific primers and corresponding probes were designed according to the sequence of the transformed disease resistance gene nib8,and the optimal concentration of probe and primer,the optimal annealing temperature of the system,to find the most appropriate template concentration were determined through experiments.Then,the copy number of nib8 genes in the nib8 transgenic wheat was detected by digital PCR;the accuracy of the above-mentioned copy number assay results were verified through using methods including the real time PCR and Southern blot;the copy number of the Wx012(waxy gene)and SSII(starch synthesis gene)were detected by using the PINb-D1b as reference gene,and copy number of nib8 were detected by using the Wx012 and SSII as reference genes in the nib8 transgenic wheat,to verify the accuracy of the results using the PINb-D1b as reference gene;the specific primers were designed in different regions of the nib8 gene to compare the difference of the copy number analysis results of the nib8 transgenic wheat.Finally,a high-throughput assay method based on digital PCR was established to detect the copy number of target gene in wheat genome.【Result】Finally,the detection system of target gene copy number in wheat genome based on digital PCR was determined.The optimal final concentration of primer and probe was 500 and 250 nmol·L-1,respectively.The optimal an

关 键 词：小麦 基因拷贝数 数字PCR 内参基因 

分 类 号：S512.1[农业科学—作物学]