TaNAC14转基因小麦外源目标基因拷贝数及遗传稳定性分析  

Analysis of Copy Number and Genetic Stability of the Transgene TaNAC14 in Transgenic Wheat(Triticum aestivum)

作　　者：张立坚 池青[1] 杜琳颖 郭利建 李玉莲[2] 刘香利[1,3] 马猛 赵惠贤[1,3] ZHANG Lijian;CHI Qing;DU Linying;GUO Lijian;LI Yulian;LIU Xiangli;MA Meng;ZHAO Huixian(College of Life Science,Northwest A&F University,Yangling,Shaanxi 712100,China;Crop Research Institute,Shandong Academy of Agricultural Sciences,Jinan,Shandong 250100,China;State Key Laboratory of Crop Stress Biology for Arid Areas,Northwest A&F University,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100 [2]山东省农业科学院作物研究所,山东济南250100 [3]西北农林科技大学旱区作物逆境生物学国家重点实验室,陕西杨凌712100

出　　处：《麦类作物学报》2020年第2期135-143,共9页Journal of Triticeae Crops

基　　金：国家自然科学基金项目(31471482)。

摘　　要：外源基因拷贝数是影响转基因植物遗传稳定性及其自身表达水平的重要因素。为了探析TaNAC14基因在小麦生长发育中的生物学功能,本研究通过农杆菌介导的遗传转化法获得了14个T0代TaNAC14转基因小麦植株,采用BASTA溶液涂抹和目标序列PCR检测相结合的方法,从14个转基因小麦植株中鉴定出9个阳性植株。通过TaqMan实时定量PCR方法,以小麦内源单拷贝基因Pinb为内参基因,对9个T0代转基因小麦阳性植株中外源目标基因拷贝数进行检测,结果表明,T0代转基因小麦再生植株中有5株为单拷贝,4株为双拷贝,其中单拷贝插入整合的比率接近55.6%。选取单拷贝转基因植株收获的种子进行种植,根据BASTA溶液涂抹鉴定对T0:1代小麦株系遗传情况进行分析,结果表明目标基因TaNAC14是可遗传的。此外,还对T1代转基因小麦目标基因表达水平进行测定,结果表明,与野生型JW1相比,各转基因小麦植株目的基因TaNAC14表达量均极显著增加。该研究结果为后续TaNAC14基因的功能研究奠定了基础。The copy number of a transgene is an important factor affecting the genetic stability of the transgenic plants and its expression levels.In order to explore the biological function of TaNAC14 gene in wheat growth and development,14 TaNAC14 transgenic wheat plants of T0 generation were obtained by Agrobacterium-mediated genetic transformation in this study.9 positive plants were identified from 14 transgenic wheat plants by identification of BASTA solution smearing combined with PCR detection of target sequence.The TaqMan real-time quantitative PCR method was used to detect the copy number of the transgene TaNAC14 in the 9 T0 transgenic wheat plants using the wheat endogenous single copy gene Pinb as an internal reference gene.The results showed that 5 single-copy and 4 double-copy transgenic plants were identified among these T0 transgenic plants,and the ratio of single-copy insertion and integration was close to 55.6%.The seed of these single-copy transgenic plants were harvested to plant to obtain T1 generation plants.According to the results of BASTA solution smearing,the genetic analysis of T0:1 wheat transgenic lines showed that the transgene TaNAC14 were heritable.In addition,the expression level of the transgene of T1 transgenic wheat plants was also determined,and the results showed that the expression level of the TaNAC14 gene of individual transgenic wheat plants was significantly increased,compared with the wild type wheat JW1.The results of this study laid the foundation for subsequent functional research of the TaNAC14 gene using these transgenic wheat lines.

关 键 词：小麦 TaNAC14基因 转基因拷贝数 转基因遗传稳定性 目标基因表达水平 

分 类 号：S512.1[农业科学—作物学] S330