摘要
水稻转化过程中,多个拷贝数整合到基因组中影响遗传稳定性和基因表达。传统的Southern杂交检测法鉴定费时费力,不适合目前高通量和自动化的检测要求。因此研究通过比较定量除草剂草铵膦抗性基因(bar)和甘油醛-3-磷酸脱氢酶GAPDH2,建立了基于实时荧光定量PCR的拷贝数检测法。利用此体系对9个独立的异天冬氨酸甲基转移酶OsPIMT1转基因T0代植株进行了荧光定量分析,其中6个为单拷贝,3个为双拷贝,经过Southern杂交和分离比分析验证,表明该方法能够快速,准确的鉴定水稻含有草铵膦抗性基因的水稻转基因植株。
Multiple copy number of target gene affects the genetic stability and gene expression level in plant transformation. Traditionally, determination of copy number was carried out by Southern analysis, which is both time-consuming and labour-intensive and not suitable for high-throughput screening. In this study, qPCR-based system for copy number identification of transgenic rice was established by comparative quantification of a pairs of the anti- phosphinotricin （ herbicide ） gene bar and rice Glyceraldehyde- 3-phosphate dehydrogenase2 GAPDH2. By using this system, the copy number of 90sPIMT1 transgenic clones were determined by qPCR, of which 6 were single copy, and 3 were double copies. The result was further verified by Southern blot and segregation ratio analysis, which shown this qPCR-based system can be an alternative to fast and accurately identify copy number of anti- phosphinotricin gene in flee.
出处
《福建稻麦科技》
2017年第4期39-42,共4页
Fujian Science and Technology of Rice and Wheat
基金
福建省自然科学基金项目(2015J05059)
国家自然科学基金项目(31401471
31501387)
福建省科技计划项目--省属公益类科研院所基本科研专项(2017R1021-6)
福建省农业科学院青年英才计划项目(YC2016-16
YC2015-14)
福建省农业科学院杰出青年人才基金(JC2018-2)
