基于二代测序技术的玉米内标准基因扩增子的筛选与评估  

Screening and Evaluation of Amplicons of Maize Endogenous Reference Genes Based on Next-Generation Sequencing Technology

作　　者：陈利红 周俊飞[1] 梁晋刚 李甜甜 王颢潜 方治伟 陈红 彭海[1] CHEN Lihong;ZHOU Junfei;LIANG Jingang;LI Tiantian;WANG Haoqian;FANG Zhiwei;CHEN Hong;PENG Hai(College of Life Science,Jianghan University,Wuhan 430056,China;Development Center of Science and Technology,Ministry of Agriculture and Rural Affairs,Beijing 100176,China)

机构地区：[1]江汉大学生命科学学院,湖北武汉430056 [2]农业农村部科技发展中心,北京100176

出　　处：《食品科学》2023年第20期146-154,共9页Food Science

基　　金：农业生物育种重大项目(2022ZD04020);湖北省自然科学基金面上项目(2021CFB563);武汉市科技局应用基础前沿专项(2020020601012234)。

摘　　要：通过对先前研究报道的7个内标准基因的核苷酸序列分析,设计13个扩增区域(扩增子);利用208个普通玉米品种(系)对这13个扩增子进行高通量测序与分析,并对其序列保守性、特异性、检出稳定性及其动态检测范围进行评估,以获得合适的基于二代测序技术定量检测玉米DNA含量或拷贝数的内标准基因扩增子。结果显示,7个内标准基因的13个扩增子在208个玉米样品中的检出率为94.7%~100%,而在水稻、大豆、棉花、白菜等25个非玉米样品中均未被检出,基本符合内标准基因种内高度一致与种间高度特异的标准;种内保守性分析显示E3-UBI-1扩增子不含单核苷酸多态性(single nucleotide polymorphism,SNP)和插入缺失(insertion-deletion,InDel),而剩余12个扩增子至少含有一个SNP或InDel;稳定性分析结果显示最不稳定的内标准基因扩增子是IVR,最稳定的是ADH1-2;它们的动态检测范围为0.2~200 ng。综合上述结果,ADH1-2与E3-UBI-1比较适合作为二代测序技术平台中定量检测玉米DNA含量或转基因玉米成分的内标准基因扩增子。In this study,13 amplification regions(amplicons)were designed by analyzing the nucleotide sequences of seven endogenous reference genes reported in previous studies.Totally 208 common maize varieties(lines)were used for highthroughput sequencing analysis of these 13 amplicons,and their sequence conservation,specificity,detection stability and dynamic detection range were evaluated to select appropriate amplicons of the endogenous reference genes for quantitative detection of maize DNA content or copy number based on next-generation sequencing technology.The results showed that the detection rates of the 13 amplicons of the seven endogenous reference genes were 94.7%–100%in the 208 maize samples,while they were not detected in 25 non-maize samples of rice,soybean,cotton and Chinese cabbage,indicating that these amplicons met the standards of high intraspecies consistency and interspecies specificity.Intraspecies conservation analysis showed that E3-UBI-1 did not contain single nucleotide polymorphism(SNP)or insertion deletion(InDel),while the remaining 12 amplicons contained at least one SNP or InDel.Stability analysis showed that the most unstable amplicon in these endogenous reference genes was IVR,and the most stable one was ADH1-2.Their dynamic detection range was 0.2-200 ng.Based on these results,ADH1-2 and E3-UBI-1 are suitable amplicons of the endogenous reference genes for quantitative detection of maize DNA content or transgenic maize components on the next-generation sequencing platform.

关 键 词：玉米 二代测序 内标准基因 定量检测 稳定性 

分 类 号：TS201.6[轻工技术与工程—食品科学]