[1]袁翠平,齐广勋,王玉民,等.大豆胞囊线虫侵染下野生大豆根组织实时定量PCR分析中内参基因的筛选[J].大豆科学,2018,37(01):22-31.[doi:10.11861/j.issn.1000-9841.2018.01.0022]
　YUAN Cui-ping,QI Guang-xun,WANG Yu-min,et al.Reference Gene Identification for Real-time Quantitative PCR Analysis in Glycine soja Roots Infected with Heterodera glycine[J].Soybean Science,2018,37(01):22-31.[doi:10.11861/j.issn.1000-9841.2018.01.0022]
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大豆胞囊线虫侵染下野生大豆根组织实时定量PCR分析中内参基因的筛选

《大豆科学》[ISSN:1000-9841/CN:23-1227/S] 卷: 第37卷 期数: 2018年01期 页码: 22-31 栏目: 出版日期: 2018-01-20
Title:
Reference Gene Identification for Real-time Quantitative PCR Analysis in Glycine soja Roots Infected with Heterodera glycine
作者:
袁翠平1; 齐广勋2; 王玉民1; 赵洪锟2; 刘晓冬1; 王英男1; 李玉秋1; 董英山1
（1.吉林省农业科学院 大豆研究所/大豆国家工程研究中心，吉林 长春 130033； 2.吉林省农业科学院 作物资源研究所，吉林 公主岭 130033）
Author(s):
YUAN Cui-ping 1;  QI Guang-xun 2;  WANG Yu-min 1;  ZHAO Hong-kun 2;  LIU Xiao-dong 1;  WANG Ying-nan 1;  LI Yu-qiu 1;  DONG Ying-shan 1
(1.Soybean Research Institute, Jilin Academy of Agricultural Sciences / National Engineering Research Center for Soybean, Changchun 130033, China; 2. Crop Germplasm Institute, Jilin Academy of Agricultural Sciences, Gongzhuling 136199, China)
关键词:
内参基因; 实时定量PCR; 大豆胞囊线虫
Keywords:
Glycine soja;  Reference gene;  Real-time quantitative PCR;  Heterodera glycine
分类号:
S565.1；S432.1
DOI:
10.11861/j.issn.1000-9841.2018.01.0022
文献标志码:
A
摘要:
以高抗和高感HG 0大豆胞囊线虫的野生大豆种质为试验材料，采用qRT-PCR技术检测了ACT11（Glyma.18G290800）、Tubulin_motif（Glyma.19G194800）等24个候选持家基因在不同大豆胞囊线虫侵染时期（接种后9，15和20 d）野生大豆根系中的基因表达情况，除Actin（Glyma.08G182200）出现了非特异扩增外，其它23个持家基因的qRT-PCR扩增效果均理想，扩增特异性高。分析上述23个持家基因的表达丰度，并利用BestKeeper、geNorm和Normfinder对其表达稳定性进行评价。结果表明：持家基因间的表达丰度不尽相同，而且有些持家基因在不同品种间或不同处理间（接种虫卵和接种水）也存在表达丰度的差异。23个持家基因的表达稳定性也不相同，综合来看，表达最不稳定的基因为Cons9（Glyma.10G152200）、Cons2（Glyma.17G138500）、Cons1（Glyma.15G270900）和Tubulin_motif(Glyma.20G136000），本试验条件下它们不适宜作内参基因；其余持家基因相对稳定，本试验条件下可选择Tubulin_motif（Glyma.15G132200），Cons6/SKIP16（Glyma.12G051100）或Tubulin_motif（Glyma.05G110200）作为内参基因，它们表达量较高，表达最为稳定，其Ct平均值分别为25.7、26.5和25.0，标准偏差分别为0.540、0.575和0.490，geNorm软件评价其稳定性的M值分别为0.698、0.715和0.727。该研究为野生大豆抗胞囊线虫相关基因的表达分析提供了参考。
Abstract:
Real-time quantitative PCR (qRT-PCR) has been widely used in crops for gene expression analysis, and reference genes are required in qRT-PCR analysis to minimize influences of RNA quality and quantity and efficiency of reverse transcription. Housekeeping genes are often selected as reference genes, however many of the housekeeping genes provide stable expression under only certain environments. So it is necessary to identify and select suitable reference genes for a given experiment. In our study, 24 candidate housekeeping genes were selected for their gene expression analysis, there happened nonspecific amplification products for qRT-PCR of Actin(Glyma.08G182200). The other 23 candidate housekeeping genes were evaluated for their expression level and stability in roots of susceptible and resistant wild soybean accessions under soybean cyst nematode HG 0 infection at 9, 15 and 20 days after inoculation. Housekeeping genes had different expression level, and some of them had different expression level between wild soybean accessions, or between different treatments. There also existed difference in expression stability among the 23 housekeeping genes. As a whole, Cons9 (Glyma.10G152200), Cons2 (Glyma.17G138500), Cons1 (Glyma.15G270900) and Tubulin_motif (Glyma.20G136000) were the top four least stable genes, and they were not suitable for the study. The three housekeeping genes of Tubulin_motif (Glyma.15G132200), Cons6/SKIP16 (Glyma.12G051100) and Tubulin_motif (Glyma.05G110200) were the most stable genes with M value of 0.698, 0.715 and 0.727 respectively and with their expression level of Ct=25.7, 26.5 and 25.0 respectively, which indicated that they are ideal reference genes for qRT-PCR analysis under our experiment conditions.

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备注/Memo

收稿日期：2017-08-07

基金项目：吉林省科技厅国际合作项目(20170414009GH)；国家自然科学基金青年科学基金项目（31200240）；吉林省农业科学院创新工程（CXGC2017ZD014）。
第一作者简介：袁翠平（1978-），女，博士，副研究员，主要从事野生大豆优异基因发掘与利用研究。E-mail：cpyuan2004@126.com。
通讯作者：董英山（1963-），男，研究员，主要从事野生大豆资源研究与利用研究。E-mail：ysdong@cjaas.com。

更新日期/Last Update: 2018-03-13