|Table of Contents|

Reference Gene Identification for Real-time Quantitative PCR Analysis in Glycine soja Roots Infected with Heterodera glycine(PDF)

《大豆科学》[ISSN:1000-9841/CN:23-1227/S]

Issue:
2018年01期
Page:
22-31
Research Field:
Publishing date:

Info

Title:
Reference Gene Identification for Real-time Quantitative PCR Analysis in Glycine soja Roots Infected with Heterodera glycine
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)
Keywords:
Glycine soja;  Reference gene;  Real-time quantitative PCR;  Heterodera glycine
PACS:
S565.1；S432.1
DOI:
10.11861/j.issn.1000-9841.2018.01.0022
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.

References:

［1］ 黄志熊, 王飞娟, 蒋晗, 等. 两个水稻品种镉积累相关基因表达及其分子调控机制［J］. 作物学报, 2014, 40(4): 581-590. (Huang Z X, Wang Z X, Jiang H, et al. Comparison of cadmium-accumulation-associated genes expression and molecular regulation mechanism between two rice cultivars (Oryza sativa L. subspecies japonica) ［J］. ACTA Agronomica Sinica, 2014, 40(4): 581-590.)

［2］ 崔洪秋, 冯乃杰, 孙福东, 等. DTA-6对大豆花荚脱落纤维素酶和GmAC基因表达的调控［J］.作物学报, 2016, 42(1): 51-57. (Cui H Q, Feng N J, Sun F D, et al. Regulation of DTA-6 by abscission cellulase and GmAC gene expression in flowers and pods of soybean［J］. ACTA Agronomica Sinica, 2016, 42(1): 51-57.)
［3］ 曹鑫, 邓梅, 张正丽, 等. 小麦分蘖角度TaTAC1基因同源克隆及表达分析.植物遗传资源学报［J］, 2017, 18: 125-132. (Cao X, Deng M, Zhang Z L, et al. Molecular characterization and expression analysis of TaTAC1 gene in Triticum aestivum L［J］. Journal of Plant Genetic Resources, 2017, 18: 125-132.)
［4］ 史庆玲, 董永彬, 周强, 等. 玉米转录因子ZmERF1的克隆及表达分析［J］.生物技术进展.2017,7,38-42. (Shi Q L, Dong Y B, Zhou Q, et al. Cloning and expression analysis of ZmERF1 in maize［J］. Current Biotechnology, 2017, 7: 38-42.)
［5］ 王艳, 武林, 孙梦阳, 等. 不同生育时期大豆异黄酮合成相关酶基因表达的分析［J］.大豆科学, 2012, 31: 887-893. (Wang Y, Wu L, Sun M Y, et al. Analysis of gene expression underlying soybean isoflavone synthesis relative enzymes at different growth stages［J］. Soybean Science, 2012, 31: 887-893.)
［6］ 李媛媛, 南海洋, 刘宝辉, 等. 大豆GmFDL06基因抗干旱及耐盐性研究［J］. 大豆科学, 2017, 36(3): 351-359. (Li Y Y, Nan H Y, Liu B H, et al. Study on drought resistance and salt tolerance of soybean gene GmFDL06［J］. Soybean Science, 2017, 36(3): 351-359.)
［7］ 王骁. 基于实时荧光定量PCR的大豆内参基因筛选［D］. 太古: 山西农业大学, 2016: 27-44. (Wang X. Selection of candidate reference genes for gene expression studies［D］. Taigu: Shanxi Agricultural University, 2016: 27-44.)
［8］ 陈玉连, 张涛, 董登峰. 低磷胁迫大豆qRT-PCR内参基因的筛选及谷胱丙肽合成代谢酶的表达［J］. 广西植物. 2016, DOI:10.11931/guihai.gxzw201609013. (Chen Y L, Zhang T, Dong D F. Selection of reference genes for qRT-PCR and expression of genes involved in homoglutathione anabolism in soybean under stress of phosphorus deficiency［J］. GuiHaia, 2016, DOI:10.11931/guihai.gxzw201609013.)
［9］ Ma S, Niu H, Liu C, et al. Expression stabilities of candidate reference genes for RT-qPCR under different stress conditions in soybean［J］. Plos One, 2013, 8: e75271.
［10］刘伟灿, 王骐, 周永刚, 等. 大豆干旱胁迫下miRNA与mRNA荧光定量PCR内参基因的筛选［J］.西北农林科技大学学报(自然科学版), 2016, 44(2):61-67. (Liu W C, Wang Q, Zhou Y G, et al. Selection of reference genes for quantitative polymerase chain reaction of miRNA and mRNA in soybean under drought stress［J］. Journal of Northwest A & F University (Natural Science Edition), 2016, 44(2): 61-67.)
［11］Marcolinogomes, J, Rodrigues, F A, Fugantipagliarini, R, et al. Transcriptome-wide identification of reference genes for expression analysis of soybean responses to drought stress along the day［J］.Plos One, 2015, 10(9):e0139051.
［12］Hu R, Fan C, Li H, et al. Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR［J］. BMC Molecular Biology,2009, 10: 93.
［13］侯高杰. 铝毒胁迫下大豆内参基因的筛选及相关基因表达分析［D］.南宁: 广西大学, 2015: 21-32. (Hou G J. Selection of reference genes and analysis some related gene expression in soybean under aluminum toxicity［D］. 2015:21-32.)
［14］曾文韬, 柴春月, 窦道龙. 适用于大豆实时荧光定量PCR分析的内参基因的筛选和验证［J］.南京农业大学学报, 2015, 38(5): 787-95. (Zeng W T, Zhai C Y, Dou D L. Selection and validation of reference genes for quantitative RT-PCR analysis in soybean［J］. Journal of Nanjing Agricultural University, 2015, 38(5): 787-95.)
［15］Costa J H, Saraiva K D C, Morais V D, et al. Reference gene identification for real-time PCR analyses in soybean leaves under fungus (Cercospora kikuchii) infection and treatments with salicylic and jasmonic acids［J］. Australasian Plant Pathology, 2016, 45: 191-199.
［16］Miranda V D J, Coelho R R, Viana A A B, et al. Validation of reference genes aiming accurate normalization of qPCR data in soybean upon nematode parasitism and insect attack［J］. BMC Research Notes, 2013, 6: 196.
［17］宋美静, 朱晓峰, 王东, 等. 我国大豆主产区大豆胞囊线虫群体分布及致病性分化研究［J］.大豆科学, 2016, 35(4): 630-636. (Song M J, Zhu X F, Wang D, et al. Population distribution and pathogenicity differentiation of soybean cyst nematode in main soybean production areas of china［J］. Soybean Sciences, 2016, 35(4): 630-636.)
［18］刘鹤. 大豆抗胞囊线虫组织病理学及相关基因表达研究［D］.沈阳: 沈阳农业大学, 2016:40-50 (Liu H. Study on histopathology and related genes of soybean resistant to Heterodera glycines［D］. Shenyang: Shenyang Agricultural University, 2016:40-50.)
［19］赵思阳, 王媛媛, 朱晓峰, 等. 大豆与胞囊线虫互作中GmPRs的表达研究［J］. 中国油料作物学报. 2017,39(2): 213-220. (Zhao S Y, Wang Y Y, Zhu X Y, et al. Expression of pathogenesis-related proteins (GmPRs) in interactions between soybean and soybean cyst nematode［J］. Chinese Journal of Oil Crop Sciences, 2017,39(2): 213-220.)
［20］Niblack T, Tylka G L, Arelli P, et al. A standard greenhouse method for assessing soybean cyst nematode resistance in soybean: SCE08 (standardized cyst evaluation 2008) ［J］. Plant Health Progress, 2009, 10. doi:10.1094/PHP-2009-0513-01-RV.
［21］Kim H K, Kang Y J, Kim D H, et al. RNA-Seq analysis of a soybean near-isogenic line carrying bacterial leaf pustule-resistant and -susceptible alleles［J］. DNA Research. 2011, 18, 483-497.
［22］Libault M, Thibivilliers S, Bilgin D D, et al. Identification of four soybean reference genes for gene expression normalization［J］. The plant genome, 2008, 1(1): 44-54.
［23］Jian B, Liu B, Bi Y, et al. Validation of internal control for gene expression study in soybean by quantitative real-time PCR［J］. BMC Molecular Biology,2008, 9, 1-14.
［24］Sun X, Sun M, Jia B, et al. A Glycine soja methionine sulfoxide reductase B5a interacts with the Ca2+/CAM-binding kinase GsCBRLK and activates ROS signaling under carbonate alkaline stress［J］. Plant Journal, 2016, 86: 514-529.
［25］Stolf-Moreira R, Gertrudes de Macedo Lemos E, Vilela-Abdelnoor R, et al. Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean［J］. Pesquisa Agropecuária Brasileira, 2011, 46: 58-65.

Memo

Memo:
-
Last Update: 2018-03-13