水稻缺素胁迫下实时荧光定量RT-PCR的内参基因的选择  

Selection of Reference Genes for Real-time Quantitative RT- PCR in Rice(Oryza sativa L. ssp. japonica) under Nutrient Deficiency

作　　者：魏毅东[1,2] 陈玉[2] 郭海萍[2] 谢华安[1,2] 张建福[1,2] 王宗华[1,2] 

机构地区：[1]福建农林大学生命科学学院,福州350002 [2]农业部华南杂交水稻种质创新与分子育种重点实验室/福州(国家)水稻改良分中心/福建省作物分子育种工程实验室/福建省水稻分子育种重点实验室/福建省作物种质创新与分子育种省部共建国家重点实验室培育基地/杂交水稻国家重点实验室华南基地/水稻国家工程实验室/福建省农业科学院水稻研究所,福州350003

出　　处：《农业生物技术学报》2013年第11期1302-1312,共11页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展规划(973)项目(No.2010CB723003);国家转基因农作物新品种培育重大专项资助项目(No.2008ZX08001-006;No.2011ZX08001-006和No.2011ZX08001-004);福建省财政专项-福建省农业科学院创新团队项目(No.CXTD-1-1301)

摘　　要：营养元素的吸收和利用是影响水稻产量的重要决定因素。利用qRT-PCR进行缺素胁迫下水稻功能基因的表达分析时,应当筛选适合于缺素胁迫下qRT-PCR分析的内参基因。本研究以缺乏氮、磷、钾、钙、镁、硫6种大量元素1周和2周的水稻(Oryza sativa L.ssp.japonica)幼苗组成的缺素样品池为材料,选取12个水稻管家基因作为缺素条件下qRT-PCR的候选内参基因,利用geNorm软件分析缺素条件下各基因总体表达的稳定性并筛选出适合于缺素处理下qRT-PCR分析的内参基因eIF4a,确定用于缺素条件下内参校正的最佳基因数量组合为2个。根据比较分析多种内参基因组合在不同缺素处理下的实际校正结果,证明内参基因的最佳组合对于缺素条件下qRT-PCR分析的重要性以及校正结果的有效性。最后,验证了eIF4a单基因校正的可行性与有效性,为geNorm软件提供了有益的改进和补充,这为水稻缺素胁迫下qRT-PCR分析的提供了参考和借鉴。Mineral nutrient assimilation and utilization play a critical role in rice yield. Reference genes suitable for nutrient deficiency must be selected, prior to qRT-PCR analysis of rice functional gene. In this study, a total of 12 rice housekeeping genes were selected as candidate reference genes for qRT-PCR of rice under nutrient deficiency. Total sample pool consisted of rice（Oryza sativa L. ssp. japonica） plants grown under 1-week and 2-week nutrient deficiency of 6 macro elements, including nitrogen, phosphorus, potassium, calcium, magnesium, sulphur. Stability of candidate reference genes under nutrient deficiency was analysed and reference gene eIF4a was identified as the most suitable gene using geNorm, and two genes with most stable expression were found to be optimal for reliable normalisation, based on the result of actual normalisation effect under different nutrient deficiency using various reference gene groups, reference gene selection was been proved to be of importance on qRT-PCR results under nutrient deficiency treatment of rice, and effectiveness of gene stability analysis using geNorm was further confirmed. Finally, single gene normalisation of eIF4a was validated as an improvement and complement for geNorm software. Taken together, all the results provide good reference for further research in qRT-PCR under nutrient deficiency.

关 键 词：荧光定量RT-PCR 内参基因 水稻 缺素 GENORM 

分 类 号：S943[农业科学—水产养殖]