高温胁迫香葱内参基因筛选与稳定表达分析  

Screening of Suitable Reference Genes for RT-qPCR Normalization in Chives under Heat Stress

作　　者：郭元元[1] 蒋月喜 车江旅[2] 张力[1] 陈琴 宋焕忠[1] 李洋 秦龙妹 甘立[1] 陈振东[1] Guo Yuanyuan;Jiang Yuexi;Che Jianglü;Zhang Li;Chen Qin;Song Huanzhong;Li Yang;Qin Longmei;Gan Li;Chen Zhendong(Vegetable Research Institute,Guangxi Academy of Agricultural Sciences,Nanning,530007;Achievement Transformation Department,Guangxi Academy of Agricultural Sciences,Nanning,530007;Guangxi Key Laboratory of Vegetable Breeding and New Technology Development,Vegetable Research Institute,Guangxi Academy of Agricultural Sciences,Nanning,530007)

机构地区：[1]广西农业科学院蔬菜研究所,南宁530007 [2]广西农业科学院成果转化处,南宁530007 [3]广西农业科学院蔬菜研究所,广西蔬菜育种与新技术研究重点实验室,南宁530007

出　　处：《分子植物育种》2023年第22期7341-7350,共10页Molecular Plant Breeding

基　　金：广西自然科学基金面上项目(2020GXNSFAA297125);第三次全国农作物种质资源普查与收集行动(11172130-1354052035-5,1120162130135252038-7);广西农业科学院基本科研业务专项(桂农科2021YT01)共同资助。

摘　　要：香葱(Allium fistulosum L.var.Caespitosum Makino)响应高温胁迫过程中,差异表达基因的鉴定及表达验证对深入了解香葱的高温应答分子响应机理具有重要意义。为筛选高温胁迫下香葱稳定表达的内参基因,本研究以香葱耐热品种‘AF60’和不耐热品种‘AF35’的叶片为材料,分别进行高温胁迫不同时间处理,选取13个候选内参基因(Ac18S,Af18S,Af28S,AcActin,AcActin,AfTUA,AfTUB,AsSAND,AfMYH,AfGAPDH,AfUBQ,AfEF1α,AfCYC),利用实时荧光定量RT-qPCR技术检测香葱高温胁迫处理候选内参基因的表达,采用GeNorm、NormFinder、BestKeeper软件评价其稳定性,并进行综合分析。结果表明:13个内参基因在不同高温处理下的表达稳定性存在差异,综合分析香葱内参AcActin表达最稳定,其次为AfEF1α、Af28S;而AcActin表达最不稳定;同时,结果显示利用香葱转录组开发的内参基因普遍稳定性高于葱属其他作物开发的内参。该结果为香葱基因的表达分析提供了可供选择的内参基因。The identification and expression verification of differentially genes has great significance for further understanding of the molecular response mechanism of Allium fistulosum L.var.Caespitosum Makino in response to high temperature stress.In order to screen the stable reference genes under high temperature,the leaves of the heat resistant'AF60'and non-heat resistant'AF35'were used as materials and treated at different time lengths under high temperature stress respectively.13 candidate reference genes(Ac18S,Af18S,Af28S,AcActin,AcActin,AfTUA,AfTUB,AsSAND,AfMYH,AfGAPDH,AfUBQ,AfEF1α,AfCYC)were selected,and their expression levels were detected by real-time fluorescence quantitative(RT-qPCR).The stability of the 13 reference genes was evaluated by combining GeNorm,NormFinder and BestKeeper software,and a comprehensive analysis was conducted at last.The results showed that there were significant differences in the expression stability of 13 reference genes under different treatments,and AcActin expression was the most stable,followed by AfEF1 and Af28S.AcActin expression was the most unstable.At the same time,the results showed that the reference genes developed by Allium fistulosum L.var.Caespitosum Makino transcriptome were generally more stable than those developed by other crops.The results provided an alternative reference gene for the expression analysis of Allium fistulosum L.var.Caespitosum Makino genes.

关 键 词：香葱 定量RT-qPCR 内参基因 高温胁迫 

分 类 号：S633[农业科学—蔬菜学]