干旱胁迫下蒙农杂种冰草RT-qPCR内参基因的筛选  

Selection of Reference Genes of Agropyron cristatum×A.desertorum cv.Hycrest-Mengnong for RT-qPCR Analysis under Drought Stress

作　　者：王晓宇 刘秉毅 詹圆 伏兵哲[2] 米福贵 张志强 WANG Xiao-yu;LIU Bing-yi;ZHAN Yuan;FU Bing-zhe;MI Fu-gui;ZHANG Zhi-qiang(College of Grassland,Resources and Environment,Inner Mongolia Agricultural University/Key Laboratory of Grassland Resources,Ministry of Education P.R.of China,Hohhot 010011,China;College of Agriculture,Ningxia University/Ningxia Grassland and Animal Husbandry Engineering Technology Research Center,Yinchuan 750021,China;College of Grassland,Resources and Environment,Inner Mongolia Agricultural University/Technology Engineering Center of Drought and Cold-resistant Grass Breeding in North of the National Forestry and Grassland Administration,Hohhot 010011,China)

机构地区：[1]内蒙古农业大学草原与资源环境学院/草地资源教育部重点实验室,内蒙古呼和浩特010011 [2]宁夏大学农学院/宁夏草牧业工程技术研究中心,宁夏银川750021 [3]内蒙古农业大学草原与资源环境学院/国家林业草原北方抗旱耐寒草品种育繁工程技术研究中心,内蒙古呼和浩特010011

出　　处：《中国草地学报》2022年第8期1-8,共8页Chinese Journal of Grassland

基　　金：宁夏科技厅重大专项子课题(2019NYYZ0403);自然科学基金青年科学基金项目(31702180)资助。

摘　　要：为了筛选出蒙农杂种冰草基因表达分析研究的最适内参基因,基于前期蒙农杂种冰草转录组数据(NCBI临时登录号:PRJNA777813),以不同干旱胁迫下蒙农杂种冰草茎和叶2个器官为材料,用RT-qPCR技术分析6个候选内参基因(CYP、GAPDH、G6PD、UBQ11、ACT11和TUB4)的表达情况,并利用Ct值和3个软件分析候选内参基因的表达稳定性。Ct值分析结果表明,6个候选内参基因在干旱胁迫下蒙农杂种冰草茎和叶中的Ct值为24~35,表达丰度适中,适合进行下一步筛选。geNorm、NormFinder及ReFinder综合分析后结果表明,GAPDH是干旱胁迫下蒙农杂种冰草不同器官基因表达分析的最佳内参基因,TUB4次之,CYP最差。为了提高准确性,干旱胁迫下蒙农杂种冰草也可同时使用两个内参基因组合,GAPDH和G6PD是最稳定的内参基因组合。The study aimed to select the most suitable reference genes for gene expression of Agropyron cristatum×A.desertorum cv.Hycrest-Mengnong,based on the obtained transcriptome data(NCBI access number:PRJNA777813).The expression of candidate reference genes(Cytochrome P450,CYP;Glyceraldehyde phosphate dehydrogenase,GAPDH;Glucose 6 phosphate dehydrogenase G6PD;Ubiquitin-11,UBQ11;Actin-11,ACT11;Tubulin-4,TUB4)were analyzed by RT-qPCR technique using the stems and leaves of Agropyron cristatum×A.desertorum cv.Hycrest-Mengnong under different drought stress conditions.Ct value and three software analyzed the expression stability of candidate reference genes.The results showed that Ct values of 6 candidate reference genes were between 24 and 35 in the stem and leaf of Agropyron cristatum×A.desertorum cv.Hycrest-Mengnong under drought stress.The expression abundance was optimum and suitable for future analysis.The geNorm,NormFinder,and ReFinder analyses showed that GAPDH was the most appropriate reference gene,followed by TUB4 and CYP.In conclusion,it is beneficial to use the combination of two reference genes(GAPDH and G6PD)of Agropyron cristatum×A.desertorum cv.Hycrest-Mengnong to improve the RT-qPCR accuracy.

关 键 词：蒙农杂种冰草 内参基因 实时荧光定量PCR 

分 类 号：S543.9[农业科学—作物学]