柳枝稷根组织实时定量PCR分析中内参基因的选择  

Reference Gene Selection for Real-time Quantitative PCR Normalization in Switchgrass(Panicum virgatum L.) Root Tissue

作　　者：蒋晓梅[1] 张新全[1] 严海东[1] 张瑜杨 盛婷 黄琳凯[1] 

机构地区：[1]四川农业大学动物科技学院草业科学系,雅安625014

出　　处：《农业生物技术学报》2014年第1期55-63,共9页Journal of Agricultural Biotechnology

基　　金：科技部“十二五”国家高技术研究发展计划(863)(No.2012AA101801-02);国家自然基金(No.31201845);四川农业大学大学生创新计划(No.121062603);动物类实验教学示范中心

摘　　要：选择表达稳定性高的内参基因是提高实时荧光定量PCR分析(qRT-PCR)准确性的重要条件。本研究以能源植物柳枝稷(Panicum virgatum L.)根组织为材料,通过不同非生物条件胁迫,利用qRT-PCR技术检测UBQ1(泛素基因)、ACT2(肌动蛋白基因)、UBQ2(泛素基因)、TUB(β微管蛋白基因)、GAPDH(甘油醛-3-磷酸-脱氢酶基因)、CBP20(类胡萝卜素结合蛋白基因)、UCE2(泛素接合酶基因)、18S rRNA1(18S核糖体RNA基因)、NAC(NAC域蛋白基因)和CYP2(亲环蛋白基因)10个内参基因的mRNA的表达情况,并运用Delta-Ct method,Genorm(ver.3.5)、Bestkeeper(ver.1.0)和NormFinder(ver.0.953)软件综合分析10个内参基因的表达稳定性。结果显示,在不同的非生物环境胁迫条件下,最适合作为柳枝稷根组织研究的内参基因各不相同。10个内参基因平均表达稳定由高到低排序为:ACT2,TUB,UCE2,CBP20,CYP2,UBQ2,18S rRNA1,NAC,UBQ1,GAPDH。在干旱胁迫条件下,ACT2基因表达稳定性最高;在盐胁迫和热胁迫条件下,18S rRNA1基因表达稳定性最高;在冷胁迫和涝胁迫条件下,ACT2基因表达稳定性最高。经软件综合分析显示,ACT2、TUB和UCE2基因最适合作为柳枝稷非生物胁迫研究的内参基因;UBQ1和GAPDH基因最不合适作为内参基因。该研究结果可用于柳枝稷非生物胁迫下各种基因表达的进一步研究。The selection of a suitable reference gene is a critical condition for improving the precision of gene expression analyzing by quantitative Real-time PCR （qRT-PCR）. In this study, switchgrass（Panieum virgatum L.） root tissues were used as materials. Under different abiotic treatment conditions, mRNA expression stability of ten candidate reference genes, including UBQl（ubiquitin conjugating enzymel）, ACT2 （actin）, UBQ2 （ubiquitin conjugating enzyme2）, TUB（tubulin）, GAPOH（glyceraldehyde- 3- phosphate dehydrogenase）, CBP20（cap binding protein 20）, UCE2（E2 ubiquitin-conjugating enzyme）, 18S rRNAI（18S ribsomal RNA protein）, NAC（NAC domain protein） and CYP2（cyclophilin） was detected by qRT-PCR technology. Delta-Ct method, Genorm（ver. 3.5）, Bestkeeper（ver. 1.0） and NormFinder （ver. 0.953） were used to analyze the all data. The results showed that the most candidate reference genes of switchgrass had some differences in the various abiotic stresses. In the switchgrass root tissues the ranking order of ten referencegenes from better to average was as follows： ACT2, TUB, UCE2, CBP20, CYP2, UBQ2, 18S rRNA1, NAC, UBQ1 and GAPDH. Among them, ACT2 gene had the highest expression stability under the drought stress in the root tissue. The 18S rRNA1 gene was the best gene under salt and heat stresses. Under cold and waterlogging stresses, ACT2 was the most comparatively suitable genes. The results of software comprehensive analysis indicated that ACT2, TUB and UCE2 genes were the most stable reference genes which were suitable for switchgrass abiotic stress studies. UBQ1 and GAPDH were not suitable reference genes. The research results can be used for further gene expression study under various abiotic stresses in switchgrass.

关 键 词：非生物胁迫 柳枝稷 实时荧光定量PCR 内参基因 组织 

分 类 号：S543.9[农业科学—作物学]