TSA诱导橡胶树次生乳管分化过程qRT-PCR内参基因的筛选  

Selection of Reference Genes for Normalization of Quantitative Real-time PCR Analysis in Secondary Laticifer Differentiation Induced by Trichostatin A in Rubber Tree(Hevea brasiliensis Muell. Arg.)

作　　者：吴绍华[1] 张世鑫[1] 杨署光[1] 田维敏[1] WU Shaohua;ZHANG Shixin;YANG Shuguang;TIAN Weimin(Rubber Research Institute,Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Biology and Genetic Resources of Rubber Tree,Ministry of Agriculture and Rural Affairs/State Key Laboratory Breeding Base of Cultivation and Physiology for Tropical Crops,Haikou,Hainan 571101,China)

机构地区：[1]中国热带农业科学院橡胶研究所/农业农村部橡胶树生物学与遗传资源利用重点实验室/省部共建国家重点实验室培育基地-海南省热带作物栽培生理学重点实验室,海南海口571101

出　　处：《热带作物学报》2020年第3期504-512,共9页Chinese Journal of Tropical Crops

基　　金：国家天然橡胶产业技术体系育种技术与方法岗位科学家项目(No.CARS-34-YZ1);国家自然科学基金青年科学基金项目(No.31800577)。

摘　　要：橡胶树乳管分化过程中差异表达基因的鉴定对于进一步认识乳管分化的分子机理具有重要的作用。实时荧光定量PCR(qRT-PCR)是进行基因表达分析的常用技术手段,合适内参基因的选择是准确进行基因表达定量的前提。以组蛋白去乙酰化抑制剂曲古抑菌素A(trichostatin A, TSA)诱导橡胶树次生乳管分化的实验系统,采用qRT-PCR技术分析TSA处理橡胶树萌条及对照内层树皮中22个候选内参基因的表达情况。结果显示:TSA诱导橡胶树萌条次生乳管分化的过程中,稳定性最高的3个基因分别为UBC3、UBC4和eIF1Aa,而稳定性最差的3个基因分别为ROC3、PTP、CYP2。以UBC3、Actin和ROC3基因为内参,分析组蛋白去乙酰化酶基因HDA1和HDA2在TSA处理下树皮中表达量相对于对照的变化,结果初步证实TSA诱导橡胶树萌条次生乳管分化的过程中,UBC3是最佳的内参基因,ROC3不适合作为内参基因。The identification of differentially expressed genes associated with laticifer differentiation is crucial for understanding the molecular mechanism of laticifer differentiation. The selection of a suitable reference gene is a precondition for accurate gene expression analysis by quantitative real-time PCR(qRT-PCR). In the present study, the expression stability of 22 candidate reference genes were evaluated on the basis of TSA-induced secondary laticifer differentiation using geNorm and NormFinder algorithms. According to the analysis of geNorm and NormFinder, UBC3, UBC4, eIF1 Aa were the top three stable genes and ROC3, PTP, CYP2 were the last three stable genes in the process of TSA-induced secondary laticifer differentiation. The expression patterns of HDA1 and HDA2 in TSA treated bark were analyzed based on UBC3, Actin and ROC3 as the reference gene respectively. The results showed that UBC3 could serve as a qRT-PCR reference gene to analyze the gene expression pattern in TSA-induced secondary laticifer differentiation. And ROC3 was not suitable as reference genes for qRT-PCR.

关 键 词：巴西橡胶树 乳管分化 曲古抑菌素A(TSA) 内参基因 

分 类 号：S794.1[农业科学—林木遗传育种]