蝴蝶兰PhPP2Aa基因作为低温胁迫内参基因的研究  

PhPP2Aa as Reference Gene in Phalaenopsis under Low-temperature Stress

作　　者：梁芳[1] 许申平[1] 张燕[1] 王默霏[1] 崔波[1] LIANG Fang;XU Shenping;ZHANG Yan;WANG Mofei;CUI Bo(Bioengineering Research Center,Zhengzhou Normal University,Zhengzhou,Henan 450044,China)

机构地区：[1]郑州师范学院生物工程研究中心,河南郑州450044

出　　处：《热带作物学报》2022年第7期1338-1346,共9页Chinese Journal of Tropical Crops

基　　金：河南省科技攻关计划项目(No.212102110116,No.222102110470);河南省高校重点科研项目(No.22A210025)。

摘　　要：实时荧光定量PCR(qPCR)技术因其具有简单灵敏、准确高效等诸多优点,成为目的基因表达水平研究最常用的技术手段。而结果的可靠性取决于很多因素,其中使用合适的内参基因是qPCR技术最基本的应用前提。许多研究表明没有一种内参基因可以在任何条件下都能稳定地表达。目前尚未见到关于蝴蝶兰低温生长条件下最佳内参基因选择的有关报道。蛋白磷酸酶2A(PP2A)是真核生物体内一种主要的细胞内源丝氨酸/苏氨酸蛋白磷酸酶。本研究根据蝴蝶兰低温转录组测序结果克隆得到1个PP2A的A亚基基因,其cDNA开放阅读框(ORF)长度为1764 bp,编码1个含有587个氨基酸的蛋白,将该基因命名为PhPP2Aa,GenBank登录号为MW847782。序列分析结果表明,该基因与其他植物PP2A核苷酸序列相似性均在80%以上,氨基酸序列与小兰屿蝴蝶兰PP2A序列相似性为99.66%。基于氨基酸序列进化分析结果表明,蝴蝶兰PhPP2Aa与小兰屿蝴蝶兰和铁皮石斛的亲缘关系最近。将蝴蝶兰PP2A与其他7种候选内参基因(TUA、TUB、ACTIN、F-box、RPL19、RPL36及RPL41)进行实时荧光定量PCR,用3种常用内参基因分析软件对各个基因Ct值进行稳定性分析,结果表明蝴蝶兰8个候选内参基因在低温胁迫条件下表达水平最稳定的内参基因为PP2A,其次为ACTIN;最不稳定的基因为F-box,其次为TUA。以蝴蝶兰PP2A作为内参基因探讨低温胁迫响应基因PhNAC1的表达情况,结果显示蝴蝶兰PhNAC1的表达模式符合低温胁迫条件下的表达特性。该结果表明蝴蝶兰PhPP2Aa基因可作为低温胁迫条件下目的基因转录水平研究的内参基因。Quantitative real-time polymerase chain reaction(qPCR) analysis, with the benefits of simplicity, high sensitivity, accuracy and high-throughput characteristics, has been used in many fields to quantify the transcript levels of target genes. There are many rules that must be followed to ensure the reproducible and accurate expression profiles of target genes using qPCR. Among them, the use of a reliable internal control known as a reference gene for data normalization is the elementary prerequisite for valid results and proper analysis. Numerous studies have suggested that no single reference gene is always expressed stably under any condition. There are no reports on the selection of optimal reference genes for Phalaenopsis under low temperature conditions. Protein phosphatase 2A(PP2A) is a major intracellular serine/threonine protein phosphatase in eukaryotes. A subunit gene of PP2A was cloned by the results of the transcriptome sequencing of Phalaenopsis hybrid under cold stress, which was named PhPP2Aa and the GenBank accession number was MW847782. The coding region(ORF) of PhPP2Aa was 1764 bp, encoding 587 amino acids. Homologous alignment showed that it shared over 80% nucleotide sequence similarity with PP2A in other plants, and that it shared 99.66% amino acid sequence similarity with P. equestris. The phylogenetic tree analysis based on the amino acid suggested that the relationship of PP2A between Phalaenopsis hybrid, P. equestris and Dendrobium catenatum was close, which belonged to the same branch. Three conventional software(geNorm, NormFinder, BestKeeper) were used to analyze the expression stability of 8 candidate reference genes(TUA, TUB, ACTIN, F-box, PP2A, RPL19, RPL36 and RPL41) from Phalaenopsis. The results showed that the most stable reference gene was PP2A, followed by ACTIN. And,the most unstable was F-box, followed by TUA. Using PP2A of Phalaenopsis(PhPP2Aa) as the reference gene to explore transcriptional profile of the target gene PhNAC1, the results demonstrated that the expression pat

关 键 词：蝴蝶兰 蛋白磷酸酶2A 内参基因 低温胁迫 序列分析 

分 类 号：Q785[生物学—分子生物学] Q786