美洲南瓜α-微管蛋白CpTUA基因的分离及其作为内参基因的应用  

Isolation of α-Tubulin CpTUA Gene from Cucurbita pepo and It's Application as An Internal Standard

作　　者：朱海生[1] 刘建汀 温文旭 叶新如 王彬[1] 李永平[1] 陈敏氡[1] 林珲[1] 温庆放[1] Zhu Haisheng;Liu Jianting;Wen Wenxu;Ye Xinru;Wang Bin;Li Yongping;Chen Mindong;Lin Hui;Wen Qingfang(Crops Research Institute,Fujian Academy of Agricultural Sciences,Vegetable Research Center,Fufian Academy of Agricultural Sciences,Fujian Engineering Research Center for Vegetables,Fuzhou 350013,China)

机构地区：[1]福建省农业科学院作物研究所,福建省农业科学院蔬菜研究中心,福建省蔬菜工程技术研究中心,福州350013

出　　处：《中国细胞生物学学报》2018年第12期2040-2050,共11页Chinese Journal of Cell Biology

基　　金：福建省属公益类科研院所基本科研专项(批准号:2018R1026-5);福建省农业科学院“青年科技英才百人计划”(批准号:YC2017-5);福建省农业科学院蔬菜科技创新团队(批准号:STIT2017-1-2);中央引导地方科技发展专项(批准号:2018L3005);国家大宗蔬菜产业技术体系福州综合试验站(批准号:CARS-23-G-53)资助的课题~~

摘　　要：为了获得美洲南瓜α-tubulin基因,并设计合适的荧光定量PCR内参引物,该研究通过转录组测序和RT-PCR方法获得了1条长达1 863 bp的c DNA,命名为CpTUA, GeneBank登录号为:MH310440。生物信息学分析表明,该序列包含1个开放读码框(open reading frame, ORF),大小为1353 bp,预测共编码450个氨基酸,理论分子大小约为49.57 kDa,蛋白质等电点为4.84。Motif Scan分析显示, CpTUA蛋白质的氨基酸序列49―246位和248―393位分别为Tubulin和Tubulin-C保守区域。同源性分析表明,基因编码的蛋白质与同为南瓜属的中国南瓜和印度南瓜同源蛋白的相似性达到99%,具有高度的保守性。在此基础上,设计了1对的荧光定量PCR引物,该引物具有较高的特异性和重复性。RT-PCR和qRT-PCR分析表明, CpTUA基因在美洲南瓜不同组织和不同胁迫处理下均能稳定表达,适合在美洲南瓜基因表达研究中作为内参基因。该研究为开展美洲南瓜重要功能基因的表达模式和调控机制的研究奠定基础。The study was to obtain the α-tubulin gene of zucchini and design the qRT-PCR primers. A 1 863 bp cDNA was obtained by transcriptome sequencing and RT-PCR. This gene was named CpTUA and the GenBank accession was MH310440. Bioinformatics analysis results indicated that the sequence contained a size of 1 353 bp open reading frame(ORF) encoding 450 amino acids, with a theoretical molecular weight of 49.57 kDa and a protein isoelectric point(PI) of 4.84. Motif Scan analysis showed that CpTUA protein had the Tubulin and Tubulin-C domains of conserved actin in the position of 49―246 and 248―393 sites, respectively. Homol-ogy analysis revealed that CpTUA shared 99% identity with the homologous proteins from Cucurbita moschata and Cucurbita maxima which were also belong to Cucumber the same as Cucurbita pepo, proving that it was highly conservative. A pair of qRT-PCR primers were then derived from the Cp TUA gene sequence which had the high specificity and repeatability. The RT-PCR and qRT-PCR indicated that the Cp TUA gene was stable expression in different tissues and under different stress treatments, so it was suitable as a reference gene for the analysis of gene expression patterns in zucchini. The present study has provided an important reference for analysis the expression of critical genes zucchini.

关 键 词：美洲南瓜 内参基因 Α-微管蛋白 表达分析 

分 类 号：S642.1[农业科学—蔬菜学]