珙桐CAC基因的克隆及其作为内参基因的评价

Cloning of a CAC Gene in Dove Tree(Davidia involucrata Baill.)and Evaluating its Potential as a Novel Reference Gene

从珙桐(Davidia involucrata Baill.)中克隆到一个编码网格蛋白衔接蛋白复合物(Clathrin adaptor complexes,CAC)的基因,命名为DiCAC(Gen Bank登录号为KX268525)。该基因开放阅读框全长1 317 bp,编码438个氨基酸。同源序列比对与聚类分析表明,该基因与其它15种植物的CAC氨基酸序列的相似度达到94%以上,其中与葡萄CAC的氨基酸序列同源性最高。为评价该基因作为珙桐内参基因的可行性,结合5个珙桐管家基因(ACT7、EF1a、GAPDH、β-TUB、18S rRNA),采用半定量PCR和实时荧光定量PCR对DiCAC基因在珙桐不同组织以及不同发育阶段苞片中的表达稳定性进行分析,并与常见管家基因的稳定性进行比较,结果表明,该基因在珙桐不同组织以及不同发育阶段苞片中均为稳定表达,且表达稳定性优于常见管家基因,可以作为相关基因表达研究中的内参基因。首次克隆到珙桐CAC基因,并且明确了其作为内参基因的可行性,为深入研究珙桐相关基因的表达分析提供了候选内参基因资源。

In this research,a gene named as DiCAC（Gen Bank accession number KX268525）,encoding the clathrin adaptorcomplexes（CAC）,has been cloned from dove tree（Davidia involucrata Baill.）. The sequence of DiCAC includes a ORF（open readingframe）of 1317 bp,which encodes a protein with 438 amino acids. Homologous alignment and cluster analysis show that DiCAC sharedover 94% amino acid sequence identity with CAC genes in other 15 plants,and it has the highest amino acid homology with grape（Vitis vinifera）CAC gene. To evaluate the feasibility of using DiCAC as a reference gene in dove tree,combined with five housekeeping genes ofdove tree（ACT7,EF1 a,GAPDH,β-TUB and 18 S rRNA）,we analyzed the expression stability of DiCAC in different tissues and in bractsof different developmental stages. Furthermore,expression analysis of DiCAC detected by semi-quantitative PCR and qPCR showed that theexpression levels of DiCAC are stable in both different tissues and bracts of different developmental stages. Remarkably,the expression of DiCAC showed more stability than the expression of common housekeeping genes. Therefore,it could be used as a reference gene of relatedgene expression research in dove tree. In this study,we have cloned the CAC gene from dove tree firstly and have confirmed the feasibility of itsutilization as a reference gene. Our finding provides a novel candidate reference gene resources for the further research gene expression analysisin dove tree.