盐生植物四翅滨藜Actin基因片段的克隆及表达分析

摘要

在研究植物功能基因表达模式中肌动蛋白(Actin)编码基因是最常用的内参基因之一.本研究根据藜科植物Actin基因编码区保守序列设计一对简并引物,以盐生植物四翅滨藜(Atriplex canescens)4周龄幼苗叶片的总RNA为模板,采用逆转录PCR(reverse transcription PCR,RT-PCR)方法扩增出Actin基因片段并连接到pMD19-T载体上,所得阳性克隆经PCR检测后进行测序.序列分析结果表明,该基因片段长度为598 bp,编码198个氨基酸;该序列与Genbank中登录的高等植物Actin基因核苷酸序列同源性在84％以上,氨基酸序列同源性在94％以上.说明其为Actin基因片段,具有高度保守性,命名为AcACT.序列进化分析显示,AcACT与甜菜(Beta vulgaris)和碱蓬(Suaeda glauca)肌动蛋白编码基因的亲缘关系最近.荧光定量PCR分析结果表明,AcACT在盐处理下各组织中的表达量恒定.表明AcACT可作为内参基因来研究四翅滨藜耐盐相关基因的表达模式.%The Actin gene is one of the most commonly used reference genes for studying functional gene expression patterns.In this study,total RNA was extracted from the leaves of 4-week-old Atriplex canescens seedlings,and an Actin gene fragment was obtained by reverse transcription-polymerase chain reaction (RTPCR) and linked into pMD19-T vector using primers designed based on conserved Actin gene sequences from similar plant species.The positive clones identified by PCR were sequenced,and the results showed that the Actin gene fragment of A.canescens contains 598 bp encoding 198 amino acids.Homology comparison with Actin genes from other higher plants in GenBank indicate that this gene fragment shared more than 84％ nucleotide sequence similarity and 94 ％ amino acids sequence homology with other plants,suggesting strong conservation of this gene.Our results confirmed that the cloned gene was an Actin gene fragment,which we named as AcACT.Phylogenetic analysis showed a close relationship between AcACT and the Actin genes from Beta vulgaris and Suaeda glauca.Meanwhile,AcACT expression was constant in different tissues under salt treatment based on quantitative PCR analysis,which suggests that AcACT can be used as a reference gene for studying the expression patterns of salt tolerance-related genes of A.canescens.