滨梅2个肌动蛋白cDNA片段的克隆与表达

cDNA Fragment Cloning and Expression Analysis of Two Actin Genes in Beach Plum(Prunus maritima Marshall)

目的:克隆耐盐碱果树滨梅的肌动蛋白基因actin,为该物种优异性状基因的功能鉴定提供内参。方法:利用一对actin简并引物克隆滨梅actin cDNA片段,对其进行序列和表达分析。结果:得到2条743 bp的cDNA片段,两条核苷酸序列相似性为82%,命名为PmAct1和PmAct2并在GenBank登录(分别为JX855160和JX855161);序列比对发现2条actin片段氨基酸序列与其他植物同源性均在96%以上;根据不同果树Actin相似性构建进化树,表明2个滨梅actin明显分为两种类型,但均与蔷薇科果树亲缘关系较密切;半定量RT-PCR表达谱发现PmAct1可能为组成型表达类型,而PmAct2特异地在根和叶组织中表达量较高。结论:首次获得了2个滨梅actin基因,为该物种其他功能基因的挖掘和表达分析奠定了基础。

Objective： The study aimed to clone actin genes in salt - tolerant fruit tree beach plum as the reference for functional identifica- tion of excellent genes. Method： Using a pair of degenerate primers, actin cDNA fragments were isolated from beach plum and sequence a- lignment and semi -quantitative RT- PCR analysis were then performed. Result： Two actin cDNA fragments of 743 bp were ob- tained. There was a consistency of 82% at the nucleotide level of the two fragments and were named PmActl and PmAct2 with GenBank ac- cession number JX855160 and JX855161 respectively. Sequence homology of the two both shared over 96% amino acid sequence similari- ty. Phylogenetic analysis based on Actin of fruit trees found PmActl and PmAct2 belonged to two types, but each had close genetic relation- ship with Rosaceae fruit trees. Semi quantitative RT- PCR analysis showed that expression of PmActl was possibly constitutive type,while PmAct2 specifically had higher expression level in root and leaf tissues. Conclusion：This was not only the first report on cloning of actin genes from beach plum, but also provided internal standard reference for other genes＇ expression analysis.