果子蔓凤梨Actin基因的克隆与序列分析

持家基因Actin常被用作定量、半定量PCR试验的内参,以确定目标基因的相对表达量。基于前期从全长cDNA文库中获得Actin基因的EST单克隆,进行Primer Walking测序,获得一个Actin基因的全长cDNA序列,序列长1625bp,ORF为1131bp,可编码377个蛋白氨基酸,命名为Goactin1(GenBank登录号:HQ184438)。蛋白质理论分子质量为41.7kD,等电点pI为5.31,包含一个Actin superfamily保守区,二级结构主要由随机卷曲、Alpha螺旋、延伸链和Beta转角组成。此外以2BTF A链为基础建立起了Goactin1蛋白的三级结构图。系统进化树分析表明Goactin1蛋白与马铃薯、棉花、烟草、短柄草、拟南芥等的Actin蛋白聚为一类,它们的亲缘关系最近。

Cloning and Sequence Analysis of Actin Gene from Guzmania

As a house keeping gene,Actin was often used as inner control in quantitative and semiquantitative PCR tests to determine relative expressive amount of target genes.Based on EST monoclones of Actin gene obtained from Guzmania full length cDNA library,its full length cDNA sequence was obtained by Primer Walking sequencing.The gene,named for Goactin1(GenBank accession No.HQ184438),consisted of 1 625 bp cDNA sequence,1 131 bp ORF(open reading frame) which encoded a protein with 377 amino acids residues,and a putative protein which was 41.7 kD at estimated molecular weight,5.31 at isoelectric point and had a ’actin superfamily’ conservative domain.The secondary structure of the protein was composed of random coil,alpha helix,extend strand and beta turn by SPOMA analysis.Furthermore,its tertiary structure was also build based on A chain of 2BTF.Through phylogenetic tree analysis,Goactin1 was gather to a same group with Actin protein in Solanum tuberosum,Gossypium hirsutum,Nicotiana tabacum,Brachypodium sylvaticum,and Arabidopsis thaliana.