‘甘肃红豆草’肌动蛋白基因片段的克隆及序列分析

Cloning and sequence analysis of actin gene fragment from Onobrychis viciifolia ‘Gansu'

以‘甘肃红豆草’叶片总RNA为模板,通过RT-PCR方法扩增出肌动蛋白(Actin)基因片段,并克隆到pMD18-T载体,阳性克隆经PCR鉴定后测序.结果表明:序列分析表明,该片段长258bp,编码86个氨基酸,所得序列与GenBank中注册的Actin基因核苷酸序列的相似性在88%以上,与其他肌动蛋白的氨基酸序列的相似性达92%以上.注册该基因到GenBank中,注册号为KP159623.采用半定量RT-PCR方法,分析Actin基因在甘肃红豆草不同组织器官中的表达量相对恒定,而编码花青素还原酶的BAN基因在器官间的表达量差异较大,表明其可作为研究其它重要功能基因在‘甘肃红豆草’中的表达和调控的内参基因.

Total RNA of Onobrychis viciaefolia ‘Gansu’was extracted from leaf.The RNA was used to clone Actin gene fragment by RT-PCR,then the gene fragment was cloned into pMD1 8-T vector.The gene was sequenced after identifying the positives clone by PCR.The results revealed that the actin gene fragment from Onobrychis viciaefolia ‘Gansu’contained 258 bp and encoded a sequence of 86 amion acids.Similarity comparison showed that it shared over 88% similarity of nucleotide sequence similarity and over 92% similarity of amino acid sequence with other actin in the GenBank.The gene was submitted to GenBank and was given the accession number KP159623.Semi-quantitative PCR analysis indicated that the expression quantity of Actin gene was similar in different organs,however,the expression quantity of BAN gene encoding anthocyanin reductase was different.The results show that Actin gene can be used as the reference for analysis of gene expression in Onobrychis viciifolia ‘Gansu’.