山药Actin基因片段的克隆及表达分析  

Cloning and Expression Analysis of Actin Gene Fragment from Dioscorea opposite

作　　者：龚明霞[1,2,3] 周芸伊 王爱勤[1] 罗海玲[1,2] 何龙飞[1] 

机构地区：[1]广西大学农学院,南宁530004 [2]广西农科院作物遗传改良生物技术重点实验室,南宁530007 [3]广西农科院蔬菜研究所,南宁530007

出　　处：《生物技术通报》2016年第7期73-80,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(30760126);公益性行业(农业)科研专项(200903022)

摘　　要：通过山药Actin基因的克隆和表达分析,为研究山药生长发育中肌动蛋白的作用及其他基因的表达和调控奠定基础。根据Gen Bank中已经公布的其他植物肌动蛋白基因(Actin)的保守序列设计一对简并引物,采用RT-PCR技术从山药块茎中分离出1个Actin基因cDNA片段,命名为DoActin。片段长度为1 091 bp,编码357个氨基酸,并提交Gen Bank(登录号:KU669295)。与NCBI核酸和蛋白质数据库序列比对,该序列与其他植物Actin基因核苷酸序列的同源性均在83%以上,氨基酸序列的同源性均在97%以上。进化分析结果显示,DoActin与海枣Actin-2、木本棉Actin-7的亲缘关系最近。实时定量PCR结果显示,DoActin在山药叶片、地上茎和地下块茎以及不同发育期的块茎和叶片中表达量相对稳定,表明其适宜作为山药的内参基因。The cloning and expression analysis of Actin gene fragment from Dioscorea opposite was studied in order to provide thefoundation for studying its function and other genes expression and regulation in yam growth and development.A pair of degenerate primerswere designed based on the conserved sequences of the published Actin genes of other plants in Gen Bank.The cDNA fragment of 1 Actin genewas isolated from yam tuber by RT-PCR and designated as DoActin.It＇s length was 1 091 bp encoding a protein with 357 amino acids residues,and was deposited in Gen Bank（Accession number ：KU669295）.Homologous alignment according to the nucleotide and protein databasesshowed that it shared over 83% nucleotide sequence similarity and over 97% amino acid sequence similarity with Actins in other plants.Thephylogenetic tree reconstructed on the base of amino acid sequences suggested that the DoActin had the nearest relationship with Phoenix dactylifera Actin-2 and Gossypium arboreum Actin-7.RT-PCR results revealed that the expression levels of DoActin in different organs suchas leaf,aboveground stem and underground tuber,as well as tubers and leaves in different developmental periods were relatively stable,indicating that DoActin can be used as an appropriate reference gene for D.opposite.

关 键 词：山药 ACTIN基因 克隆 表达分析 序列分析 

分 类 号：Q943.2[生物学—植物学]