南美蟛蜞菊WtActin1基因克隆及表达稳定性分析

Cloning and expression stability analysis of WtActin1 gene from Wedelia trilobata

【目的】克隆南美蟛蜞菊肌动蛋白基因(WtActin1),并进行生物信息学及表达稳定性分析,为南美蟛蜞菊耐热耐冷功能基因表达及调控研究提供可靠的内参基因。【方法】RACE克隆WtActin1基因,通过在线生物信息学分析软件对其氨基酸序列进行分析,并利用实时荧光定量PCR检测该基因在不同组织及温度胁迫下的表达情况。【结果】克隆获得的WtActin1基因cDNA全长1682 bp(GenBank登录号MN485900),其中5'非编码区(5'-UTR)170 bp,3'非编码区(3'-UTR)378 bp,开放阅读框(ORF)1134 bp,编码377个氨基酸残基。WtActin1蛋白分子量为41725.81 Da,理论等电点(pI)为5.31,不稳定性指数(II)为37.48,亲水性的平均值(GRAVY)为-0.166,为稳定的亲水性蛋白,无跨膜结构域和无信号肽,主要存在于细胞质中,具有Actin蛋白家族的典型特征结构,其二级结构主要由α-螺旋、β-转角、延伸链和无规则卷曲组成,三级结构同源模型为嵌合肌动(3ci5.1.A),与莴苣、橡胶草和洋蓟的同源蛋白三级结构高度相似。WtActin1基因与其他植物Actin基因核苷酸序列相似性在86.4%以上,其推导氨基酸序列相似性在94.0%以上。从基于Actin氨基酸序列相似性构建的系统发育进化树可看出,WtActin1与同为菊科的莴苣LsActin7亲缘关系最近,与传统分类学相吻合,其次是菊科的橡胶草TkActin1和锦葵科的棉花GhActin。WtActin1基因在南美蟛蜞菊根、茎、叶和花中的表达量均无显著差异(P>0.05,下同),且在高、低温胁迫下与正常温度处理的表达量也均无显著差异。【结论】WtAtin1基因属于Actin基因家族成员,在不同组织及温度胁迫下均能稳定表达,可作为内参基因用于南美蟛蜞菊耐热耐冷功能基因研究。

【Objective】In this study,actin gene WtActin1 was cloned from Wedelia trilobata and its bioinformatics and expression stability were analyzed to provide an appropriate reference gene for the expression and regulation of functional genes with cold and heat resistance in W.trilobata.【Method】The WtActin gene from W.trilobata was cloned by RACE,its amino acid sequence was analyzed by bioinformatics software,and the expression of WtActin gene in different tissues and under temperature stress was detected by real-time quantitative PCR.【Result】The full-length cDNA of WtActin1 gene was 1682 bp(GenBank accession number:MN485900),contained a 1134 bp open reading frame encoding(ORF)377 amino acids residues,with 170 bp in the 5’non-coding region(5’-UTR)and 378 bp in the 3’-UTR.WtActin1 protein had a molecular weight of 41725.81 Da and a theoretical isoelectric point(pI)of 5.31,instability index(II)of 37.48,average hydrophilicity(GRAVY)of-0.166.It was a stable hydrophilic protein without transmembrane domain and signal peptide.It mainly existed in cytoplasm,and had typical characteristic structure of Actin gene family.Its secondary structure was mainly composed ofα-helix,β-turn,extended chain and irregular coil,the three-level structure homology model was chimeric myokinesis(3 ci5.1.A),which was highly similar to that of Lactuca sativa,Taraxacum kok-saghyz and Cynara cardunculus var.scolymus.Homology comparison with other plant Actin nucleotide sequences showed that it shared over86.4%nucleotide sequence identity and 94.0%amino acid sequence identity with Actin in other plants.Based on phylogenetic tree of Actin amino acid sequence similaruty,WtActin1 was closely related to L.sativa(LsActin7)of Asteraceae,which was consistent with traditional taxonomy,followed by T.kok-saghyz(TkActin1)of Asteraceae and Gossypium hirsutum(GhActin)of Malvaceae.The expression level of WtActin1 gene was stable in root,stem,leaf and flower of W.trilobata and there was no significant difference among them(P>0.05,the same below),and there was no significant difference between high or low temperature stress and normal temperature.【Conclusion】The WtActin1 gene cloned in this study is a member of Actin gene family and could be stably expressed in different tissues and under temperature stress.It can be used as an internal reference gene for the study of heat and cold tolerance genes of W.trilobata.