黄连重金属ATP酶基因CcHMA3的克隆和表达分析

Cloning and Expression Analysis of Heavy Metal ATPase Gene CcHMA3 in Coptis chinensis

目的:克隆黄连重金属ATP酶基因CcHMA3,并对其编码蛋白进行生物信息学以及差异表达分析。方法:根据黄连基因组和转录组数据,利用PCR扩增CcHMA3的全长cDNA并进行生物信息学分析。利用实时荧光定量PCR法(qRT-PCR)检测CcHMA3基因在黄连植株中的表达差异。结果:克隆获得CcHMA3基因,开放阅读框全长为3030 bp,编码1009个氨基酸;蛋白相对分子量为109.03 kD,理论等电点为6.7;蛋白序列中含有6个跨膜结构域和2个保守结构域,亚细胞定位预测表明CcHMA3位于质膜上,具有一定亲水性。qRT-PCR结果表明,CcHMA3基因在黄连不同组织中的相对表达量由高到低依次为须根、根茎、叶柄、叶片;当镉处理后,CcHMA3基因在须根中的表达量显著高于其他组织(P<0.05),且对不同镉胁迫处理时间存在明显不同的响应表达。结论:该研究为进一步深入研究CcHMA3基因在黄连重金属镉代谢过程中的调控机制,进而寻找可能的阻断方式奠定了实验基础。

Objective:To clone the heavy metal ATPase gene CcHMA3 of Coptis chinensis and analyze its encoding protein bioinformatics and differential expression.Methods:Based on the genome and transcriptome data of Coptis chinensis,the full-length cDNA of CcHMA3 was amplified by PCR and bioinformatic analysis was performed.Real-time quantitative PCR(qRT-PCR)was used to detect the expression difference of CcHMA3 gene in Coptis chinensis.Results:CcHMA3 gene was cloned.The open reading frame length was 3030 bp,encoding 1009 amino acids.The relative molecular weight of the protein was 109.03 kD and the theoretical isoelectric point was 6.7.The protein sequence contained 6 transmembrane domains and 2 conserved domains.Subcellular localization prediction indicated that CcHMA3 was located on the plasma membrane and had certain hydrophilicity.The results of qRT-PCR showed that the relative expressions of CcHMA3 gene in different tissues of Coptis chinensis were from high to low as fibrous root,rhizome,petiole and leaf.After Cd treatment,the expression of CcHMA3 gene in fibrous roots was significantly higher than that in other tissues(P<0.05),and the expression of CCHMA3 gene was significantly different in response to different Cd stress treatment times.Conclusion:This study lays an experimental foundation for further research on the regulatory mechanism of CcHMA3 gene in the heavy metal cadmium metabolism of Coptis chinensis,and further search for possible blocking methods.