温郁金萜类合成酶基因CwTPS05、CwTPS10的克隆与表达分析

Cloning and expression analysis of terpene synthase CwTPS05 and CwTPS10 from Curcuma wenyujin

目的克隆温郁金Curcuma wenyujin萜类合成酶基因(CwTPS05 CwTPS10)并进行生物信息学及表达分析,为今后利用代谢工程生产温郁金萜类成分提供依据。方法根据前期温郁金转录组测序数据,利用PCR技术从根茎cDNA文库中克隆CwTPS05和CwTPS10序列;生物信息学分析其核苷酸和氨基酸序列特征;qRT-PCR检测基因在不同组织中的表达水平。结果克隆获得CwTPS05 (GenBank登入号:MT814867)基因,开放阅读框全长1446 bp,编码481个氨基酸,预测相对分子质量为55 990;CwTPS10 (GenBank登入号:MT814863)基因,开放阅读框全长1494 bp,编码497个氨基酸,预测相对分子质量为58 550。CwTPS05和CwTPS10均属于酸性、非跨膜、无转运肽的亲水性蛋白。同源进化树分析表明,CwTPS05和CwTPS10基因被归类为植物萜类合成酶TPSa亚家族。qRT-PCR分析表明,CwTPS05在根茎中特异性表达,CwTPS10在根茎和块根中特异性表达。结论成功克隆获得温郁金CwTPS05和CwTPS10基因并进行生物信息分析,为研究温郁金萜类成分的生物合成途径提供参考。

Objective To clone genes encoding the terpene synthases(CwTPS05 and CwTPS10) from the total RNA of the rhizome of Curcuma wenyujin, investigate their expression patterns, and then provide a basis for the production of C. wenyujin terpenoids through metabolic engineering platform. Methods According to the transcriptome sequencing data previously sequenced, PCR technology was used to obtain the sequences of two specifically expressed genes CwTPS05 and CwTPS10 in rhizome;bioinformatics software was used to analyze their nucleotide and amino acid sequences;qRT-PCR was used to detect their expression levels in different tissues. Results The full sequence of CwTPS05(accession: MT814867) gene was obtained, with the open reading frame(ORF) length of 1446 bp, encoding 481 amino acids;The predicted molecular weight was 55 990. Similarly, the sequence of CwTPS10(accession: MT814863) gene was also obtained with the length of 1494 bp ORF, encoding 497 amino acids, the predicted molecular weight was 58 550. CwTPS05 and CwTPS10 were both acidic, non-transmembrane, and non-transit peptide hydrophilic proteins. The homologous phylogenetic tree analysis showed that the CwTPS05 and CwTPS10 genes were classified as TPSa subfamily. The qRT-PCR analysis showed that CwTPS05 was specifically expressed in the rhizome, and CwTPS10 was specifically expressed in the rhizome and tuber. Conclusion The CwTPS05 and CwTPS10 genes of C. wenyujin are successfully cloned and analyzed by bioinformatics, which provide a solid basis for studying the biosynthetic pathways of terpenoids in C. wenyujin.