阳春砂基于转录组的萜类合酶基因挖掘及一个单萜合酶基因的克隆

Mining of Genes Involved in Terpenoid Synthases Based on Transcriptome Analysis and Cloning of Monoterpene Synthase from Amomum villosum Lour.

【目的】深入挖掘阳春砂转录组中挥发性萜类合酶相关基因,为全面认识阳春砂挥发性萜类代谢途径和分子调控模式奠定基础。【方法】整理前期工作获得的阳春砂2个转录组的数据,筛选已注释的及利用本地blast方法再注释的萜类合成途径的unigene;并通过对部分候选unigene的表达量与挥发性萜类化合物含量的相关性分析及生物信息学分析,进一步筛选出相关基因;采用PCR及重组载体构建的方法克隆其中一个单萜合酶基因,并对其编码蛋白序列进行分析。【结果】筛选得到10个挥发性萜类合成上游途径相关unigene及11个下游萜类合酶基因;相关性分析证明候选基因与阳春砂主要挥发性萜类有较强相关性;并成功克隆得到Av TPS1基因,其序列包含1 803 bp的开放阅读框,编码600个氨基酸;其编码蛋白含有单萜合酶蛋白特有的DDXXD、RRX8W和NSE/DTE等保守基序,N端含有一段叶绿体转运肽;系统进化分析表明Av TPS1属于TPS-b亚家族。【结论】结合转录组、表达谱数据的深入挖掘和与挥发性萜类化合物数据的关联,有效筛选出了阳春砂多个值得后续研究的萜类合酶基因;其中对Av TPS1的克隆及生物信息学分析为后续功能鉴定提供了基础。

Objective To dig out the related volatile terpene synthase genes from transcriptome of Amomum villosum Lour., so as to lay the foundation for comprehensive understanding of volatile terpenoid biosynthesis pathway and molecular regulation mode. Methods Based on the obtained results of the two transcriptome of Amomum villosum Lour.,we screened out the unigenes involved in volatile terpenoid biosynthesis pathway which had been annotated or was reannotated by blast method. After analyzing the correlation of the expression of partial candidate unigenes with the contents of volatile terpenoids and their bioinformatics,we selected out the relative unigenes. One monoterpene synthase gene of them was cloned by polymerase chain reaction（PCR）and recombinant vector construction methods, and then the protein coding sequences of the monoterpene synthase gene were analyzed. Results Ten candidate terpenoid biosynthesis upstream pathway unigenes and 11 candidate downstream pathway genes were obtained from the transcriptome of Amomum villosum Lour.. The data proved that the expression of the candidate genes had a strong relationship with main volatile terpenes of Amomum villosum Lour.. Av TPS1 had been cloned successfully, which contained 1 803 bp open reading frame（ORF）,encoding 600 amino acids. The encoded protein of Av TPS1 contained the specific conservative sequences DDXXD, RRX8 W and NSE/DTE which were special for terpene synthase protein, and N-terminal of the encoded protein had a chloroplast transit peptide. Phylogenetic analysis showed that Av TPS1 belonged to the subfamily of TPS-b terpene synthase. Conclusion Based on the results of transcriptome and gene expression profile of Amomum villosum Lour.,and on the correlation of gene expression profile with the content of volatile terpenoids,we found out several valuable candidate terpene synthases genes from Amomum villosum Lour.. And the analysis results of clone and bioinformatics of Av TPS1 will supply evidence for the future study of gene function characterization.