白木香倍半萜合成酶基因As-SesTPS的克隆及生物信息学与表达分析

Cloning of sesquiterpene synthase gene As-SesTPS from Aquilaria sinensis and analysis its bioinformatics and expression

目的从白木香Aquilaria sinensis总RNA中克隆倍半萜合成酶基因,并对其进行生物信息学及表达分析。方法对白木香总RNA进行反转录聚合酶链式反应(reverse transcription-PCR,RT-PCR)和cDNA末端快速扩增(rapid amplification of cDNA ends,RACE),获得完整的开放阅读框(ORF)。运用生物信息学的方法对该序列进行相似性比较和同源性分析,预测编码蛋白,并对其进行各种理化性质分析。通过半定量PCR检测该基因在白木香树干中不同部位的表达情况。结果获得As-SesTPS基因,ORF长1 629 bp,编码542个氨基酸,与葡萄Vitis vinifera的大根香叶烯-D合成酶[(-)-germacrene D synthas]相似性最高,且包含RRx8W和DDxxD的保守序列。As-SesTPS蛋白无跨膜区域,定位于细胞质中,且仅在白木香结香部位表达。结论首次从白木香中克隆得到可能编码大根香叶烯-D合成酶的基因,为白木香倍半萜生物合成代谢途径的研究提供参考。

Objective To clone the sesquiterpene synthase gene As-SesTPS from total RNA ofAquilaria sinensis and to analyze the bioinformatics and gene expression. Methods The gene containing intact open reading frame （ORF） was cloned by reverse transcription-PCR （RT-PCR） and rapid amplification of cDNA ends （RACE）. The similarity comparison and honaology analysis of the sequence were carried out using bioinformatic method, the coding protein was predicted and the physicochemical： properties were analyzed. The expression of the gene in different locations of A. sinensis trunk was determined by semiquantitative PCR using gene-specific primers. Results The As-SesTPS gene, containing a 1 629 bp ORF that encoded 542 amino acids, was cloned. The deduced protein sequence had the most similarity to the （-）-germacrene-D synthase in Fitis vinifera and exhibited two conserved motifs （RRxsW and DDxxD）. Without transmembrane domain, As-SesTPS was located in cytoplasm and expressed only in the agarwood part. Conclusion The As-SesTPS gene probably encoding （-）-germacrene-D synthase ofA. sinensis is successfully cloned for the first time, which would provide a reference for the study of sesquiterpene biosynthase pathway in A. sinensis.