基于转录组数据对七叶树中三萜皂苷合成途径的研究

Synthesis of triterpenoid saponins from Aesculus chinensis based on transcriptome data

七叶树是七叶树科七叶树属高大乔木,集药用与观赏于一身。七叶树种子是其主要的药用部位,主要有效成分为齐墩果烷型三萜皂苷类化合物七叶皂苷。为了解七叶树三萜化合物生物合成的分子基础,该研究采用Illumina Hi Seq 2000高通量测序获得七叶树种子与花的转录组数据,并对其代谢途径相关基因进行挖掘;使用Trinity软件实现unigene的de novo拼接;并将转录组的测序结果运用KEGG数据库进行注释,从而预测七叶树三萜代谢的具体途径。结果显示七叶树转录组共有2个三萜代谢途径相关数据集,分别为萜类物质前体代谢途径(途径编号为ko00900)和倍半萜与三萜代谢途径(途径编号为ko00909),对应的基因分别有47,27条。进一步根据催化酶与活性产物的总结比较分析发现萜类物质前体代谢途径相关酶共有8种,三萜代谢途径相关酶有3种。该研究首次在七叶树中解析出5条与三萜环化酶对应的基因,它们可能参与β-香树精合成进而形成七叶皂苷。通过种子与花的转录组表达差异分析显示,ko00900和ko00909途径相关的差异基因有33个;相对于花器官,种子中有17个unigenes表达量上调,16个unigenes表达量下调。该研究运用qRT-PCR实验对SQE(Unigene25806),HMGS(Unigene36710),β-AS(Unigene33291) 3个差异显著的关键酶基因的表达进行了试验验证,实验结果qRT-PCR结果与转录组数据一致。该研究所发现的七叶树的三萜皂苷合成相关候选基因能对七叶树三萜代谢合成与调控方面提供理论依据。

Aesculus chinensis belongs to Hippocastanaceae family,bears medicinal and ornamental values. The oleanane type triterpenoid saponin aescin is regarded as active ingredient and accumulated in seed. In order to understand its molecular basis of the triterpenoid biosynthesis,we used high-throughput sequencing under Illumina Hi Seq 2000 platform to obtain the transcriptome data of seed and flower from A. chinensis to further mine the genes involved in its metabolic pathway. Unigene’s de novo splicing was performed using Trinity software;the transcriptome results were annotated with KEGG database to predict the specific pathways of the aescin triterpenoid metabolism. Terpenoid and triterpenoid pathways were found from transcriptome data,and forty seven and twenty seven corresponding genes were uncovered respectively. It was found that there are eight kinds of enzymes related to the terpenoid metabolism pathway precursors and three kinds of enzymes related to the triterpenoid metabolism pathway. In this study,five genes corresponding to triterpene cyclase were analyzed in A. chinensis for the first time,which may participate in the synthesis of triterpenoid. It’s revealed that there were thirty three differential genes associated with the ko00900 and ko00909 pathways by analysis on the difference in transcriptome expression between seeds and flowers;seventeen unigenes were up-regulated and sixteen unigenes were down-regulated in the seeds relative to flowers. In this study, qRT-PCR experiments were used to verify the expression of three key enzyme genes of SQE( Unigene25806),HMGS( Unigene36710),and β-AS( Unigene33291). The results of qRT-PCR were consistent with the transcriptome data. The candidate genes related to triterpenoid saponin aescin synthesis in A. chinensis found in this study can provide theoretical basis for the metabolism synthesis and regulation of aescin.