盾叶薯蓣转录组分析及其皂苷元生物合成关键酶基因的挖掘

Analysis of transcriptome of Dioscorea zingiberensis and excavation of key enzyme genes of saponin biosynthesis

目的分析比较盾叶薯蓣根茎与叶片的转录组,挖掘与盾叶薯蓣中皂苷元生物合成途径相关的关键酶基因。方法利用Illumina Hi Seq2000高通量测序技术对盾叶薯蓣根茎与叶片进行转录组测序,对测序结果进行注释分析;并结合根茎与叶片中皂苷元的含量测定结果,挖掘与盾叶薯蓣中皂苷元生物合成途径相关的关键酶基因;最后利用实时荧光定量PCR(q RT-PCR)技术对部分候选基因的表达模式进行分析。结果共获得了81 660个Unigene,有64.33%在NT、NR、Swiss-Prot、KOG、GO、KEGG数据库中得到注释;根据其表达量与代谢通路分析筛选出29种共227个可能参与盾叶薯蓣中皂苷元生物合成途径的催化酶基因,部分催化酶基因的表达模式与皂苷元含量具有一定相关性;并发现5个盾叶薯蓣内生菌基因。结论研究初步获得了盾叶薯蓣中参与皂苷元生物合成的候选关键酶基因,部分候选酶基因可能参与盾叶薯蓣中皂苷类成分的后修饰过程,并发现盾叶薯蓣根茎中内生菌可能参与其皂苷元的生物合成,为进一步阐明盾叶薯蓣中皂苷元生物合成的分子机制奠定基础。

Objective To compare and analyze the transcriptome of rhizome and leaves of Dioscorea zingiberensis, and excavate the key enzyme genes related to the saponin biosynthetic pathway in D. zingiberensis. Methods The transcriptome of rhizome and leaves of D. zingiberensis were sequenced by Illumina Hi Seq2000 high-throughput sequencing technique. According to sequence annotate results to find the differentially expressed genes. Then the key enzyme genes related to the biosynthesis of diosgenin were identified according to the content of saponins in rhizomes and leaves of D. zingiberensis. The expression levels of some candidate genes were analyzed by real-time fluorescence quantitative PCR（q RT-PCR）. Results A total of 81 660 Unigenes were gained and 64.33% of them were annotated in NT, NR, Swiss-Prot, KOG, GO, and KEGG databases. Based on their expression and KEGG annotation, totally 227 catalytic enzyme genes of 29 kinds that may participate in D. zingiberensis saponin biosynthetic pathway were screened. The expression pattern of some catalytic enzymes was correlated with the content of saponin. Also were found five D. zingiberensisendophyte genes. Conclusion This experiment obtained candidate key enzyme genes tentatively that involved in the biosynthesis of saponin. Some candidate enzyme genes may participate in the post-modification process of steroidal saponins in D. zingiberensis. In additon, it was found that D. zingiberensis endotrophic bacteria may be involved in the saponin biosynthesis. The results laid a foundation to further elucidate the molecular mechanism of sapogenin synthesis pathway.