[关键词]
[摘要]
目的 获得药用植物阳春砂Amomum villosum萜类合酶基因AvBPPS(bornyl diphosphate synthase)、AvLIS(linalool synthase)和AvHUS(humulene synthase)的启动子,探究其顺式作用调控元件参与萜类合成的调控。方法 从阳春砂叶片基因组DNA(genomic DNA,gDNA)中分别克隆获得AvBPPS、AvLIS和AvHUS的gDNA序列,据此设计特异引物,再通过FPNI-PCR(fusion primer and nested integrated PCR,FPNI-PCR)方法分别克隆其启动子并进行生物信息学分析,结合转录组及对应萜类含量数据,分析其较为关键的顺式作用调控元件与萜类调控的关系。结果 获得AvBPPS、AvLIS和AvHUS的gDNA长度分别为2374、2295、2362 bp,均包含相应基因的完整编码区和7个外显子。进一步克隆获得470、934、762 bp的启动子。AvBPPS启动子含有参与胚乳表达的顺式作用调控元件GCN4-motif,结合基因表达量的分析,该顺式作用调控元件调控了AvBPPS在阳春砂药用部位种子中的特异表达,进而影响了主要药效萜类成分龙脑、乙酸龙脑酯等在种子中的积累。AvLIS和AvHUS启动子均含有参与茉莉酸甲酯(methyl jasmonate,MeJA)反应的顺式作用调控元件,转录组和萜类含量的分析显示AvLIS响应高浓度MeJA的诱导,而AvHUS没有表现出对MeJA的响应。结论 首次从药用植物阳春砂的启动子中发现了参与胚乳表达和MeJA反应的调控元件,为深入探究萜类合成关键酶的种子表达特异性和对MeJA响应的机制提供了基础,为萜类代谢的调控提供了操作元件。
[Key word]
[Abstract]
Objective To obtain the promoters of terpenoid synthase genes AvBPPS (bornyl diphosphate synthase), AvLIS (linalool synthase) and AvHUS (humulene synthase) of medicinal plants Amomum villosum, and investigate the regulation of cis-acting regulatory elements involved in terpenoid synthesis. Methods The gDNA of AvBPPS, AvLIS, and AvHUS were cloned from genomic DNA (gDNA) of leaves from A. villosum, and then specific primers were designed accordingly. The promoters were cloned by FPNI-PCR subsequently and bioinformatics analysis was performed. The relationship between key cis-acting regulatory elements and terpene regulation was analyzed by combining the transcriptome data and terpenoid contents. Results The gDNA lengths of AvBPPS, AvLIS and AvHUS were 2374 bp, 2295 bp and 2362 bp, respectively, and all contained complete coding regions of the corresponding genes and seven exons. The promoters of AvBPPS, AvLIS and AvHUS were cloned, respectively. The promoter of AvBPPS contained GCN4-motif, a cis-acting regulatory element involved in endosperm expression. Combined with the analysis of gene expression, it was speculated that this cis-acting regulatory element regulated the specific expression of AvBPPS in seeds, and then affected the accumulation of major terpenoids such as borneol and borneol acetate in seeds. Both AvLIS and AvHUS promoters contained cis-acting regulatory elements involved in methyl jasmonate (MeJA) reaction. Transcriptome and terpene content analysis showed that AvLIS responded to the induction of high-concentration-MeJA, while AvHUS showed no response to MeJA. Conclusion Cis-acting regulatory elements involved in endosperm expression and MeJA response were discovered from promoters of medicinal plants A. villosum, which provided a basis for in-depth exploration of seed expression specificity of key enzymes of terpene synthesis and the mechanism of response to MeJA, and provided operating elements for regulation of terpene metabolism.
[中图分类号]
R282.12
[基金项目]
国家自然科学基金面上项目(81872954)