UV-C处理穿心莲转录组分析及穿心莲内酯合成相关基因挖掘

Transcriptome analysis and discovery of genes related to andrographolide synthesis of Andrographis paniculata under UV-C treatment

  • 摘要: 【目的】对紫外线C(UV-C)处理的穿心莲叶片进行转录组测序分析,挖掘穿心莲内酯合成相关基因,为深入探究穿心莲内酯合成相关基因的调控机制和生物学功能提供理论参考。【方法】选用生长60 d的穿心莲幼苗为材料,经UV-C照射2 h后,利用Illumina HiSeq二代测序平台进行转录组测序,并结合生物信息学软件和实时荧光定量PCR (qRT-PCR)对所得序列进行功能注释和相对表达水平验证,挖掘响应UV-C的穿心莲内酯合成相关基因。【结果】共注释到21545个穿心莲基因,有2137个基因呈显著差异表达模式,其中,1147个基因显著上调表达,990个基因显著下调表达。GO功能注释结果显示,UV-C处理造成了氧化胁迫,线粒体氧化磷酸化电子传递链的NADH脱氢酶基因上调表达。KEGG代谢通路富集结果显示,二萜类物质穿心莲内酯的前体(E,E,E)-香叶基香叶基二磷酸(GGPP)合成途径甲羟戊酸(MVA)途径和2-C-甲基-D-赤藓糖醇-4-磷酸(MEP)途径差异表达基因显著富集。转录组测序结果和实时荧光定量PCR(qRT-PCR)检测结果均显示,MVA途径的4种合成酶基因HMGS(CXN00016849)、HMGR(CXN00000058)、MVK(CXN00002900)和MVD(CXN00004398)及GGPP合成关键酶基因FPS的表达量显著上调;MEP途径中,仅DXS(CXN00013302)的表达量显著下调。蛋白互作预测结果显示,2个途径中的差异表达基因编码蛋白之间存在互作,HMGSCYP71家族成员之间,以及FPS1与UGT71UGT73UGT76之间的互作网络丰富。【结论】 UV-C照射调控穿心莲重要生命进程,其中,次生代谢、氧化磷酸化、光合作用等途径基因转录本的相对表达量呈UV-C特异性响应模式。合成穿心莲内酯前体GGPP的细胞质MVA途径基因显著上调表达,推测UV-C诱导穿心莲内酯合成的主要场所为细胞质。穿心莲MVA途径基因及CYP71UGT71UGT73UGT76可作为穿心莲内酯合成途径研究的候选分子靶标。

     

    Abstract: 【Objective】Transcriptome sequencing was performed on the leaves of Andrographis paniculata treated with ultraviolet C(UV-C),and the genes related to andrographolide synthesis were excavated to provide reference for further exploring the regulatory mechanism and biological functions of andrographolide synthesis-related genes.【Method】 A.paniculata seedlings grown for 60 days were selected as experimental materials. After 2 h of UV-C irradiation,their transcriptome was sequenced using the 2nd-generation sequencing platform,andwith bioinformatics software and qRTPCR, the sequence were evaluated by gene expression analysis and functional annotation to identify genes related to andrographolide synthesis in response to UV-C.【Result】A total of 21545 A.paniculata genes were annotated,and 2137 genes showed significant differential expression(SDE)patterns. Among them,1147 genes were significantly up-regulated and 990 genes were significantly down-regulated. GO annotation results showed that UV-C treatment caused oxidative stress,and the expression of NADH dehydrogenase genes in mitochondrial oxidative phosphorylation electron transport chain were significantly up-regulated. KEGG metabolic pathway enrichment analysis showed that the precursor of andrographolide(E, E, E)-geranylgeranyl diphosphate(GGPP)synthesis pathway(Mevalonate,MVA pathway and 2-Cmethyl-D-erythritol-4-phosphate, MEP pathway)SDE genes were significantly enriched. According to the results of RNAseq and real-time quantitative PCR(qRT-PCR),the expression of four synthase genes in the MVA pathway,HMGS (CXN00016849),HMGR(CXN00000058),MVK(CXN00002900)and MVD(CXN00004398),as well as GGPP synthesis key enzyme FPS were significantly up-regulated. In the MEP pathway,only the expression of DXS (CXN00013302)was significantly down-regulated. The type of alternative splicing(AS)that commonly occurs in the genes of the two pathways was exon skipping(SE). The protein interaction prediction showed that there were interactions between the differentially expressed genes(DEGs)of the two pathways,and the interaction networks between HMGS and CYP71 family members,and the ones among FPS1,UGT71,UGT73 and UGT76 were enriched.【Conclusion】UV-C irradiation regulates important life processes of A. paniculata. The relative expression of gene transcripts in secondary metabolism,oxidative phosphorylation,photosynthesis and other pathways show UV-C-specific response pattern. The expressions of cytoplasmic MVA pathway genes are significantly up-regulated. It is speculated that the main site of UV-Cinduced andrographolide synthesis is the cytoplasm. A. paniculata MVA pathway genes, CYP71,UGT71,UGT73 and UGT76 can be used as candidate molecular targets for andrographolide synthesis pathway research.

     

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