重金属胁迫下白骨壤数字基因表达谱分析

Digital Gene Expression Profiling Analysis of Avicennia marina under Heavy Metal Stress Treatment

[目的]从转录组水平研究重金属污水胁迫处理下白骨壤的基因表达变化,以揭示白骨壤响应重金属污染胁迫的机制。[方法]采用基于高通量测序的数字基因表达谱(DGE)技术对重金属污水胁迫处理组和对照组白骨壤材料进行转录组测序分析。[结果]与对照组相比,重金属处理下白骨壤共有10 459个差异表达基因,其中,8 685个表达量上升,1 774个表达量下降。Me V聚类表明:大部分基因在重金属处理样本中的表达量受到极大的诱导。GO功能注释发现,差异表达基因主要定位于叶绿体以及细胞内各种膜结构,参与"细胞代谢"、"细胞组分的组合和生物合成"、"对刺激的响应"等过程。KEGG代谢通路分析显示,差异表达基因分布于126条Pathways,涉及"核糖体"、"光合作用"、"乙醛酸盐代谢"、"丙酮酸代谢"等途径。重金属胁迫还促进萜类、黄酮类化合物生物合成的关键酶基因(牻牛儿基焦磷酸合酶(0009238)、黄酮醇合成酶(0001833))的表达,进而促进白骨壤有效成分的积累;显著诱导细胞分裂素水解酶编码基因CKX7(0057124)、油菜素内脂信号转导组分基因BSK(0043741)等的表达,进而提高白骨壤对重金属胁迫的适应能力。此外,转录因子分析发现,b HLH、NAC、MYB-related和WRKY在重金属胁迫中发挥重要作用。实验选取8个与环境刺激响应密切相关的基因,通过qRT-PCR验证了它们在重金属污水胁迫处理下的表达差异,结果与数字基因表达谱分析的结果较一致,证实了差异表达基因数据的有效性。[结论]重金属处理影响了白骨壤大量的新陈代谢、光合作用、小分子酸合成、激素合成与信号转导及次生代谢产物合成相关基因的表达。

Transcriptome sequencing was perform to study the molecular mechanism in response to heavy metal stress in Avicennia marina.[Method]Digital Gene Expression Profiling （DGE） technique was used to analyze the differences in gene expressions of A. marina under artificial heavy metal stress.[Result]Compared to the controls, 10459 differentially expressed genes （DEGs） were detected, including 8685 up-regulated genes and 1774 down-regulated genes. MeV cluster analysis indicated that the expression level of most genes was largely induced in artificial heavy metal treatment sample than that in control sample. Gene Ontology analysis showed that most DEGs locate in ＂organelle envelope＂, ＂plastid envelope＂ and ＂chloroplast part＂, and were enriched in the biological process of ＂response to stimulus＂, and ＂localization＂, etc. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that these genes are distributed in 126 pathways involved in ＂ribosome＂, ＂photosynthesis＂, and various metabolism pathways, such as pyruvate and glyoxylate. Several terpenoid and flavonoid biosynthesis-related enzymes, such as geranylgeranyl pyrophosphate synthase （Unigene0009238） and flavonol synthase （Unigene0001833）, were up-regulated by heavy metal stress, which might be the reason for the enhancement for the active ingredients accumulation in A. marina. Furthermore, heavy metal stress significantly induced the cytokinin dehydrogenase 7（CKX7） （Unigene0057124） and serine/threonine-protein kinase （BSK） （Unigene0043741）. Besides, the transcription factor analysis demonstrated that bHLH, NAC, MYB-related and WRKY play vital roles in response to heavy metal stress.Furthermore, eight randomly selected genes were used to confirm the accuracy of DGE by qRT-PCR method.[Conclusion]Heavy metal stresses have effects on the expression of related genes involved in the metabolism, photosynthesis, small molecule acid synthesis, hormone synthesis and signal transduction, and secondary metabolites synthesis of A. marina