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镉胁迫下中辽1号杨转录组分析
李晓宇1, 李文颖1, 杨成超,等1
辽宁省杨树研究所
摘要:
【目的】分析镉胁迫下中辽1号杨的转录水平,筛选差异表达基因,挖掘与镉胁迫相关的功能基因,为深入探索中辽1号杨对镉胁迫响应的分子机制提供理论依据。【方法】以中辽1号杨为试验材料,采用盆栽试验的方法将其扦插于土壤Cd含量为20 mg/kg(M20)的花盆中,以不加Cd为对照(CK),80 d后采集不同处理中辽1号杨叶片进行转录组测序,使用DESeq2软件筛选CK与M20处理中辽1号杨的差异表达基因,将得到的差异基因在基因本体数据库(gene ontology,GO)、京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)、蛋白相邻类聚簇数据库(clusters of orthologous groups of proteins,COG)中进行注释,分析差异表达基因在不同数据库中的注释信息。随机挑选6个差异表达基因(超氧化物歧化酶(SOD)、丝氨酸/苏氨酸蛋白激酶(STK)、脱落酸(ABA)、T复合蛋白1(TCP1)、MYB基因(MYB)、丝裂原活蛋白激酶12(MAPK12)),利用Primer 5软件设计特异性引物进行实时荧光定量PCR(RT-qPCR)试验,检测这6个基因表达水平的变化,验证转录组结果的准确性。【结果】在CK和M20处理的中辽1号杨中,共发现3 812 个差异表达基因,其中上调表达的有2 209 个,下调表达的有1 603 个。GO 分析发现,差异表达基因注释到3大类49个功能组中,其中与代谢过程有关基因最多。转录组KEGG代谢通路分析发现,差异表达基因主要在信号转导途径、代谢途径和生物合成途径富集。COG分析发现,1 455个差异基因被注释到22种分类中,其中一般功能预测基因注释最多,其次是信号转导机制基因。结合各数据库分析结果发现,Cd胁迫下中辽1号杨转录组的注释结果中代谢过程和信号转导过程相关基因较多。镉胁迫下差异表达基因最多的家族是ABC、MYB、WRKY、bHLH和NAC基因家族,挖掘出与镉胁迫相关基因WRKY家族基因47(WRKY47)、硝酸盐转运蛋白基因(NRT)、ABC家族转运蛋白基因2(ABC2)、苹果酸脱氢酶(MDH)、谷胱甘肽S-转移酶基因(GSTs)、NAC家族基因2(NAC2)的表达量显著上调,MYB家族基因44(MYB44)、重金属相关异戊二烯化植物蛋白基因39(HIPP39)的表达量显著下调。RT-qPCR结果显示,随机挑选6个差异表达基因表达量的变化趋势与转录组测序结果一致。【结论】镉胁迫后中辽1号杨转录组的差异表达基因主要富集在代谢过程和信号转导过程,挖掘出与镉胁迫相关基因,分别是WRKY47、NRT、ABC2、MDH、GSTs、NAC2、MYB44、HIPP39,为深入探索杨树对镉胁迫响应的分子机制提供了理论依据。
关键词:  中辽1号杨  镉胁迫  转录组测序  差异基因
DOI:
分类号:
基金项目:辽宁省自然科学基金项目(2022-MS-066);辽宁省农业科学院基本科研业务费项目(2022DD227439)
Transcriptome analysis of Populus×canadensis cv.‘Zhongliao 1’ under cadmium stress
LI Xiaoyu,LI Wenying,YANG Chengchao,et al
Abstract:
【Objective】This study analyzed the transcription level of P.×canadensis cv. ‘Zhongliao 1’ under cadmium stress,screened differentially expressed genes,and explored functional genes related to cadmium stress to provide basis for in-depth exploration of molecular mechanisms related to response of P.×canadensis cv. ‘Zhongliao 1’ poplar to cadmium stress.【Method】P.×canadensis cv. ‘Zhongliao 1’ was cut into flower pots for planting with Cd content of 20 mg/kg (M20) in soil using the pot experiment method and no Cd (CK) addition as the control.After 80 days,leaf slices of P.×canadensis cv.‘Zhongliao 1’ from different treatments were collected for transcriptome sequencing,and DESeq2 software was used to screen differentially expressed genes.The obtained differential genes were annotated in Gene Ontology (GO),Kyoto Encyclopedia of Genes and Genomes (KEGG),and clusters of orthologous groups of proteins (COG),and the annotation information of differential expression genes in different databases was analyzed.Six differentially expressed genes including superoxide(SOD),serine/threonine protein kinase (STK),abscisic acid (ABA),T complex protein 1 (TCP1),MYB gene (MYB) and mitogen activated protein kinase 12 (MAPK12) were randomly selected and real-time fluorescence quantitative PCR (RT-qPCR) testing was applied using specific primers designed by Primer 5 software to detect changes in their expression levels and verify the transcriptome accuracy. 【Result】A total of 3 812 differentially expressed genes were found from P.×canadensis cv. ‘Zhongliao 1’ in CK and M20 treatments,of which 2 209 were upregulated and 1 603 were downregulated.GO analysis found that differentially expressed genes were annotated into three major categories and 49 functional groups,with the highest number related to metabolic processes.The analysis of KEGG metabolic pathway in transcriptome showed that differentially expressed genes were mainly enriched in signal transduction pathway,metabolic pathway and biosynthetic pathway.COG analysis found that 1 455 differentially expressed genes were annotated into 22 classifications,and general functional prediction genes were annotated the most,followed by signal transduction mechanism genes.Combined with the analysis of various databases,the annotation results of P.×canadensis cv. ‘Zhongliao 1’ transcriptome under Cd stress included more annotation of metabolic process and signal transduction process.The families with the most differential genes under cadmium stress were ABC,MYB,WRKY,bHLH and NAC.The WRKY family gene 47 (WRKY47),nitrate transporter gene (NRT),ABC family transporter gene 2 (ABC2),malic dehydrogenase (MDH),glutathione S-transferase gene (GSTs),and NAC family gene 2 (NAC2) related to cadmium stress were excavated and their expression levels were significantly upregulated under cadmium stress.The expression levels of MYB family gene 44 (MYB44) and heavy metal related isoprene plant protein gene 39 (HIPP39) were significantly downregulated.The RT-qPCR showed that the expression changing trend of six randomly selected differentially expressed genes was consistent with the transcriptome sequencing results.【Conclusion】The differentially expressed genes in P.×canadensis cv. ‘Zhongliao 1’ transcriptome after cadmium stress were mainly enriched in metabolic and signal transduction processes.Identified genes related to cadmium stress included WRKY47,NRT,ABC2,MDH,GSTs,NAC2,MYB44 and HIPP39.This study provides theoretical basis for further exploring molecular mechanisms of poplar response to cadmium stress.
Key words:  Populus×canadensis cv.‘Zhongliao 1’  cadmium stress  transcriptome sequencing  differential gene