烟草叶片响应镉胁迫的差异表达基因鉴定及分析

Transcriptome analysis of tobacco in response to cadmium stress

烟草具有超富集镉的能力,严重降低烟叶品质,影响其经济价值。为了阐释烟草响应镉胁迫的分子机制,本研究采集了镉浓度为0和500μmol L^(–1)培养条件下的烟草叶片进行转录组测序。共获得76.94 Gb有效数据(Clean data),Q30碱基百分比均达到95.43%以上;在镉胁迫的烟草叶片中,共筛选出7735个差异表达基因,其中4833个基因表达上调,2902个基因表达下调,并通过qRT-PCR分析验证了转录组数据的可靠性。对差异转录本进行GO和KEGG富集分析,GO注释表明差异基因涉及代谢过程、应激反应、细胞结构体、催化活性和转录调节活性等;KEGG富集分析表明上调差异基因主要富集在氨基酸的生物合成、碳代谢、氧化磷酸化和柠檬酸循环等通路,下调差异基因则主要富集在光合作用、次生代谢产物的生物合成、代谢途径和植物激素信号转导途径。进一步分析植物激素信号转导通路发现,共有8条植物激素途径以不同的表达方式参与烟草对镉胁迫的响应。激素喷施烟草的实验结果表明,叶片通过调控赤霉素、油菜素内酯和茉莉酸途径以应对镉胁迫;拟南芥激素信号缺失突变体验证实验表明赤霉素、油菜素内酯、茉莉酸和乙烯途径均响应镉胁迫。综上所述,本文以转录组分析探究了烟草叶片响应镉胁迫的调控网络,以期为提高作物抗逆性的遗传改良提供理论依据。

With the development of industrialization process in society,the problem of cadmium(Cd)pollution in soil is increasing.However,Nicotiana tabacum has a strong Cd enrichment capacity in leaves,which seriously affects its economic value.To investigate the mechanism by which tobacco responds to Cd stress,tobacco leaves were harvested from the culture solution with Cd concentrations of 0μmol L^(–1)and 500μmol L^(–1)for subsequent transcriptome sequencing.In this study,a total of 76.94 Gb clean data was obtained,with Q30 base percentage exceeding 95.43%.The results showed that 7735 differentially expressed genes(DEGs)were screened under Cd stress conditions,including 4833 up-regulated genes and 2902 down-regulated genes.The reliability of transcriptome data was verified by qRT-PCR analysis to detect the expression patterns of candidate gene.Gene ontology(GO)annotation as well as Kyoto encyclopedia of genes and genomes(KEGG)pathway analysis were performed on differentially expressed transcripts.GO functional enrichment revealed that the differentially expressed genes were mainly distributed in metabolic processes,response to stimulus,cellular anatomical entity,catalytic activity,and transcription regulator activity.Meanwhile,KEGG analysis showed that the up-regulated differentially expressed genes were mainly involved in biosynthesis of amino acids,carbon metabolism,oxidative phosphorylation,and citrate cycle.Down-regulated differentially expressed genes were primarily enriched in photosynthesis,biosynthesis of secondary metabolites,metabolic pathways,and plant hormone signal transduction.Further analysis of plant hormone signal transduction pathways revealed that there were eight plant hormone pathways involved in response to cadmium stress in tobacco,and the relative expression patterns of different hormone gene member were also different.Experimental results from plant hormone application on tobacco leaves demonstrated that the regulation of gibberellins,brassinosteroids,and jasmonic acid pathways played roles in tobacco’s response to cadmium stress.The experimental results of Arabidopsis hormone signal mutant showed that plants respond to cadmium stress by regulating ethylene,gibberellin,brassinosteroid,and jasmonic acid pathways.In conclusion,this study not only explores the regulatory network of tobacco resistance to Cd stress,but also lays a theoretical foundation for the genetic improvement of crop resistance.