利用RNA-Seq鉴定甘蓝型油菜叶片干旱胁迫应答基因

Identification of Drought Stress-Responsive Genes in Leaves of Brassica napus by RNA Sequencing

【目的】利用RNA Sequencing(RNA-Seq)技术比较2种不同生长条件下甘蓝型油菜苗期叶片转录组,鉴定油菜叶片干旱胁迫应答相关基因,从转录组水平揭示油菜适应干旱胁迫环境的分子机制。【方法】提取正常生长(ZY)和自然失水处理(ZY8D)的六叶期甘蓝型油菜中油821的叶片总RNA,以Illumina Hiseq 2000平台进行RNA-Seq分析。利用NGSQCTookit v2.3.3去除低质量和包含模糊碱基的reads。以甘蓝型油菜亲本物种白菜染色体v1.5和甘蓝Scaffold v1.0为参考序列,采用Top Hat2-Cufflinks-Cuffmerge-Cuffdiff标准流程进行差异表达基因(differential expressed genes,DEGs)筛选。对上调和下调DEGs分别采用Cytoscape v3.1.0中的Bi NGO和KOBAS2.0进行基因本体(gene ontology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)代谢途径富集分析。选择上调和下调DEGs各3个,以实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)验证RNA-Seq结果的可靠性。【结果】过滤低质量reads后,ZY和ZY8D分别保留了26 192 312和28 378 899对高质量reads用于DEGs筛选,其中86.6%和85.8%的reads能准确比对到参考序列上,说明RNA-Seq结果和参考序列可靠。DEGs鉴定结果表明3 657个基因受干旱胁迫诱导差异表达,其中上调表达基因1 431个,下调表达基因2 226个。GO富集分析发现上调表达基因主要与非生物胁迫响应和化学刺激响应相关,其中,参与水分胁迫响应和脱落酸(abscisic acid,ABA)刺激响应的基因分别有127和141个,而下调表达基因与植物病原菌防御、蛋白激酶活性和水杨酸(salicylic acid,SA)刺激相关。KEGG富集分析表明上调表达基因主要富集于苯丙烷和类胡萝卜素的生物合成及淀粉与蔗糖代谢途径,而下调表达基因主要富集于植物-病原菌互作和植物激素ABA、SA和茉莉酸(jasmonic acid,JA)信号转导途径。q RT-PCR检测6个DEGs的表达模式与RNA-Seq分析结果一致,证实了RNA-Seq结果的可靠性。【结论】RNA-Seq分析鉴定出3 657个甘蓝型油菜叶片干旱胁迫应答基因。GO和KEGG代谢途径分析明确了差异表达基因富集的分子功能与代谢途径。

【Objective】 To identify the candidate genes involved in drought stress response in leaves of Brassica napus thereby exploring the molecular mechanism of drought stress adaptation of it,the transcriptomes of B.napus seedlings leaves under two different conditions were compared using RNA sequencing（RNA-Seq）.【Method】 Total RNA were extracted from leaves of B.napus cultivar ZY821 at six-leaf stage under normal（ZY） and natural water loss（ZY8D） conditions,and then were used for RNA-Seq analysis on the Illumina Hiseq 2000 platform.Ambiguous reads and low-quality reads were filtered using NGSQCTookit v2.3.3.The Top Hat2-Cufflinks-Cuffmerge-Cuffdiff standard pipeline was applied to identify the differentially expressed genes（DEGs）,taking the B.rapa chromosome v1.5 and B.oleracea Scaffold v1.0 as reference.In order to perform GO term and KEGG enrichment analyses,the up- and down-regulated DEGs were further analyzed using the Bi NGO plugin in Cytoscape v3.1.0 and KOBAS2.0,respectively.Subsequently,the q RT-PCR assays were implemented to verify the expression patterns of three representatives of the up- and down-regulated DEGs,respectively.【Result】 After filtration,a total of 26192312 and 28378899 high-quality reads were acquired in ZY and ZY8 D for screening DEGs,86.6% and 85.8% of the filtered reads derived from ZY and ZY8 D could be accurately mapped to the reference sequence,demonstrating the high confidence of the RNA-Seq and the reference.Of the 3 657 DEGs,1 431 and 2 226 genes were detected to be up- and down-regulated,respectively.GO enrichment analysis indicated that the up-regulated genes were mainly enriched in response to abiotic stress and chemical stimulus,and 127 and 141 out of these DEGs were involved in response to water deprivation and ABA stimulus,respectively.However,down-regulated DEGs were mainly overrepresented in defense response to plant pathogen,protein kinase activity and response to SA stimulus.KEGG enrichment analysis showed that up-regulated genes were significantly associated with phenylpropanoid and carotenoid biosynthesis pathways,and starch and sucrose metabolism,while the down-regulated DEGs mainly enriched in plant-pathogen interaction and signal transduction pathways of ABA,SA and jasmonic acid（JA）.The results of q RT-PCR analysis of six DEGs were consistent with those of RNA-Seq data,further confirming the reliability of RNA-Seq results.【Conclusion】 In total,3 657 drought stress-responsive genes were identified using RNA-Seq.GO and KEGG pathway analyses identified the overrepresented molecular function categories and pathways of DEGs.