利用RNA-Seq鉴定甘蓝型油菜叶片干旱胁迫应答基因

doi:10.3864/j.issn.0578-1752.2015.04.02

Abstract

Abstract: 【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 TopHat2-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 BiNGO plugin in Cytoscape v3.1.0 and KOBAS2.0, respectively. Subsequently, the qRT-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 ZY8D for screening DEGs, 86.6% and 85.8% of the filtered reads derived from ZY and ZY8D 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 qRT-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.

LU Kun, ZHANG Lin, QU Cun-ming, LIANG Ying, TANG Zhang-lin, LI Jia-na. Identification of Drought Stress-Responsive Genes in Leaves of Brassica napus by RNA Sequencing[J].Scientia Agricultura Sinica, 2015, 48(4): 630-645.

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

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