D3基因在抗病防卫反应中的转录调控研究

doi:10.3969/j.issn.1004-1524.20221667

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (12): 2763-2774.DOI: 10.3969/j.issn.1004-1524.20221667

D3基因在抗病防卫反应中的转录调控研究

徐悦¹^,²(), 汪少敏³, 谭晓菁¹^,², 罗英杰¹^,², 常婧一², 邓会², 刘秀丽², 崔维军², 周洁², 吴月燕¹, 严成其¹^,⁴^,^*(), 王栩鸣²^,^*()

1.浙江万里学院生物与环境学院,浙江宁波 315100
2.浙江省农业科学院病毒学与生物技术研究所,农产品质量安全危害因子与风险防控国家重点实验室,浙江杭州 310021
3.余姚市农业技术推广服务总站,浙江余姚 315400
4.宁波市农业科学研究院,浙江宁波 315100

收稿日期:2022-11-22 出版日期:2023-12-25 发布日期:2023-12-27
作者简介:徐悦(1997—),女,浙江衢州人,硕士研究生,主要从事水稻抗病育种研究。E-mail: xu1839372725@163.com
通讯作者: *严成其,E-mail: yanchengqi@163.com;王栩鸣,E-mail: xmwang@zaas.ac.cn
基金资助:
宁波市“科技创新2025”重大专项(2019B10004);浙江省重点研发计划(2021C02053);浙江省自然科学基金(LQ23C140004);余姚市科技创新项目(2022JH03010082)

Effects of the D3 gene on transcriptional regulation and its role in defense responses

XU Yue¹^,²(), WANG Shaomin³, TAN Xiaojing¹^,², LUO Yingjie¹^,², CHANG Jingyi², DENG Hui², LIU Xiuli², CUI Weijun², ZHOU Jie², WU Yueyan¹, YAN Chengqi¹^,⁴^,^*(), WANG Xuming²^,^*()

1. College of Biology and Environment, Zhejiang Wanli University, Ningbo 315100, Zhejiang, China
2. State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
3. Agricultural Technology Extension and Service Station of Yuyao City, Yuyao 315400, Zhejiang, China
4. Ningbo Academy of Agricultural Sciences, Ningbo 315100, Zhejiang, China

Received:2022-11-22 Online:2023-12-25 Published:2023-12-27

摘要/Abstract

摘要：

水稻白叶枯病(bacterial leaf blight, BLB)是由水稻黄单胞菌属水稻致病变种(Xanthomonas oryzae pv. oryzae, Xoo)引起的细菌性病害,严重影响水稻产量。随着基因技术的发展,编辑抗病基因是提高水稻抗病性降低该病害的发生的一种行之有效的方法。本研究首先对D3基因编辑株系和野生型进行白叶枯病菌接种实验来确定D3基因对于白叶枯病菌的抗性,其次利用高通量测序技术对已筛选出的遗传纯合D3基因编辑株系进行转录组分析,同时结合基因差异表达分析、基因家族分析和富集分析挖掘其在抗病相关通路上的潜在作用,并通过实时荧光定量PCR(qRT-PCR)技术对部分差异表达基因(differential expressed genes, DEGs)进行验证。结果表明:接种白叶枯病菌后D3基因被显著诱导,同时D3基因编辑株系均表现出不同程度的白叶枯病抗性。转录组数据显示,与野生型相比,在转录组水平共鉴定到8 184个差异表达基因,包含4 201个上调基因和3 983个下调基因,3组均呈现差异表达的相关基因共有359个。GO功能富集分析表明,D3基因编辑株系与野生型在生物学途径、细胞组分和分子功能上的相关基因均呈显著差异表达。KEGG代谢途径富集分析发现,DEGs显著富集于植物与病原菌互作、植物激素信号转导以及苯丙烷生物合成途径。本研究丰富了水稻D3基因在抗病相关基因转录调控方面的信息,为D3基因抗病功能研究拓展了思路。

关键词: 水稻, 白叶枯病, D3基因, 转录组分析, 差异表达基因

Abstract:

Bacterial leaf blight is a bacterial disease caused by Xanthomonas oryzae pv. oryzae (Xoo). Bacterial leaf blight can severely reduce rice yields. With the development of molecular biotechnology, editing potential disease resistance genes has become a method to improve rice disease resistance and reduce the loss of the disease. In this study, D3 gene edited lines and wild-type inoculation experiments were used to determine D3 gene resistance and high-throughput sequencing technology was employed to analyze the transcriptomes of three different D3 gene-edited lines, and the results showed that compared with the wild type, D3 gene was significantly induced, while the D3 edited lines showed different degrees of blight resistance and a total of 8 184 DEGs were identified at the transcriptome level, including 4 201 up-regulated genes and 3 983 down-regulated genes, among which 359 DEGs were found simultaneously differentially expressed in the three groups. After that, this part of differentially expressed genes (DEGs) was verified by quantitative PCR. GO functional enrichment analysis showed that the genes related to biological pathways, cellular components and molecular functions were significantly differentially expressed in D3 gene-edited lines compared with the wild-type. KEGG pathway enrichment analysis found that DEGs were significantly enriched in plant-pathogen interaction, plant hormone signal transduction and phenylpropane biosynthesis pathways. Further experiments showed that, compared with the wild type, pathogen-related genes (PRs) in D3 gene-edited lines were significantly induced after inoculation with Xoo, and D3 gene-edited lines showed different degrees of bacterial blight resistance, thus further confirmed the role of D3 in bacterial blight resistance. This study enriches the role of the rice D3 gene in affecting transcriptional regulation, especially its impact on plant disease resistance-related genes. At the same time, this study expands the ideas for the research on the disease resistance mechanism of D3 gene.

Key words: rice, bacterial blight, D3 gene, transcriptome analysis, differentially expressed genes

中图分类号:

S435.111.1

徐悦, 汪少敏, 谭晓菁, 罗英杰, 常婧一, 邓会, 刘秀丽, 崔维军, 周洁, 吴月燕, 严成其, 王栩鸣. D3基因在抗病防卫反应中的转录调控研究[J]. 浙江农业学报, 2023, 35(12): 2763-2774.

XU Yue, WANG Shaomin, TAN Xiaojing, LUO Yingjie, CHANG Jingyi, DENG Hui, LIU Xiuli, CUI Weijun, ZHOU Jie, WU Yueyan, YAN Chengqi, WANG Xuming. Effects of the D3 gene on transcriptional regulation and its role in defense responses[J]. Acta Agriculturae Zhejiangensis, 2023, 35(12): 2763-2774.

图/表 10

表1 D3基因编辑转基因GEM株系

Table 1 D3 gene-edited transgenic GEM line

株系 Line	突变方式 Mutation	处理组 Treatment group	对照组 Control group(Nip)
GEM1	-AT	A-1、A-2、A-3	CK-1
GEM2	-AGT	B-1、B-2、B-3	CK-2
GEM3	-CAGTTGCG	C-1、C-2、C-3	CK-3

表2 qRT-PCR的引物序列

Table 2 Primers used for qRT-PCR

基因Gene	正向引物Forward primer(5'→3')	反向引物Reverse primer(3'→5')
Os02g0462800	AAGGCTCGCCTTATCCACGC	CAGTCGTCCTGTGCTCCGTC
Os01g0884300	GAAGCCGGTGGCGATCAAGA	GAAGCCGGTGGCGATCAAGA
Os04g0118800	TGGTTTTGCAAACGAGCGAG	ACCTTCACACCTGCCAACAA
Os06g0521900	CACAGCTGCGAGCACTACGA	TTGTCGAACTTGGCTGGCGT
Os12g0528801	CGCCACCTTCTCTCCGGTTC	GCTGAGCAGCAGGAGACACT

图1 D3基因在不同时间点受Xoo诱导的表达量

Fig.1 The expression levels of the D3 gene induced by Xoo at different time points

图2 编辑株系和日本晴的病斑表型对比图

Fig.2 Edit plots of disease spot phenotypes between lines and Nip

图3 样本间关系的整体特征 A, 基因编辑组之间差异基因的韦恩图;B,基因编辑组之间的差异表达基因数;C,基因编辑组与对照组之间差异基因相对表达量的qRT-PCR分析。

Fig.3 Overall characterization of sample relationship A, Venn diagrams of differentially expressed genes among the gene editing groups and the control group; B, Numbers of differentially expressed genes among the gene editing groups and the control group; C, qRT-PCR analysis of relative expression levels of differentitally genes among the gene editing groups and the control group.

图4 对差异表达显著的DEGs的GO富集分析差异表达显著的基因被总结为3个主要的GO类别(细胞成分、生物过程和分子功能)。橙色为上调,绿色为下调。

Fig.4 GO enrichment analysis of DEGs with significant expression difference These genes with significant difference are summarized into three main GO categories (biological processes, molecular function, and cellular components). Orange is the up regulated, green is the down regulated.

图5 基因编辑处理组与对照组之间差异表达基因的KEGG富集途径分析圆圈的大小代表在相应途径中富集的DEG数量,圆圈的颜色代表相应路径。

Fig.5 KEGG enrichment pathway analysis of differentially expressed genes among gene editing treatment groups and control group The size of the circle represents the number of DEG enriched in the corresponding pathway, and the color of the circle represents the corresponding pathway.

图6 苯丙烷生物合成途径中基因编辑处理组与对照组之间差异表达基因分析

Fig.6 Analysis of differentially expressed genes among gene editing treatment groups and control group in the phenylpropane biosynthetic pathway

图7 植物病原菌互作中基因编辑处理组与对照组之间差异表达基因分析

Fig.7 Differentially expressed gene analysis among gene editing treatment groups and control group in plant pathogen interactions

图8 信号转导途径中基因编辑处理组与对照组之间差异表达基因分析

Fig.8 Analysis of differentially expressed genes among gene editing treatment groups and control group in signal transduction pathways

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D3基因在抗病防卫反应中的转录调控研究

Effects of the D3 gene on transcriptional regulation and its role in defense responses

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