转录因子CsWRKY61对柑橘溃疡病抗性的影响

doi:10.3864/j.issn.0578-1752.2020.08.006

摘要/Abstract

摘要：

【背景】柑橘溃疡病（citrus bacterial canker,CBC）是世界柑橘产业上危害最严重的病害之一,由柑橘黄单胞杆菌柑橘亚种（Xanthomonas citri subsp. citri,Xcc）引起,在国内外被列为检疫对象。由于柑橘分子病理研究相对滞后,导致可供利用的抗性基因资源相对匮乏。WRKY转录因子参与植物抵御生物和非生物胁迫反应,前期研究发现柑橘WRKY转录因子可能在调控寄主抗病反应中起着重要作用。【目的】通过对超量表达CsWRKY50、CsWRKY61和CsWRKY72转基因晚锦橙（Citrus sinensis）的溃疡病抗性进行评价,明确这些基因在柑橘响应溃疡病菌侵染中的生物学功能和抗病育种价值。进一步利用RNA-Seq解析CsWRKY61调控的信号通路。【方法】利用农杆菌介导法进行柑橘遗传转化,获得超量表达CsWRKY50、CsWRKY61和CsWRKY72的晚锦橙;利用实时荧光定量PCR（qRT-PCR）分析转基因植株中目的基因的表达水平以及拷贝数;以非转基因植株为对照,采用离体针刺接种评价转基因植株对溃疡病的抗性;通过比较超量表达CsWRKY61和野生型植株的转录组测序结果,探究CsWRKY61提高柑橘溃疡病抗性的内在机制。【结果】分别构建了CAMV 35S启动子控制CsWRKY50、CsWRKY61和CsWRKY72表达的植物表达载体,通过GUS组织化学染色和PCR鉴定分别获得了6、8和6株转基因晚锦橙。转基因植株中目的基因的表达量有不同程度的提高,大部分转基因植株中外源基因的拷贝数为1,只有超量表达CsWRKY61的转基因植株溃疡病抗性显著增强,其病斑面积明显小于野生型植株,而超量表达CsWRKY50和CsWRKY72的转基因植株抗病性与野生型相比无明显差异。转录组分析结果显示,超量表达CsWRKY61的转基因植株中生物胁迫相关途径（包括病原入侵的感知、活性氧爆发、转录因子、防御基因、激素、细胞壁和次生代谢等）和信号转导相关途径（主要是激酶受体）均被显著激活。【结论】超量表达CsWRKY61能够激活与生物胁迫和信号转导相关的途径,增强柑橘对溃疡病的抗性;CsWRKY61在柑橘抗病育种中存在潜在的应用价值。

关键词: 柑橘黄单胞杆菌柑橘亚种, 柑橘溃疡病, CsWRKY61, 超量表达, 抗病性, 转录组测序

Abstract:

【Background】Citrus bacterial canker (CBC) caused by Xanthomonas citri subsp. citri (Xcc) is one of the most serious citrus diseases in the world, which is a quarantine disease. Due to the relatively backward research of citrus molecular pathology, the available resistance gene resources are relatively scarce. WRKY transcription factor is involved in plant responses to biotic and abiotic stress. The previous study has found that citrus WRKY transcription factor may play an important role in regulating host disease resistance response.【Objective】The objective of this study is to evaluate the canker resistance of transgenic citrus (Citrus sinensis) with over-expression of CsWRKY50, CsWRKY61 and CsWRKY72, clarify the biological function and disease resistance breeding value of these genes in citrus in response to Xcc. RNA-seq was further used to analyze the signaling pathway regulated by CsWRKY61.【Method】Agrobacterium-mediated method was used to obtain transgenic citrus plants with over-expression of CsWRKY50, CsWRKY61 and CsWRKY72. Real-time quantitative PCR (qRT-PCR) was used to analyze the expression level and copy number of the target genes. In vitro pinprick inoculation was used to evaluate the resistance of transgenic plants to canker disease. The molecular mechanism of CsWRKY61 improving citrus bacterial canker resistance was investigated by transcriptome sequencing analysis of over-expression CsWRKY61 and wild-type (WT) plants.【Result】The plant expression vectors of CAMV 35S promoter controlling the expression of CsWRKY50, CsWRKY61 and CsWRKY72 were constructed, and 6, 8 and 6 transgenic lines were obtained by GUS staining and PCR identification, respectively. The expression of the target gene in transgenic plants increased in different degrees. The copy number of exogenous genes in most transgenic plants was 1. Only the transgenic plants with over-expression of CsWRKY61 had significantly enhanced canker disease resistance, and the lesion area was significantly smaller than that of WT plants, while over-expression of CsWRKY50 and CsWRKY72 transgenic plants had no significant difference in disease resistance compared with WT. Transcriptomics analysis showed that biotic stress related pathways (including pathogen recognition, respiratory burst, transcription factors, defense genes, hormones, cell wall and secondary metabolism, etc.) and signal transduction-related pathways (mainly kinase receptors) were significantly activated in over-expression of CsWRKY61 transgenic plants. 【Conclusion】Over-expression of CsWRKY61 can activate pathways related to biotic stress and signal transduction, enhance citrus bacterial canker resistance. It is suggested that CsWRKY61 has potential application value in citrus disease resistance breeding.

Key words: Xanthomonas citri subsp. citri (Xcc), citrus bacterial canker, CsWRKY61, over-expression, disease resistance, transcriptome sequencing

龙琴,杜美霞,龙俊宏,何永睿,邹修平,陈善春. 转录因子CsWRKY61对柑橘溃疡病抗性的影响[J]. 中国农业科学, 2020, 53(8): 1556-1571.

LONG Qin,DU MeiXia,LONG JunHong,HE YongRui,ZOU XiuPing,CHEN ShanChun. Effect of Transcription Factor CsWRKY61 on Citrus Bacterial Canker Resistance[J]. Scientia Agricultura Sinica, 2020, 53(8): 1556-1571.

图/表 11

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表4

转基因植株中生物胁迫相关的差异基因情况"

通路Pathway	基因编号Gene ID	描述Description	Log₂fold change
感知Recognition	cs7g19210	抗病蛋白家族Disease resistance protein (TIR-NBS-LRR class) family	2.464
	orange1.1t04440	抗病蛋白家族Disease resistance protein (TIR-NBS-LRR class) family	2.839
	orange1.1t05036	Toll-Interleukin-Resistance (TIR) domain family protein	3.153
	cs5g18230	Toll-Interleukin-Resistance (TIR) domain family protein	3.639
活性氧爆发Respiratory burst	cs4g06920	NADPH/respiratory burst oxidase protein D (RbohD)	1.962
活性氧爆发Respiratory burst	cs8g12000	核黄素合成酶样超家族蛋白Riboflavin synthase-like superfamily protein	2.631
信号转导 Signaling transduction	orange1.1t03802	alpha/beta-Hydrolases superfamily protein	3.683
	cs5g04790	alpha/beta-Hydrolases superfamily protein	2.423
	cs6g07420	PAR1 protein	3.644
	cs7g07000	假定的配体门控离子通道亚家族Putative ligand-gated ion channel subunit family	3.786
	cs1g16140	富亮氨酸重复I Leucine rich repeat I	4.144
	orange1.1t03347	富亮氨酸重复II Leucine rich repeat II	3.177
	orange1.1t04450	富亮氨酸重复VII Leucine rich repeat VII	3.279
	cs2g29910	富亮氨酸重复XII Leucine rich repeat XII	4.256
	orange1.1t04450	富亮氨酸重复XI Leucine rich repeat XI	3.279
	cs2g29890	富亮氨酸重复XIII Leucine rich repeat XIII	2.459
	cs2g13280	奇异果甜蛋白受体激酶Thaumatin like receptor kinases	3.153
	cs2g13360	奇异果甜蛋白受体激酶Thaumatin like receptor kinases	3.199
	cs1g11930	DUF 26受体激酶DUF 26 receptor kinases	3.791
	cs1g11960	DUF 26受体激酶DUF 26 receptor kinases	3.052
	cs8g12370	豆科凝集素Legume-lectin	5.351
	cs2g13360	Wheat LRK10 like	3.199
	cs1g11930	S位点糖蛋白S-locus glycoprotein like	3.791
	cs1g11960	S位点糖蛋白S-locus glycoprotein like	3.052
	cs8g01090	豆科凝集素Legume-lectin	2.194
	cs8g14010	胞壁相关激酶Wall associated kinase	3.464
	cs9g12300	胞壁相关激酶Wall associated kinase	3.013
	cs9g14490	胞壁相关激酶Wall associated kinase	3.918
	cs9g08050	赖氨酸基序Lysine motif	1.079
	cs2g02680	赖氨酸基序Lysine motif	2.853
	cs7g31060	褶皱样Crinkly like	2.283
	cs5g17510	褶皱样Crinkly like	1.589
	cs9g12040	Ralf-like 32 (RALFL32)	5.157
	cs9g12160	Ralf-like 32 (RALFL32)	3.365
	cs7g27120	钙调蛋白结合蛋白Calmodulin binding protein-like	3.590
	cs9g02980	钙调蛋白结合蛋白Calmodulin binding protein-like	3.336
	cs2g21150	蛋白激酶激酶Mitogen-activated protein kinase kinase	2.077
	cs3g27320	受体样胞浆激酶VII Receptor like cytoplasmatic kinase VII	3.278
	cs9g15620	受体样胞浆激酶VII Receptor like cytoplasmatic kinase VII	4.530
	cs6g20470	AAA ATPase 1	5.065
	cs5g18300	天冬氨酸蛋白酶Aspartate protease	4.689
	orange1.1t03718	半胱氨酸蛋白酶Cysteine protease	3.775
	cs2g19970	F-box和相关的相互作用域包含蛋白质 F-box and associated interaction domains-containing protein	4.369
	cs2g31260	F-box和相关的相互作用域包含蛋白质 F-box and associated interaction domains-containing protein	7.072
转录因子 Transcription factor	orange1.1t02158	富含亮氨酸的重复单位Leucine-rich repeat	1.487
	cs5g24240	抗病蛋白Disease resistance protein (TIR-NBS-LRR class)	3.080
	cs5g04160	WRKY61 DNA结合蛋白WRKY DNA-binding protein 61	10.442
	cs1g03870	WRKY51 DNA结合蛋白WRKY DNA-binding protein 51	7.885
	cs7g06330	WRKY40 DNA结合蛋白WRKY DNA-binding protein 40	3.707
	cs4g07780	R2R3 MYB 转录因子基因家族R2R3 MYB transcription factor gene family	6.169
	cs8g12680	转录因子RLTR1 Transcription factor RLTR1	2.614
防御基因 Defense gene	orange1.1t04673	抗病蛋白Disease resistance protein	3.487
	orange1.1t04706	抗病蛋白Disease resistance protein (TIR-NBS-LRR class)	2.892
	orange1.1t04750	Toll-Interleukin receptor	3.454
	orange1.1t05036	抗病蛋白Disease resistance protein (TIR-NBS-LRR class)	3.153
	orange1.1t05250	Toll-Interleukin receptor	3.423
	cs2g10790	NPR1-like protein 3	1.251

表4

图7

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)
WRKY50-f	ATC GGATCC ATGTCTAATATAAATCTTAT
WRKY50-r	CTG GTCGAC CTAAGGATTGCTTTGGTGAG
WRKY61-f	ACC AGATCT ATGGAGGAGAAAAGAGCTAT
WRKY61-r	TTA GTCGAC TCAGTCAGTGTGCTCTCTAT
WRKY72-f	TAG GGATCC ATGGAGGTTTTATTGAAAATG
WRKY72-r	CAT GTCGAC TCAACTGCTTTGATCCTTGT
NPTII-f	GGATTGCACGCAGGTTCTCCG
NPTII-r	TCAGAAGAACTCGTCAAGAAGGCG
s-f	TCTTCGTCAACATGGTGGAGCACGA
50-r	ACTCATCAAAGGTCAAGTACTCAG
61-r	TCTGGCAGTTTCAAGCTGATCATC
72-r	ACAAGTTCAGACTCCATGATTTGAC

株系 Line	2^-ΔΔCt值2^-ΔΔCt value		预测拷贝数 Estimated copy number
株系 Line	GUS	NPTII	预测拷贝数 Estimated copy number
W50-1	1.32	1.39	1
W50-3	1.50	1.26	1
W50-5	1.90	1.64	2
W50-9	3.56	3.72	4
W50-10	2.84	2.76	3
W50-11	2.84	2.51	3
W61-1	1.07	1.47	1
W61-3	1.14	1.55	1
W61-5	1.07	0.98	1
W61-6	1.33	1.63	1
W61-7	1.13	1.14	1
W61-9	0.82	1.05	1
W61-11	0.97	0.82	1
W61-12	1.22	1.32	1
W72-1	2.57	2.41	2
W72-2	1.62	1.90	2
W72-4	2.68	2.96	3
W72-5	1.28	1.30	1
W72-7	1.32	1.49	1
W72-10	1.04	1.12	1