超量表达CsGH3.6通过抑制生长素信号转导 增强柑橘溃疡病抗性

doi:10.3864/j.issn.0578-1752.2019.21.009

摘要/Abstract

摘要：

背景由柑橘黄单胞杆菌柑橘亚种（Xanthomonas citri subsp. citri,Xcc）引起的柑橘溃疡病是柑橘生产上最具毁灭性的一种病害。植物生长素在调控柑橘溃疡病菌引起的寄主侵染部位脓疱形成中起重要作用。生长素早期响应基因GH3通过酰基化吲哚-3-乙酸（indole-3-acetic acid,IAA）调控植物激素动态平衡。前期研究发现柑橘CsGH3.6在调控生长素响应溃疡病侵染中起着重要作用。目的通过对超量表达CsGH3.6转基因晚锦橙的抗病性、植株表型、细胞和激素变化进行分析,利用RNA-Seq解析CsGH3.6调控的信号通路,探明CsGH3.6调控激素动态平衡影响柑橘溃疡病抗性的内在机制。方法利用针刺法对离体转基因叶片接种溃疡病菌Xcc,统计接种第10 天时病斑面积和病情指数,以野生型为对照,评价转基因植株的抗性水平;提取感病前后叶片内源激素,利用高效液相色谱技术(high performance liquid chromatography,HPLC )检测转基因植株中激素含量变化;温室中观察转基因植株表型变化;通过测量叶片纵径、横径和厚度分析转基因植株叶型变化特征;制备叶片表皮切片,显微观察表皮细胞和气孔,并统计转基因植株表皮细胞长度和气孔密度;采用RNA-Seq测序技术研究转基因植株转录组变化情况,并利用Nr、Nt、Pfam、COG、SwissProt和gene ontology (GO)数据库注释基因功能,进一步利用KEEG数据库和MapMan软件解析超量表达CsGH3.6影响的重要基因、功能和途径,阐明CsGH3.6调控柑橘溃疡病抗性的分子机制。结果超量表达CsGH3.6显著增强转基因植株的溃疡病抗性;转基因植株分枝增多且下垂,叶片向上卷曲,变小,颜色浅;转基因植株气孔密度增加,表皮细胞变短;激素含量分析显示,转基因植株自由生长素（IAA）和茉莉酸（jasmonic acid,JA）含量显著降低,而水杨酸（salicylic acid,SA）含量显著增加;转录组测序分析表明,超量表达CsGH3.6显著抑制生长素信号转导相关基因表达,特别是所有注释的Aux/IAA家族基因均下调表达,相反,与生物胁迫相关基因的表达为上调,其中绝大部分基因为病程相关蛋白基因。结论超量表达CsGH3.6通过酰基化自由IAA抑制生长素信号转导,调控JA和SA的动态平衡,改变细胞和植株的形态建成,从而增强柑橘对溃疡病的抗性。研究结果暗示调控激素动态平衡在柑橘抗病育种中具有潜在价值。

关键词: 柑橘黄单胞杆菌柑橘亚种, 柑橘溃疡病, 生长素, CsGH3.6, 抗病性

Abstract:

【Background】 Citrus canker, induced by Xanthomonas citri subsp. citri (Xcc), is one of the most destructive disease in citrus production. Auxin plays an important role in regulating Xcc-induced pustule formation in citrus. GH3, an early auxin response gene, regulates plant hormone homeostasis through acylating indole-3-acetic acid (IAA). The previous study found that CsGH3.6 had a vital role in response to Xcc infection. 【Objective】 Here, to explore the internal mechanism of CsGH3.6 in regulating the dynamic balance of auxin and affecting the plant resistance to citrus canker, disease resistance evaluation, phenotype analysis, hormone determination and transcriptome sequencing of Wanjingcheng orange (Citrus sinensis) overexpressing CsGH3.6 were performed in this study.【Method】 To evaluate resistance to citrus canker in transgenic plants overexpressing CsGH3.6, fully expanded intact leaves gathered from transgenic plants were infected with Xcc by the pin-prick inoculation, and the diseased areas and disease severity index were investigated 10 d after inoculation, the wild type (WT) plant was used as the control. To detect hormone levels in transgenic plants, different hormones were isolated from leaves before and after Xcc infection and their contents were determined through high performance liquid chromatography (HPLC). Compared with WT plant, changes of phenotypes (plant types and leaf longitudinal diameter, transverse diameter and thickness) in transgenic plants were investigated in the greenhouse, and the length of epidermal cell and stomata density were further analyzed using optical microscopy. RNA sequencing was performed to investigate transcript changes in transgenic plant, and gene functions were annotated based on Nr, Nt, Pfam, COG, SwissProt and gene ontology (GO) databases. To elucidate the molecular mechanism of CsGH3.6 in regulating the canker resistance in citrus, the important genes, functions and pathways affected significantly by overexpressing CsGH3.6 were investigated using the KEGG database and MapMan software.【Result】 Overexpression of CsGH3.6 significantly enhanced citrus canker resistance in transgenic plants. The branches of transgenic plants increased and drooped, the leaves curled upward, became smaller and lighter in color. The stomata density of transgenic plants increased and the length of epidermal cells became shorter. Hormone analyses showed that the contents of free auxin IAA and jasmonic acid (JA) in transgenic plants decreased significantly, while the content of salicylic acid (SA) increased significantly. Transcriptome sequencing showed that overexpression of CsGH3.6 significantly inhibited the expression of auxin signal transduction related genes, especially the expression level of all the predicted Aux/IAA genes was down-regulated in transgenic plant. Conversely, the expression level of genes related to biological stress was up-regulated, most of which were pathogenesis-related genes. 【Conclusion】 Overexpression of CsGH3.6 can inhibit auxin signal transduction through acylating free IAA, regulate the homeostasis of JA and SA, change the morphogenesis of cells and plants, and finally enhance the plant resistance to citrus canker. The results suggest that the regulation of hormone homeostasis has potential value in citrus disease resistance breeding.

Key words: Xanthomonas citri subsp. citri (Xcc), citrus canker, auxin, CsGH3.6, resistance

邹修平,龙俊宏,彭爱红,陈敏,龙琴,陈善春. 超量表达CsGH3.6通过抑制生长素信号转导增强柑橘溃疡病抗性[J]. 中国农业科学, 2019, 52(21): 3806-3818.

ZOU XiuPing,LONG JunHong,PENG AiHong,CHEN Min,LONG Qin,CHEN ShanChun. Overexpression of CsGH3.6 Enhanced Resistance to Citrus Canker Disease by Inhibiting Auxin Signaling Transduction[J]. Scientia Agricultura Sinica, 2019, 52(21): 3806-3818.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.21.009

https://www.chinaagrisci.com/CN/Y2019/V52/I21/3806

图/表 11

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图2

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表1

b19转基因植株中生长素相关差异表达基因统计分析"

基因编号Gene ID	推测的功能Putative function	差异倍数Log₂ fold change*
生长素合成-降解Auxin synthesis-degradation
Cs7g08110	IAA β-葡萄糖基转移酶 IAA β-glucosyltransferase	1.832
Orange1.1t02388	IAA β-D-葡萄糖基转移酶 IAA β-D-glucosyltransferase	-1.153
Cs3g19760	IAA氨基酸共轭水解酶 IAA amino acid conjugate hydrolase	1.573
Cs7g08080	IAA氨基酸共轭水解酶 IAA amino acid conjugate hydrolase	1.074
生长素运输Auxin transport
Orange1.1t00089	生长素输出载体PIN1 Auxin efflux carrier PIN1	-1.022
Cs2g16620	生长素输出载体PIN3 Auxin efflux carrier PIN3	-2.790
Cs3g19250	生长素运输相似蛋白3 Auxin transporter-like protein 3	-1.385
生长素信号转导Auxin signal transduction
Cs5g29060	AUX/IAA家族基因IAA11 AUX/IAA family IAA11	-1.570
Cs9g09120	AUX/IAA家族基因IAA13 AUX/IAA family IAA13	-1.286
Cs5g30380	AUX/IAA家族基因IAA16 AUX/IAA family IAA16	-3.052
Cs5g30390	AUX/IAA家族基因IAA4 AUX/IAA family IAA4	-3.905
Cs7g05540	AUX/IAA家族基因IAA29 AUX/IAA family IAA29	-3.067
Cs9g08100	AUX/IAA家族基因IAA29 AUX/IAA family IAA29	-4.314
Cs9g08110	AUX/IAA家族基因IAA4 AUX/IAA family IAA14	-5.958
Cs1g13970	AUX/IAA家族基因IAA22 AUX/IAA family AUX22	-6.028
Cs4g18240	AUX/IAA家族基因IAA29 AUX/IAA family IAA29	-3.097
cs1g13960	AUX/IAA家族基因 AUX/IAA family	-1.775
cs3g10920	AUX/IAA家族基因IAA22 AUX/IAA family IAA22	-1.656
cs3g10930	AUX/IAA家族基因IAA16 AUX/IAA family IAA16	-1.831
cs3g16750	AUX/IAA家族基因IAA9 AUX/IAA family IAA9	-1.089
Orange1.1t04221	SAUR-like相似蛋白ARG7 SAUR-like protein ARG7	-4.445
Cs8g16440	生长素响应因子ARF10 Auxin response factor ARF10	-1.931
cs4g04520	生长素响应因子ARF7 Auxin response factor ARF7	-1.111
cs4g12720	SAUR-like相似蛋白 SAUR-like protein	3.141
orange1.1t00825	IAA酰基化酶GH3.5 IAA-amido synthetase GH3.5	-1.200
cs4g11890	IAA酰基化酶CsGH3.6 IAA-amido synthetase CsGH3.6	7.325

表1

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图9

表2

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基因编号Gene ID	推测的功能Putative function	差异倍数Log₂ fold change
纤维素合成Cellulose synthesis
Cs4g02000	木质素特异纤维素合酶 Xylem-specific cellulose synthase	7.304
细胞壁蛋白Cell wall protein
Cs8g16830	成束蛋白样阿拉伯半乳聚糖蛋白12 FASCICLIN-like arabinogalactan-protein 12 FLA12	5.439
细胞壁修饰Cell wall modification
Cs4g03060	木葡聚糖内糖基转移酶相关蛋白 Xyloglucan endotransglycosylase-related protein (XTR6)	2.591
Cs4g03130		2.031
Cs4g03140		2.227
Cs5g28330	角质素合成酶相关基因LCAC Cutin synthesis-related gene LCAC	1.576
Orange1.1t00556	植物蜡质合成相关基因KCS1 Wax biosynthesis-related gene KCS1	1.408
Cs5g07854	延展蛋白EXLB1 Expansin EXLB1	-1.393