柑橘感染褐斑病过程中BAG1的作用

doi:10.16420/j.issn.0513-353x.2023-0273

摘要/Abstract

摘要：

本研究旨在探究柑橘BAG1参与褐斑病菌（Alternaria alternata）侵染的调控机制。从‘秤砣红橘’（Citrus tangerina Hort.ex Tanaka）、‘宫本温州蜜柑’（C. unshiu Macf）、‘爱媛38’杂柑（C. reticulata Blanco）和‘金秋砂糖橘’（C. reticulata Blanco）叶片中扩增BAG1基因。BAG1蛋白相对保守，均具有Ubiquitin泛素结构域和BAG结构域，‘爱媛38’的BAG结构域存在3个新的氨基酸变动，导致侧链基团变动。上游启动子序列显示4个柑橘品种均包含相同的激素和胁迫相关顺式作用元件。系统分析显示4个柑橘品种与宽皮柑橘类BAG1蛋白聚为一支，相似度最高，与甜橙类、柚类亲缘关系较远。qRT-PCR显示在褐斑病菌THJ菌株侵染的抗、感品种上BAG1的表达差异显著。BAG1受外源MeJA、ABA和SA诱导上调表达。烟草亚细胞定位显示BAG1存在于细胞核和细胞膜中。BAG1沉默能够增强对A. alternata的抗性，过表达对A. alternata的抗性降低。表明BAG1通过参与活性氧激发和ABA、JA或SA信号途径，参与柑橘对褐斑病菌的调控。

关键词: 柑橘, BAG1, 褐斑病菌, 感病基因, 活性氧

Abstract:

In this paper，the regulatory mechanism of citrus BAG1 gene in response to Alternaria alternata infection was explored. BAG1 was amplified from the leaves of‘Chengtuo Hongju’（Citrus tangerina Hort.ex Tanaka），‘Miyagawa Wase Unshiu’（C. unshiu Macf），‘Ehime 38’（C. reticulata Blanco）and‘Jinqiu Shatangju’（C. reticulata Blanco）. BAG1 protein was relatively conserved and had Ubiquitin domain and BAG domain. There are only three amino acid changes in the BAG domain of Ehime 38，resulting in changes in the side chain groups. The upstream promoter sequence showed that the four citrus cultivars contained the same hormone and stress-related cis-acting elements. Systematic analysis showed that the four citrus cultivars had the highest similarity to the BAG1 protein of mandarin，and had a distant relationship with sweet oranges and pomelo. qRT-PCR showed that the expression of BAG1 gene was significantly different in the resistant and susceptible cirtus cultivars during infected by A. alternata strain THJ. Hormonal stress treatment showed that BAG1 was up-regulated by exogenous plant hormones MeJA，ABA and SA. Trasient expression of tobacco showed that BAG1 protein was localized to the cell nucleus and cell membrane. BAG1 gene silencing and overexpression showed that BAG1 silencing could enhance the resistance of citrus to A.alternata. Overexpression reduced the resistance to A.alternata. The results showed that citrus BAG1 was involved in the regulation of A. alternata by participating in ROS excitation and ABA and JA signaling pathways.

Key words: Citrus, BAG1, Alternaria alternata, susceptibility gene, reactive oxygen species

周慧珍, 张嘉, 胡军华, 李白雪, 曹立, 余歆, 王福生, 邹修平, 周彦. 柑橘感染褐斑病过程中BAG1的作用[J]. 园艺学报, 2024, 51(5): 956-970.

ZHOU Huizhen, ZHANG Jia, HU Junhua, LI Baixue, CAO Li, YU Xin, WANG Fusheng, ZOU Xiuping, ZHOU Yan. The Role of BAG1 in the Infection of Citrus with Alternaria Brown Spot[J]. Acta Horticulturae Sinica, 2024, 51(5): 956-970.

图/表 10

表1 本研究中的引物信息

Table 1 Information of primers in this study

名称 Name	上游引物序列（5′-3′） Forward primer sequence	下游引物序列（5′-3′） Reverse primer sequence
CiBAG1	ATGCTTTCATTGGCAAGAAGC	TCAATCAAAAGTTTCCCATTTTG
qCiBAG1	TAACCGAGAAAGACGGTGCC	TCCGTAACCTGCTGAGCAAA
TRV-BAG1	GCTCTAGAGCGGAAATTAGGCCTGGAGGAATG	CGAGCTCGCCAAATCCGTAACCTGCTGAGC
PLGN-BAG1	CCCCGGGGATGCTTTCATTGGCAAGAAGC	CGGAATTCCGTCAATCAAAAGTTTCCCATTTTG
PCX-BAG1	GCGTCGACGCATGCTTTCATTGGCAAGAAGC	CCCCGGGGTCAATCAAAAGTTTCCCA TTTTG
TRV1	TTGGGTTGCTACTGATTCGACT	CTGTAAGGACCATCATACTTCGC
TRV2	ATTCACTGGGAGATGATACGCT	GAATCTAAGTCCACTCGTCCGT
QiBAG1	GCATGAGACAATTGCTTCATATGG	CCCTTAAAGCCTTCGAGTGG
Actin	GCCTTCCGTCTTGGGTCT	AGGGCGGTGATTTCCTT
NtPR1	CCGCCTTCCCTCAACTCAAC	GCACAACCAAGACGTACTGAG
NtPR2	AGGTGTTTGCTATGGAATGC	TCTGTACCCACCATCTTGC
NtPR4	GGCCAAGATTCCTGTGGTAGAT	CACTGTTGTTTGAGTTCCTGTTCCT
NtActin	ACCAGATTAATGAGCCCAAGAG	CCAACAGGGACAGTACCAATAC
CiNPR1	GGCCTTTCCCTATAGGCGTT	AAATCAACAGCCGGCCTACA
CiICS	ATTGATTGGCTCCATGCCCA	TGAGCTTTGGCCATTACCGT
CiPDF1.2	CAGTGGCAGAAGCAAAACAA	CCGGGGAAGTCGTAGTGGC
CiLOX	GCATCCTTTCTTGATCGGTTTC	GGCAGGCTCGCCATGA
CiAOC	AGATCGTGGCAGTCCAGCTT	GCTAAAAGGGACAAGATCACCAA
CiAOS	GCTATACACTCCTCGGGTTCGA	TGAAATGGAGGCGGTGATC
CiJAR1	GCACCATCAGGCATCAGACA	CAATGCGTTGAGTGTGCGTT
CiPOD	GACAGCAGAACAGCCAACAGG	GTTCGGCACTGAGCCCTTC
CiSOD	TGGAAGGGCTGTTGTAGTCC	ATGATGCCGCAAGCTACTCT
CiCAT	GTCTCAATGTGA GGCCAAGC	TAGGTGTCGGGAGCTTCTCT
Plant actin	CATCCCTCAGCACCTTCC	CCAACCTTAGCACTTCTCC

图1 柑橘5个品种BAG1与拟南芥BAG1的多重序列比较 Ci：克里曼丁；H：红橘；a：爱媛38；J：金秋砂糖橘；G：宫本；At：拟南芥。三角上为在Hsp70/ Hsc70蛋白结合中高度保守关键残基。

Fig. 1 Comparison of multiple sequences of BAG1 in five citrus cultivars with Arabidopsis BAG1 Ci：Lementina；H：Hongju；a：Ehime 38；J：Jinqiu Shatangju；G：Miyagawa；At：Arabidopsis. On the triangle are key residues that are highly conserved in Hsp70/Hsc70 protein binding.

图2 不同物种的BAG1聚类分析（邻接法）

Fig. 2 Cluster analysis of BAG1 among different species（Neighbor-Joining）

表2 CiBAG1上游2 000 bp启动子区列顺式作用元件预测

Table 2 The cis-acting elements prediction of the upstream 2 000 bp promoter region of CiBAG1

功能 Function	顺式元件 cis-Element	序列 Sequence	数量 Number
参与脱落酸响应Involved in the abscisic acid responsiveness	ABRE	ACGTG	2
赤霉素响应Gibberellin-responsive element	GARE-motif	TCTGTTG	1
厌氧诱导元件Essential for the anaerobic induction	ARE	AAACCA	2
厌氧特异性诱导Involved in anoxic specific inducibility	GC-motif	CCCCCG	1
干旱诱导性MYB binding site involved in drought-inducibility	MBS	CAACTG	1
参与低温响应Involved in low-temperature responsiveness	LTR	CCGAAA	1
部分光响应Part of a light responsive element	Box 4	ATTAAT	2
	AE-box	AGAAACTT	3
	TCT-motif	TCTTAC	4
	LS7	CAGATTTATTTTTA	1
	I-box	TGATAATGT	1
	ACE	CTAACGTATT	1
	GT1-motif	GGTTAA	1
	G-Box	CACGTT	2

图3 褐斑病菌THJ菌株诱导下‘红橘’和‘宫本’BAG1的表达模式 * 表示差异显著（P < 0.05）；*** 表示差异非常显著（P < 0.001）。

Fig. 3 The expression pattern of BAG1 in‘Hongju’and‘Miyagawa’at different time points after Alternaria alternata srain THJ infection * indicates significant difference（P < 0.05），*** indicates very significant difference（P < 0.001）.

图4 在植物生长调节剂胁迫下‘红橘’BAG1的表达模式 * 表示差异显著（P < 0.05）；** 表示差异极显著（P < 0.01）；*** 表示差异非常显著（P < 0.001）。

Fig. 4 The expression pattern of BAG1 gene in‘Hongju’under plant growth regulators stress * indicates significant difference（P < 0.05），** indicates extremely significant difference（P < 0.01），*** indicates very significant difference（P < 0.001）.

图5 ‘红橘’BAG1基因的VIGS瞬时沉默 a：BAG1基因相对表达量；b：Alternaria alternata 菌株THJ接种沉默株（pTRV2-BAG1-1/2/3）和对照株的症状；c：病斑直径。* 表示差异显著（P < 0.05）；** 表示差异极显著（P < 0.01）。

Fig. 5 ‘Hongju’VIGS transient silence a：BAG1 gene expression of control and silenced plants；b：Symptoms of control and silenced plants（pTRV2-BAG1-1/2/3）inoculated with A. alternata strain THJ；c：Average disease spot diameter of control and silenced plants after A. alternata inoculation. * indicates significant difference（P < 0.05）；** indicates extremely significant difference（P < 0.01）.

图6 ‘红橘’瞬时过表达株BAG1表达量（a）及其接种THJ菌株后的表型（b）、病斑直径（c）和相关基因表达量（d） WT：野生型；PLGN：阴性对照；PLGN-BAG1：过表达植株。ns表示差异不显著；* 表示差异显著（P < 0.05）；** 表示差异极显著（P < 0.01）。

Fig. 6 The expression level of BAG1（a）and the phenotype after inoculation with THJ strain（b），spot diameter（c）and related gene expression of‘Hongju’transient overexpression strain（d） WT：Wild-type；PLGN：Negative control；PLGN-BAG1：Overexpressed strain. ns indicates no significant difference；* indicates significant difference（P < 0.05）；** indicates extremely significant difference（P < 0.01）.

图7 瞬时过表达BAG1的烟草叶片基因表达量测定和染色观察 a：BAG1的表达量；b：Alternaria alternata看家基因Actin的表达量；c：A. alternata接种前后病程相关蛋白基因的相对表达量；d：染色观察。PCX用含有PCX空载的农杆菌侵染，PCX-BAG1用携带BAG1的PCX重组载体农杆菌侵染。（ⅰ）：叶片注射部位；（ⅱ）：注射3 d后二氨基联苯胺染色（DAB）；（ⅲ）：注射3 d后台盼蓝染色（TPB）。* P < 0.05；*** P < 0.001。

Fig. 7 Gene expression determination and staining observation of transient overexpression of BAG1 in tobacco leaves a：Expression analysis of BAG1 in Nicotiana benthamiana（tobacco）leaves；b：Quantification of A. alternata biomass in BAG1 transgenic tobacco leaves；c：Relative expression of pathogensis related protein genes in empty vector（PCX）and BAG1 overexpressed tobacco before and after inoculation of A. alternata；d：Staining observation. PCX，infiltrated with Agrobacterium containing empty vector（PCX）. PCX-BAG1，infiltrated with Agrobacterium harboring recombinant plasmid（PCX-BAG1）. （ⅰ）：Leaf injection site；（ⅱ）：Diaminobenzidine staining （DAB）of tobacco leaves three days after infiltration；（ⅲ）：Typan blue staining（TPB）of tobacco leaves three days after infiltration. * P < 0.05；*** P < 0.01.

图8 BAG1蛋白的烟草亚细胞定位

Fig. 8 Subcellular localization of BAG1 protein in tobacco leaves

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