山荆子类受体蛋白MbRLP7负调控杜梨对腐烂病的抗性

doi:10.16420/j.issn.0513-353x.2022-0938

园艺学报 ›› 2023, Vol. 50 ›› Issue (11): 2489-2498.doi: 10.16420/j.issn.0513-353x.2022-0938

山荆子类受体蛋白MbRLP7负调控杜梨对腐烂病的抗性

蒋达吉¹, 温有福², 余宏强¹, 郭志刚⁵, 左陇刚¹, 孙娥¹, 李岩妍¹, 左存武¹^,³^,^*(), 赵军营⁴^,^*()

¹ 甘肃农业大学园艺学院，兰州 730070
² 甘肃省静宁县林业和草原局，甘肃静宁 743400
³ 省部共建干旱生境作物学国家重点实验室，兰州 730070
⁴ 甘肃省林业科学研究院，兰州 730020
⁵ 天水师范学院，甘肃天水 741001

收稿日期:2023-05-04 修回日期:2023-08-17 出版日期:2023-11-25 发布日期:2023-11-28
通讯作者:
*（E-mail：zuocw@gsau.edu.cn，
362369437@qq.com）
基金资助:
甘肃省林业和草原科技创新项目(KJCX2021001); 国家自然科学基金地区基金项目(32260741); 甘肃省重点研发计划—农业类(21YF5NE199); 甘肃省重点研发计划—农业类(20YF8NA051)

The Malus baccata Receptor Like Protein MbRLP7 Negatively Regulates the Resistance of Pyrus betulifolia Bunge to Valsa Canker

JIANG Daji¹, WEN Youfu², YU Hongqiang¹, GUO Zhigang⁵, ZUO Longgang¹, SUN E¹, LI Yanyan¹, ZUO Cunwu¹^,³^,^*(), ZHAO Junying⁴^,^*()

¹ College of Horticulture，Gansu Agricultural University，Lanzhou 730070，China
² Forestry and Grassland Bureau of Jingning County，Jingning，Gansu 743400，China
³ State Key Laboratory of Aridland Crop Science，Lanzhou 730070，China
⁴ Gansu Academy of Forestry，Lanzhou 730020，China
⁵ Tianshui Normal University，Tianshui，Gansu 741001，China

Received:2023-05-04 Revised:2023-08-17 Published:2023-11-25 Online:2023-11-28
Contact:
*（E-mail：zuocw@gsau.edu.cn，
362369437@qq.com）

摘要/Abstract

摘要：

山荆子类受体蛋白MbRLP7为研究对象，结合生物信息分析、功能验证和表达量测定，分析了其在对腐烂病菌的抗性中的作用和调控机制。结果表明，MbRLP7编码的氨基酸序列与拟南芥AtRLP6和AtRLP7相似性最高，分别为43.0%和42.8%。MbRLP7启动子区域存在多个响应逆境相关信号的顺式作用元件，并响应腐烂病菌信号。将该基因导入杜梨悬浮细胞获得过表达细胞系3个。接种腐烂病病原菌Valsa pyri后，过表达细胞的发病速度显著高于野生型细胞。腐烂病菌代谢物处理后的所有过表达细胞系的细胞存活率显著低于野生型。此外，该基因的过表达增强了腐烂病菌代谢物对病原体相关分子模式（pathogen-associated molecular patterns，PAMPs）触发的免疫反应（PAMP-triggered immunity，PTI）、活性氧相关基因和病程相关蛋白PR1的诱导。综上所述，MbRLP7负调控杜梨对腐烂病的抗性，PTI、活性氧和PR1参与了该调控过程。

关键词: 杜梨, 类受体蛋白, 腐烂病, 病原体相关分子模式触发的防御反应, ROS

Abstract:

Based on bioinformatics analysis，functional verification，and expression measurement，the role of the receptor-like protein MbRLP7 in Valsa pyri resistance，and its potential regulatory mechanisms were explored. The results showed that MbRLP7 had the highest sequence similarity with AtRLP6 and AtRLP7，with 43.0% and 42.8%，respectively. There are several cis-elements in the promoter region of MbRLP7 in response to stress-related signals and Valsa pyri signals. The MbRLP7 was transformed into suspension cells of Pyrus betulifoliaBunge，and three overexpressed cell lines were obtained. After inoculation with the pathogen Valsa pyri（Vp），the growth rate of mycelia on the overexpressing cells was significantly higher than that on the wild-type cells. After Valsa pyri metabolites treatment，the cell activity of all overexpressed cell lines was significantly lower than that of wild-type. In addition，the overexpression of gene MbRLP7 enhanced the induction of Valsa pyrimetabolites on PAMP-triggered immunity（PTI），reactive oxygen species related genes and pathogenesis-related protein PR1. In conclusion，MbRLP7 negatively regulates the resistance of Pyrus betulifoliaBunge to Valsa pyri，and PTI，reactive oxygen species and PR1 are involved in this regulatory process.

Key words: Pyrus betulifolia Bunge, receptor-like protein, Valsa pyri, PAMP-triggered immunity, ROS

蒋达吉, 温有福, 余宏强, 郭志刚, 左陇刚, 孙娥, 李岩妍, 左存武, 赵军营. 山荆子类受体蛋白MbRLP7负调控杜梨对腐烂病的抗性[J]. 园艺学报, 2023, 50(11): 2489-2498.

JIANG Daji, WEN Youfu, YU Hongqiang, GUO Zhigang, ZUO Longgang, SUN E, LI Yanyan, ZUO Cunwu, ZHAO Junying. The Malus baccata Receptor Like Protein MbRLP7 Negatively Regulates the Resistance of Pyrus betulifolia Bunge to Valsa Canker[J]. Acta Horticulturae Sinica, 2023, 50(11): 2489-2498.

图/表 7

表1 PCR和qPCR引物

Table 1 Primers for PCR and quantitative real-time PCR

用途 Application	基因 Gene	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reverse prime
PCR	MbRLP7	CTGGCGCGCCATGAAAACTTTACAATCTTTTGGCC	TCCCTAGGTCAACGTCTCCACCTCTGATTTATG
qRT-PCR	MbRLP7	AACACATCTCGGGTGGCATT	AGCCGTGCAATAACCCTGAA
	PbeWRKY22	CATATCCAAGGGGATATTACAGATG	GTGACTATAAAAATATTCGGGTCGG
	PbeFRK1	TCGAGTCCAAAAAACGACAGTTTAC	TTGTCAAGTTTCTGTGATGAACTCT
	PbeOXI1	CAACTTGGAAAACTCCGAGAAGTC	TTGTCAGTATCCGATAAAACGTCTC
	TaNOX	TTGGAGTTGTTCGATGCGTTG	TCTTGTCCACCATGTCGAAGAAG
	PbePR1	AATCTTGTTCATTTTGGTGGGCC	AACAACCTGAGTATAATGCCCACAC
	PbePR4	GGTGAGAATTGTTGATCAGTGCAG	GTAGTTGACAATAAGGTGGCCTTG
	PbePR5	GACACACCCCAAGACTACCTCAAG	GTCACCAGTGCTCATGGCAAG

表2 MbRLP7启动子区顺式作用元件

Table 2 Analysis of the main cis-elements in promoter region of MbRLP7

顺式元件名称 cis-Element name	序列 Sequence	位置 Site	响应信号 Signal of response
TC-rich repeats	ATTCTCTAAC	-2 699	防御和胁迫Defense and stress
CCAAT-box	CAACGG	-2 459	转录因子MYBv1结合位点MYBv1 binding site
CGTCA-motif	CGTCA	-2 414	茉莉酸甲酯MeJA
O2-site	GATGA(C/T)(A/G)TG(A/G)	-2 065	玉米素Zeatin
GARE-motif	TCTGTTG	-1 026	赤霉素GA
ABRE	ACGTG	-1 003	脱落酸ABA
TGA-element	AACGAC	-119	生长素IAA

图1 山荆子悬浮细胞在腐烂病菌次生代谢物处理下的MbRLP7转录组数据FPKM值（A）和qRT-PCR表达量（B） * P < 0.05；** P < 0.01。下同。

Fig. 1 FPKM value（A）of MbRLP7 transcriptome data and qRT-PCR expression level（B）of MbRLP7 in Malus baccata suspension cells treated with Valsa pyri metabolites * P < 0.05；** P < 0.01. The same below.

图2 过表达MbRLP7促进杜梨细胞表面腐烂病菌（Valsa pyri）的扩散 A为野生型（WT）和过表达系中MbRLP7的相对表达量。B为杜梨细胞表面腐烂病菌接种和活性染料MTT染色试验。C为腐烂病菌菌落直径统计。

Fig. 2 Overexpression of MbRLP7 promotes the spread of Valsa pyri on the Pyrus betulifolia Bunge cells surface A is the relative expression of MbRLP7 in wild type（WT）and overexpressed lines. B is the inoculation of cell surface Valsa pyri（Vp） and MTT staining test. C is the colony diameter statistics of Valsa pyri（Vp）.

图3 不同浓度腐烂病菌次生代谢物（VpM）处理后的杜梨野生型（WT）和MbRLP7-OE7细胞存活率的变化

Fig. 3 Analysis of cell viability of wild type（WT）and MbRLP7-OE7 after Valsa pyri metabolites（VpM）treatment

图4 腐烂病菌次生代谢物处理后的杜梨野生型（WT）和MbRLP7-OE7活性氧含量的变化相对荧光比值（488nm/525nm）代表活性氧相对水平。

Fig. 4 Changes of active oxygen content of Pyrus betulifolia Bunge wild type（WT）and MbRLP7-OE7 after Valsa pyri metabolites treatment The relative fluorescence ratio represents the relative ROS level.

图5 杜梨野生型（WT）和过表达系MbRLP7-OE7经腐烂病菌次生代谢物处理后防御基因的表达量的变化

Fig. 5 Changes of defense gene expression in Pyrus betulifolia Bunge wild type（WT）and MbRLP7-OE7 overexpression line after Valsa pyri metabolites treatment

参考文献 25

[1]	Albert I, Böhm H, Albert M, Feiler C E, Imkampe J, Wallmeroth N, Brancato C, Raaymakers T M, Oome S, Zhang H, Krol E, Grefen C, Gust A A, Chai J, Hedrich R, van den A G, Nürnberge T. 2015. An RLP23-SOBIR1-BAK 1 complex mediates NLP-triggered immunity. Nature Plants, 1 (10):1-9.
[2]	Desaki Y, Miyata K, Suzuki M, Shibuya N, Kaku H. 2018. Plant immunity and symbiosis signaling mediated by LysM receptors. Innate Immunity, 24 (2):92-100. doi: 10.1177/1753425917738885 pmid: 29105533
[3]	Ding Li-na, Yang Guo-xing. 2016. Research advances in the mechanism and signal transduction of plant disease resistance. Biotechnology Bulletin, 32 (10):109-117. (in Chinese) doi: 10.13560/j.cnki.biotech.bull.1985.2016.10.013
	丁丽娜, 杨国兴. 2016. 植物抗病机制及信号转导的研究进展. 生物技术通报, 32 (10):109-117. doi: 10.13560/j.cnki.biotech.bull.1985.2016.10.013
[4]	Gao Xi-quan, Ruan Xin-sen, Ma Liang, Sun Ya-li. 2018. Regulation of programmed cell death responses in plant innate immunity. Journal of Nanjing Agricultural University, 41 (6):971-982. (in Chinese)
	高夕全, 阮辛森, 马亮, 孙亚利. 2018. 植物先天性免疫反应中的细胞死亡及其调控. 南京农业大学学报, 41 (6):971-982.
[5]	Hok S, Danchin E G J, Allasia V, Panabières F, Attard A, Keller H. 2011. An Arabidopsis(malectin-like)leucine-rich repeat receptor-like kinase contributes to downy mildew disease. Plant,Cell and Environment, 34 (11):1944-1957. doi: 10.1111/pce.2011.34.issue-11 URL
[6]	Jamieson P A, Shan L B, He P. 2018. Plant cell surface molecular cypher:receptor-like proteins and their roles in immunity and development. Plant Science, 274:242-251. doi: S0168-9452(18)30167-5 pmid: 30080610
[7]	Jehle A K, Lipschis M, Albert M, Fallahzadeh-Mamaghani V, Fürst U B, Mueller K, Felix G. 2013. The receptor-like protein ReMAX of Arabidopsis detects the microbe-associated molecular pattern eMax from Xanthomonas. Plant Cell, 25 (6):2330-2340. doi: 10.1105/tpc.113.110833 URL
[8]	Jones D A, Thomas C M, Hammond-Kosack K E, Balint-Kurti P J, Jones J D. 1994. Isolation of the tomato Cf-9 gene for resistance to Cladosporium fulvum by transposon tagging. Science, 266 (5186):789-793. doi: 10.1126/science.7973631 pmid: 7973631
[9]	Li Jiang-zhou, Wang Yao, Li Yuan-xiu, Zhuo Xin-yi, Wang Zi-xuan, Zhao Xue-li, Wang Mei-chao, Hou Yi-long, Li Xiao-yan. 2019. Research progress and prospect on Valsa mali Miyabe & Yamada. Molecular Plant Breeding, 17 (21):7222-7227. (in Chinese)
	李江舟, 王耀, 李元秀, 卓馨怡, 王子烜, 赵雪丽, 王美超, 侯义龙, 李晓艳. 2019. 苹果树腐烂病研究进展与展望. 分子植物育种, 17 (21):7222-7227.
[10]	Mengiste T. 2012. Plant immunity to necrotrophs. Annual Review of Phytopathology, 50 (1):267-294. doi: 10.1146/phyto.2012.50.issue-1 URL
[11]	Nie J J, Zhou W J, Liu J Y, Tan N, Zhou J M, Huang L L. 2020. A receptor-like protein from Nicotiana benthamiana mediates VmE 02 PAMP-triggered immunity. The New Phytologist, 229 (4):2260-2272. doi: 10.1111/nph.v229.4 URL
[12]	Shen Y P, Diener A C. 2013. Arabidopsis thaliana resistance to Fusarium oxysporum 2 implicates tyrosine-sulfated peptide signaling in susceptibility and resistance to root infection. PLoS Genetics, 9 (5):e1003525. doi: 10.1371/journal.pgen.1003525 URL
[13]	Shiu S H, Bleecker A B. 2003. Expansion of the receptor-like kinase/pelle gene family and receptor-like proteins in Arabidopsis. Plant Physiology, 132 (2):530-543. doi: 10.1104/pp.103.021964 URL
[14]	Tian Y, Wang Q, Zhang H, Zhou N, Yan H, Jian H, Li S, Xiang G, Tang K, Qiu X. 2022. Genome-wide identification and evolutionary analysis of MLO gene family in Rosaceae plants. Horticultural Plant Journal, 8 (1):110-122. doi: 10.1016/j.hpj.2020.07.007 URL
[15]	Wang C, Mao X, Zhao D, Yu H Q, Duo H, Sun E, Lu Y, Zuo C W. 2022. Transcriptomic analysis reveals that cell wall- and hypersensitive response (HR)-related genes are involved in the responses of apple to Valsa mali. Plant Biotechnology Reports, 16:539-551. doi: 10.1007/s11816-022-00763-z
[16]	Wu Jin-bin, Song Yin, Xu Shan-shan, Qiang Yi, Wang Guo-dong. 2012. Progress on the role of receptor-like protein in plant growth and development. Plant Physiology Journal, 48 (12):1121-1127. (in Chinese)
	吴锦斌, 宋银, 徐姗姗, 强毅, 王国栋. 2012. 植物类受体蛋白参与植物生长与发育的研究进展. 植物生理学报, 48 (12):1121-1127.
[17]	Wu S J, Shan L B, He P. 2014. Microbial signature-triggered plant defense responses and early signaling mechanisms. Plant Science, 228:118-126. doi: 10.1016/j.plantsci.2014.03.001 pmid: 25438792
[18]	Wu Yu-jun, Wu Wang-ze. 2021. Pattern recognition receptors and plant innate immunity. Plant Physiology Journal, 57 (2):301-312. (in Chinese) doi: 10.1111/ppl.1983.57.issue-2 URL
	吴玉俊, 吴旺泽. 2021. 植物模式识别受体与先天免疫. 植物生理学报, 57 (2):301-312.
[19]	Xu J, Xian Q, Wang K, Dong J, Zhang C, Du S, Xu X, Chen X. 2022. Screening and identification of candidate Fusarium wilt-resistance genes from pumpkin. Horticultural Plant Journal, 8 (5):583-592. doi: 10.1016/j.hpj.2021.11.011 URL
[20]	Yan Jia, Liu Ya-qiong, Hou Sui-wen. 2018. Recent advances in disease resistance proteins in plant Immunity. Chinese Bulletin of Botany, 53 (2):250-263. (in Chinese)
	闫佳, 刘雅琼, 侯岁稳. 2018. 植物抗病蛋白研究进展. 植物学报, 53 (2):250-263. doi: 10.11983/CBB17148
[21]	Yang Y, Zhang Y, Ding P, Johnson K, Li X, Zhang Y. 2012. The ankyrin-repeat transmembrane protein BDA1 functions downstream of the receptor-like protein SNC2 to regulate plant Immunity. Plant Physiology, 159 (4):1857-1865. doi: 10.1104/pp.112.197152 pmid: 22740615
[22]	Zhang L, Kars I, Essenstam B, Liebrand T W H, Wagemakers L, Elberse J, Tagkalaki P, Tjoitang D, van den Ackerveken G, van Kan J A L. 2014. Fungal endopolygalacturonases are recognized as microbe-associated molecular patterns by the Arabidopsis receptor-like protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1. Plant Physiology, 164 (1):352-364. doi: 10.1104/pp.113.230698 URL
[23]	Zhang Q, Chen T, Wang X, Wang J, Gu K, Yu J, Hu D, Hao Y. 2022. Genome-wide identification and expression analyses of homeodomain-leucine zipper family genes reveal their involvement in stress response in apple(Malus × domestica). Horticultural Plant Journal, 8 (3):261-278. doi: 10.1016/j.hpj.2021.04.003 URL
[24]	Zhang Qiuyue, Liu Changlai, Yu Xiaojing, Yang Jiading, Feng Chaonian. 2022. Screening of reference genes for differentially expressed genes in Pyrus betulaefolia plant under salt stress by qRT-PCR. Acta Horticulturae Sinica, 49 (7):1557-1570. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0376 URL
	张秋悦, 刘昌来, 于晓晶, 杨甲定, 封超年. 2022. 盐胁迫条件下杜梨叶片差异表达基因qRT-PCR内参基因筛选. 园艺学报, 49 (7):1557-1570. doi: 10.16420/j.issn.0513-353x.2021-0376 URL
[25]	Zhang W, Fraiture M, Kolb D, Löffelhardt B, Desaki Y, Boutrot F F G, Tör M, Zipfel C, Gust A A, Brunner F. 2013. Arabidopsis receptor-like protein30 and receptor-like kinase suppressor of BIR1-1/EVERSHED mediate innate immunity to necrotrophic fungi. Plant Cell, 25 (10):4227-4241. doi: 10.1105/tpc.113.117010 URL

山荆子类受体蛋白MbRLP7负调控杜梨对腐烂病的抗性

The Malus baccata Receptor Like Protein MbRLP7 Negatively Regulates the Resistance of Pyrus betulifolia Bunge to Valsa Canker

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 25

相关文章 13

编辑推荐

Metrics

本文评价

访问总数:　当日访问总数:　当前在线人数:

[1]	高云静, 王天好, 胡旺成, 洪霓, 王国平, 王利平. 梨轮纹病菌弱毒株携带产黄青霉病毒水平传染力测定[J]. 园艺学报, 2023, 50(11): 2477-2488.
[2]	李岩妍, 郭守军, 蔡敏蕊, 孙娥, 余宏强, 蒋达吉, 左存武, 王海. 苹果类受体激酶基因MdLYK1正调控对腐烂病的抗性[J]. 园艺学报, 2023, 50(11): 2499-2508.
[3]	张秋悦, 刘昌来, 于晓晶, 杨甲定, 封超年. 盐胁迫条件下杜梨叶片差异表达基因qRT-PCR内参基因筛选[J]. 园艺学报, 2022, 49(7): 1557-1570.
[4]	李泳潭，张军*，黄亚丽，范建敏，张益文，左力辉. 杜梨叶绿体基因组分析[J]. 园艺学报, 2020, 47(6): 1021-1032.
[5]	刘河，朵虎，赵丹，孙娥，马富鹏，马春玲，左存武*. 梨CRK家族基因及其腐烂病菌侵染响应成员的鉴定[J]. 园艺学报, 2020, 47(5): 963-973.
[6]	朵虎, 赵兴刚, 吕前前, 王东东, 冒霞, 刘河, 赵丹, 左存武, . 植物Gly-RLK基因家族鉴定及其在苹果中的表达分析[J]. 园艺学报, 2020, 47(10): 1883-1894.
[7]	吕前前, 赵兴刚, 王东东, 冒霞, 左存武, 杨江山, . 解淀粉芽孢杆菌BaA-007鉴定及其对苹果腐烂病的抑制作用[J]. 园艺学报, 2020, 47(10): 1895-1904.
[8]	田丹，朵虎，刘河，吕前前，左存武*. 苹果L-LEC-RLK基因家族鉴定及响应病原真菌信号的表达分析[J]. 园艺学报, 2019, 46(3): 421-432.
[9]	张欢，杨英杰，李鼎立，宋健坤，马春晖，王然*. 杜梨根茎叶特异表达基因的RNA-Seq分析[J]. 园艺学报, 2018, 45(10): 1881-1894.
[10]	吕前前，刘钰玺，李静轩，马宗桓，姜雪峰，王宝林，毛娟，褚明宇，陈佰鸿，左存武*. 苹果CR4基因家族鉴定与表达分析[J]. 园艺学报, 2018, 45(10): 2019-2029.
[11]	张美鑫, 翟立峰, 胡红菊, 张本贤, 洪霓. 梨种质资源对腐烂病抗性的室内评价[J]. 园艺学报, 2014, 41(7): 1297-1306.
[12]	李慧1,2,，李刚波1,3,，丛郁4，常有宏1,2,*，蔺经1，盛宝龙1. 杜梨类钙调磷酸酶B亚基蛋白基因PbCBL2*的克隆和功能初探[J]. 园艺学报, 2013, 40(8): 1445-1455.
[13]	胡小松;肖华志;王晓霞. 苹果α-法尼烯和共轭三烯含量变化与贮藏温度的关系[J]. 园艺学报, 2004, 31(2): 169-172.

山荆子类受体蛋白MbRLP7负调控杜梨对腐烂病的抗性

The Malus baccata Receptor Like Protein MbRLP7 Negatively Regulates the Resistance of Pyrus betulifolia Bunge to Valsa Canker

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 25

相关文章 13

编辑推荐

Metrics

本文评价

访问总数: 当日访问总数: 当前在线人数:

访问总数:　当日访问总数:　当前在线人数: