辣椒乙烯反应抑制因子Cacl-6468的克隆及其抗根结线虫作用分析

doi:10.16420/j.issn.0513-353x.2022-1220

摘要/Abstract

摘要：

Cacl-6468是新发现的辣椒乙烯反应抑制因子基因，来源于辣椒（Capsicum annuum）抗根结线虫材料HDA149，属于抑制因子Ⅰ超基因家族基因（InhibitorⅠ），与马铃薯的乙烯反应抑制因子具有较近的遗传同源关系。在抗性辣椒中Cacl-6468的表达与南方根结线虫早期侵染紧密相关，在毒性线虫侵染12 h时表达量即明显下调，到7 d时达到最低，而在非毒性线虫侵染12 h时则显著上调，3 d时达到最高。利用病毒诱导的基因沉默（VIGS）技术，证实抑制Cacl-6468的表达能促使南方根结线虫非毒群体克服辣椒HDA149的抗性，形成根结和卵块，但其发病程度显著低于毒性线虫。在VIGS测试中还证实Cacl-6468负调控乙烯信号通路，其表达下降伴随着乙烯信号通路基因的显著上调，但是对水杨酸信号通路影响不显著。通过早期病理观察及二龄线虫趋性测试，证实抑制Cacl-6468的表达可以增强辣椒根系对南方根结线虫的吸引性，促进早期侵染。利用气相质谱法检测乙烯释放量发现，抑制Cacl-6468的表达可以增强辣椒植株的乙烯释放量。通过以上结果可推测辣椒乙烯反应抑制因子基因Cacl-6468通过调控乙烯信号通路参与辣椒对根结线虫的抗性调控，抑制该基因表达可以增加辣椒对线虫的感病性，降低抗性。

关键词: 乙烯反应抑制因子, 辣椒, 根结线虫, 抗性

Abstract:

The pepper gene Cacl-6468 is a novel ethylene-responsive proteinase inhibitor gene，from the nematode-resistance pepper HDA149（Capsicum annuum），belongs to the Potato InhibitorⅠsupper gene family，and has close genetic homology with potato ethylene-responsive proteinase inhibitor. The expression levels of Cacl-6468 in HDA149 were significantly affected by the population kinds of Meloidogyne incognita. For virulence population of M. incognita，Cacl-6468 was down-regulated significantly after 12 h infection，and reached the lowest level at seven days；while for avirulence population，the expression level was significantly up-regulated after 12 h infection，and reached the highest level at three days. Using virus-induced gene silencing（VIGS）technique，it was proved that the knock-down of Cacl-6468 could promote avirulent population to overcome the resistance of pepper HDA149，and developed some galls and egg masses，but the numbers were significantly lower than that of virulent population. The VIGS test also confirmed that Cacl-6468 negatively regulated ethylene signaling pathway，but had no significant effect on salicylic acid signaling pathway. In addition，the early pathological observation and chemotaxis test showed that VIGS treatment of Cacl-6468 enhanced the attraction of pepper root to M. incognita，and promoted early stage infection. The ethylene release of pepper was detected by GC-MS，showed that inhibition of Cacl-6468 expression could enhance the ethylene release of pepper. qPCR results suggested that Cacl-6468 is involved in the resistance regulation of pepper to M. incognita through ethylene signaling pathway，decreased the expression level of Cacl-6468 can increase the susceptibility，and weaken the resistance of pepper HDA149.

Key words: ethylene-responsive proteinase inhibitor, Capsicum annuum, Meloidogyne incognita, resistance

侯雪, 王姣姣, 张雯雯, 赵建龙, 茆振川. 辣椒乙烯反应抑制因子Cacl-6468的克隆及其抗根结线虫作用分析[J]. 园艺学报, 2024, 51(4): 761-772.

HOU Xue, WANG Jiaojiao, ZHANG Wenwen, ZHAO Jianlong, MAO Zhenchuan. Cloning of Pepper Ethylene-Responsive Proteinase Inhibitor Cacl-6468 and Its Effect on Resistance to Meloidogyne incognita[J]. Acta Horticulturae Sinica, 2024, 51(4): 761-772.

图/表 8

表1 相关基因及其qRT-PCR引物序列

Table 1 related genes and their qRT-PCR primer sequences

用途 Usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence
3′RACE克隆 3′RACE clone	3′RACE	ACCTAGTAGATATACGTACA
5′RACE克隆 5′RACE clone	5′RACE	CTTAGTTTCAAGATCTACGA
Cacl-6468基因克隆 Cacl-6468 clone	Cacl-6468-F	ATGGATAAGTTAGCTCATT
Cacl-6468基因克隆 Cacl-6468 clone	Cacl-6468-R	TCAACCAACTTGGGGAGTTT
VIGS载体构建 VIGS vector construction	pTV-Cacl-6468-F	AGAAGGCCTCCATGGGGATCCGAAAAAGGACAAGTGGCCAGAA
	pTV-Cacl-6468-R	GAGACGCGTGAGCTCGGTACCAGACGAACTCGATCACATCTAAAATC
	pTV-PDS-F	GTGAGTAAGGTTACCGAATTCTAGGGTGACGGATGAGGTGTTC
	pTV-PDS-R	GAGACGCGTGAGCTCGGTACCTTCCAGTCTTCAGGCAAAAGAAG
qRT-PCR	β-Tubulin-F	GAGGGTGAGTGAGCAGTTC
	β-Tubulin-R	CTTCATCGTCATCTGCTGTC
	qCacl-6468-F	AAAAGGACAAGTGGCCAGAA
	qCacl-6468-R	TTGTGTAACTGGAGAACCATTCA
	qCaERF66-F	AGAAGGTCGAAAGAGGAT
	qCaERF66-R	GCTGAGGAATTAGGGTTA
	qCaPTI1-F	GGTTCCAACTCACCAAAGTGATC
	qCaPTI1-R	TTCTTGCCTATGGTTTGTGATGG
	qCaPR1-F	GCCGTGAAGATGTGGGTCAATGA
	qCaPR1-R	TGAGTTACGCCAGACTACCTGAGTA
	qCaNPR1-F	ACTTCTTCGCCGACGCCAAG
	qCaNPR1-R	GCCAACACATTCACCAGAGCATC

图1 辣椒乙烯反应抑制因子基因Cacl-6468保守结构域氨基酸序列与其他物种序列的进化树

Fig. 1 The evolutionary tree of the conserved domain amino acid sequence of the capsicum-ethylene response inhibitor gene Cacl-6468 and its sequence in other species

图2 HDA149辣椒接种毒性（vir）和无毒（avir）南方根结线虫后根中抗性基因Cacl-6468的表达量的变化

Fig. 2 Changes in the expression of resistance gene Cacl-6468 in roots of HDA149 pepper after inoculation with vir and avir population of Meloidogyne incognita * P < 0.05，** P < 0.01，**** P < 0.0001.

图3 HDA149辣椒抗线虫基因沉默植株及对照植株接种毒性和无毒南方根结线虫后根部根结和卵块数的变化 H2O：清水对照植株；pTV-PDS：阳性对照植株；VIGS：抗线虫基因Cacl-6468沉默植株。

Fig. 3 Changes in root knot and egg mass number in HDA149 capsicum plants after inoculation with vir and avir population of Meloidogyne incognita after gene silencing and control plants H2O：Water control plant；pTV-PDS：Positive control plant；VIGS：Nematode resistance gene Cacl-6468 silencing plants. ** P < 0.01，**** P < 0.0001.

图4 HDA149辣椒抗线虫基因沉默植株（VIGS）接种非毒性南方根结线虫对水杨酸和乙烯信号通路基因表达的影响

Fig. 4 Effects of HDA149 pepper nematode resistant gene silencing plants（VIGS）inoculated with avir population of Meloidogyne incognita on gene expression in salicylic acid and ethylene signaling pathways * P < 0.05，**** P < 0.0001.

图5 HDA149辣椒接种无毒南方根结线虫后的根组织染色及根中二龄幼虫数量统计 H2O：清水对照植株；pTV-PDS：阳性对照植株；VIGS：抗线虫基因Cacl-6468沉默植株。

Fig. 5 Staining of root tissue and the number of second-instar larvae in roots after inoculation with Meloidogyne incognita with HDA149 pepper H2O：Water control plant；pTV-PDS：Positive control plant；VIGS：Nematode resistance gene Cacl-6468 silencing plants. ** P < 0.01.

图6 无毒南方根结线虫趋性试验中辣椒根尖周围二龄线虫的分布 H2O：清水对照；pTV-PDS：阳性对照；VIGS：抗线虫基因Cacl-6468沉默处理。

Fig. 6 Distribution of second-instar nematodes around the root tip of pepper in Meloidogyne incognita tropism test H2O：Water control；pTV-PDS：Positive control；VIGS：Anti-nematode gene Cacl-6468 silencing treatment. * P < 0.05.

图7 VIGS抑制Cacl-6468表达及pTV00空载体处理对辣椒乙烯释放量的影响

Fig. 7 Effects of VIGS on the expression of Cacl-6468 gene and pTV00 empty vector treatment on the release of capsane ethylene **** P < 0.0001.

参考文献 27

[1]	Bybd D W, Kirkpatrick T, Barker K R. 1983. An improved technique for clearing and staining plant tissues for detection of nematodes. Journal of Nematology, 15 (1):142-143. pmid: 19295781
[2]	Bent A F, Hoffman T K, Schmidt J, Hartman G L, Hoffman D D, Xue P, Tucker M L. 2006. Disease-and performance-related traits of ethylene-insensitive soybean. Crop Science of America, 46:893-901.
[3]	Dahl C C, Baldwin I T. 2007. Deciphering the role of ethylene in plant-herbivore interactions. Journal of Plant Growth Regulation, 26 (2):201-209.
[4]	Dyer S, Weir R, Cox D, Cheseto X, Torto B, Dalzell J J. 2019. Ethylene response factor(ERF)genes modulate plant root exudate composition and the attraction of plant parasitic nematodes. International Journal for Parasitology, 49 (13):999-1003.
[5]	Eddaoudi M, Ammati M, Rammah A. 1997. Identification of the resistance breaking populations of Meloidogyne on tomatoes in morocco and their effect on new sources of resistance. Fundamental and Applied Nematology, 20 (3):285-289.
[6]	Elham A W, Stavros C, Eman M S, Norhan E S. 2010. Effect of two natural carotenoid sources in diets for gilthead seabream,Sparus aurata,on growth and skin coloration. Journal of Applied Aquaculture, 22 (3):216-229.
[7]	Fudali S L, Wang C L, Williamson V M. 2013. Ethylene signaling pathway modulates attractiveness of host roots to the root-knot nematode Meloidogyne hapla. Molecular Plant Microbe Interactions, 26 (1):75-86.
[8]	Glazer I, Apelbaum A, Orion D. 1985. Effect of inhibitors and stimulators of ethylene production on gall development in Meloidogyne javanica infected tomato roots. Journal of Nematology, 17 (2):145-149. pmid: 19294073
[9]	Huang J F, Shen L, Yang S, Guan D Y, He S L. 2020. CaASR1 promotes salicylic acid-but represses jasmonic acid-dependent signaling to enhance the resistance of Capsicum annuum to bacterial wilt by modulating CabZIP63. Joumal of Experimental Botany, 71 (20):6538-6554.
[10]	Jiang Li-fen, Mao Zhen-chuan, Chen Guo-hua, Yang Yu-hong, Xie Bing-yan. 2011. Fitness cost and specialization of pepper Me3virulent population in the parthenogenetic nematode Meloidogyne incognita. Acta Horticulturae Sinica, 38 (3):479-486. (in Chinese)
	蒋丽芬, 茆振川, 陈国华, 杨宇红, 谢丙炎. 2011. 南方根结线虫辣椒Me3毒性群体适合度代价及专化性分析. 园艺学报, 38 (3):479-486.
[11]	Jin J H, Zhang H X, Tan J Y, Yan M J, Li D W, Abid K, Gong Z H. 2016. A new ethylene-responsive factor CaPTI1 gene of pepper(Capsicum annuum L.) involved in the regulation of defense response to Phytophthora capsici. Frontiers in Plant Science, 1217 (6):1-12.
[12]	Jones J T, Haegeman A, Danchin E G J, Gaur H S, Helder J, Jones M J K, Kikuchi T, Manzanilla L R, Palomares R J N, Wesemael W M L, Perry R N. 2013. Top 10 plant-parasitic nematodes in molecular plant pathology. Molecular Plant Pathology, 14 (9):946-961. doi: 10.1111/mpp.12057 pmid: 23809086
[13]	Kaloshian I, Lawn D A, Williamson V M, Miyao G, Lawn D A, Westerdahl B B, Miyao G. 1996. “Resistance-breaking”nematodes identified in California tomatoes. California Agriculture, 50 (6):18-19.
[14]	Kammerhofer N, Radakovic Z, Regis J M A, Dobrev P, Vankova R, Grundler F M W, Siddique S, Hofmann J, Wieczorek K. 2015. Role of stress-related hormones in plant defence during early infection of the cyst nematode Heterodera schachtii in Arabidopsis. The New Phytologist, 207 (3):778-789.
[15]	Li S Y, Wang H, Li F Q, Chen Z L, Li X Y, Zhu L, Wang G R, Yu J J, Huang D F, Lang Z H. 2015. The maize transcription factor EREB58 mediates the jasmonate-induced production of sesquiterpene volatiles. The Plant Journal, 84 (2):296-308.
[16]	Li Deng-hui, Zhai Ming-juan, Shi Qian-qian, Ling Jian, Mao Zhen-chuan, Yang Yu-hong, Xie Bing-yan. 2018. Comparison of the chemotaxis and the infection ability of Me3 virulent and avirulent populations of Meloidogyne incognita to host root. Acta Phytopathologica Sinica, 48 (1):128-136. (in Chinese)
	李登辉, 翟明娟, 史倩倩, 凌键, 茆振川, 杨宇红, 谢丙炎. 2018. 南方根结线虫Me3毒性与非毒性群体对寄主趋向性和侵染能力的比较. 植物病理学报, 48 (1):128-136.
[17]	Liu Y, Cao H Y, Ling J, Yang Y H, Li H, Xie B Y, Zhao J L, Mao Z C. 2023. Molecular cloning and functional analysis of the pepper resistance gene Me3 to root-knot nematode. Horticultural Plant Journal, 9 (1):133-144.
[18]	Margossian L J, Federman A D, Giovannoni J J, Fischer R L. 1988. Ethylene-regulated expression of a tomato fruit ripening gene encoding a proteinase inhibitor I with a glutamic residue at the reactive site. Proceedings of the National Academy of Sciences of the United States of America, 85 (21):8012-8016.
[19]	Mazarei M, Puthoff D P, Hart J K, Rodermel S R, Baum T J. 2002. Identification and characterization of a soybean ethylene-responsive element-binding protein gene whose mRNA expression changes during soybean cyst nematode infection. Molecular Plant Microbe Interactions, 15 (6):577-586.
[20]	Mao Zhen-chuan, Xie Bing-yan, Yang Yu-hong, Feng Dong-xin, Feng Lan-xiang, Yang Zhi-wei. 2007. Analysis of pepper Me3gene resistance to root knot nematodes by SSH. Acta Horticulturae Sinica, 34 (6):1453-1458. (in Chinese)
	茆振川, 谢丙炎, 杨宇红, 冯东昕, 冯兰香, 杨之为. 2007. 辣椒Me3基因介导抗根结线虫相关基因的SSH分析. 园艺学报, 34 (6):1453-1458.
[21]	Ornat C, Verdejo-Lucas S, Sorribas F J. 2001. A population of Meloidogyne javanica in spain virulent to the Mi resistance gene in tomato. Plant Disease, 85:271-276. doi: 10.1094/PDIS.2001.85.3.271 pmid: 30832041
[22]	Roberts P A U O, Dalmasso A, Cap G B, Castagnone-Sereno P. 1990. Resistance in Lycopersicon peruvianum to isolates of Mi gene-compatible Meloidogyne populations. Journal of Nematology, 22 (4):585-589. pmid: 19287762
[23]	Studham M E, MacIntosh G C. 2012. Phytohormone signaling pathway analysis method for comparing hormone responses in plant-pest interactions. BMC Research Notes, 5:392. pmid: 22846705
[24]	Zhang Wenyue, Mao Zhenchuan, Shen Baoming, Wang Gang, Yao Yurong, Feng Dongxin, Xie Bingyan. 2014. Overexpression of pepper CaSn gene enhances resistance to Meloidogyne incognita in transgenic plants. Acta Horticulturae Sinica, 41 (1):80-88. (in Chinese)
	张文玥, 茆振川, 沈宝明, 王刚, 姚玉荣, 冯东昕, 谢丙炎. 2014. 辣椒CaSn基因超表达增强转基因烟草对南方根结线虫的抗性. 园艺学报, 41 (1):80-88.
[25]	Zheng Jing-yuan, Liu Feng, Zhu Chun-hui. 2016. Isolation and induced expression of ethylene transcription factor gene CaERF18 from Capscium annuumm. Biotechnology Bulletin, 32 (3):87-92. (in Chinese) doi: 10.13560/j.cnki.biotech.bull.1985.2016.03.014
	郑井元, 刘峰, 朱春晖. 2016. 辣椒乙烯转录因子CaERF18的分离与诱导表达. 生物技术通报, 32 (3):87-92. doi: 10.13560/j.cnki.biotech.bull.1985.2016.03.014
[26]	Zhou Y J, Shao Y, Gao C Y, Feng H, Wei L H. 2014. The white-tip nematode,Aphelenchoides besseyi,exhibits an auxin-orientated behaviour affecting its migration and propagation. Nematology, 16 (7):837-845.
[27]	Zhu Z Q, An F Y, Feng Y, Li P P, Xue L, A M, Jiang Z Q, Kim J M, To T K, Li W, Zhang X Y, Yu Q, Dong Z, Chen W Q, Seki M, Zhou J M, Guo H W. 2011. Derepression of ethylene-stabilized transcription factors(EIN3/EIL1)mediates jasmonate and ethylene signaling synergy in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 108 (30):12539-12544.