miR396b负调控茄子对黄萎病的防御反应

doi:10.16420/j.issn.0513-353x.2021-0569

摘要/Abstract

摘要：

为了研究miR396b在茄子抗黄萎病中的功能,以茄子栽培品种‘苏琦1号’为试验材料,克隆pri-miR396b基因并构建表达载体,通过农杆菌介导的遗传转化获得miR396b转基因茄子植株。在受大丽轮枝菌侵染的茄子植株中,miR396b受诱导呈下调表达。抗病性分析表明,与野生型茄子相比,miR396b过表达株系对大丽轮枝菌侵染更加敏感、病情指数为75.2,而miR396b反义抑制株系病症较轻,病情指数在16.2 ~ 25.3之间。病原菌ITS定量分析显示,miR396b过表达株系植株维管束组织中大丽轮枝菌含量较对照显著增加,而miR396b反义抑制株系植株中显著减少。抗氧化酶活性分析显示,miR396b过表达株系中超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）酶活性低于野生型茄子植株,而反义抑制株系则高于对照。以上结果表明,miR396b在茄子对黄萎病防御反应中起负调控作用。

关键词: 茄子, miR396b, 黄萎病, 功能分析

Abstract:

In order to identify the function of miR396b in eggplant resistance to verticillium wilt,pri-miR396b gene was cloned from eggplant cultivar‘Suqi 1’and miR396b transgenic eggplant lines were obtained through Agrobacterium-mediated transformation. In infected eggplant,the expression level of miR396b was down-regulated by Verticillium dahliae induction. The disease resistance analysis showed that miR396b overexpression line was more sensitive to Verticillium dahliae infection compared with the control with disease index of 75.2,while miR396b antisense lines showed more resistance with disease index of 16.2-25.3. ITS quantitative analysis showed that the content of Verticillium dahliae in the vascular tissues of miR396b overexpression plants was significantly increased compared with the wild- type eggplant,while that in miR396b antisense plants was significantly reduced. Antioxidant enzyme activity analysis showed that the activities of SOD,POD and CAT in miR396b overexpression line were lower than those in the wild-type eggplant,while they were higher in antisense lines than those in the wild-type eggplant. These results showed that miR396b might play a negative role in eggplant defense response against Verticillium wilt.

Key words: eggplant, miR396b, verticillium wilt, functional analysis

中图分类号:

S641.1

林元秘, 朱文姣, 陈敏, 薛春梅, 晋芳宇, 朱羽平, 蒋欣玥, 叶凌峰, 倪姝南伶, 杨清. miR396b负调控茄子对黄萎病的防御反应[J]. 园艺学报, 2022, 49(8): 1713-1722.

LIN Yuanmi, ZHU Wenjiao, CHEN Min, XUE Chunmei, JIN Fangyu, ZHU Yuping, JIANG Xinyue, YE Lingfeng, NI Shunanling, YANG Qing. Mir396b Negatively Regulates Eggplant Defense Response to Verticillium Wilt[J]. Acta Horticulturae Sinica, 2022, 49(8): 1713-1722.

图/表 9

表1 试验中使用的引物

Table 1 Primers used in this study

名称 Name	序列（5′-3′） Sequence	名称 Name	序列（5′-3′） Sequence
pri-miR396b-F	GAAGATCTCGAAAGAATATGTCCCAAAGTG	ITS-R	CGCCTGCGGGACTCCGATGCGAGCTGTAAC
pri-miR396b-R	GGACTAGTTGGCTGTTTGATGACGATGAC	α-tublin-F	CCACCATCAAGACTAAGCG
pri-miR396b-anti-F	GGACTAGTCGAAAGAATATGTCCCAAAGTG	α-tublin-R	AGAAGACCTCAGCAACACTC
pri-miR396b-anti-R	GAAGATCTTGGCTGTTTGATGACGATGAC	qRT-SmGRF2-F	GATCTACAAGCATTTCGTGGCA
miR396b-R	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAAAGTTC	qRT-SmGRF2-R	ATCAGTGTTGCCAGCGAAAC
miR396b-F	GAGTGTTCCACAGCTTTCTT	SmEF1a-F	CCACACTTCTCATATTGCTGTCA
Uni-R	GTGCAGGGTCCGAGGT	SmEF1a-R	ACCAGCATCACCATTCTTCAAAA
U6-F	CGGGGACATCCGATAAAA	Veri-pri-396b-F	GGCTTGATTATCTTCAAGTACTTCC
U6-R	TTGGACCATTTCTCGATTTG	Veri-pri-396b-anti-F	GGAAGTACTTGAAGATAATCAAGCC
ITS-F	CCGCCGGTCCATCAGTCTCTCTGTTTATAC	Veri-1305-R	CCGTATGTTGCATCACCTTCAC

图1 大丽轮枝菌胁迫下茄子miR396b的表达

Fig. 1 Expression analysis of eggplant miR396b under the stress of Verticillium dahliae * P﹤0.05,** P﹤0.01.

图2 pri-miR396b的克隆和载体构建 A：pri-miR396b基因PCR产物的琼脂糖凝胶电泳;B：miR396b过表达载体的构建,Ⅰ-酶切验证,Ⅱ-大肠杆菌菌液PCR验证,Ⅲ-农杆菌菌液PCR验证。

Fig. 2 Cloning of pri-miR396b and vector construction A：Agarose gel electrophoresis of PCR products of pri-miR396b gene. B：Construction of miR396b overexpression vector,Ⅰ-Enzyme digestion verification,Ⅱ-PCR identification of recombinant plasmid in transformed E. coli,Ⅲ-PCR identification of recombinant plasmid in transformed Agrobacterium.

图3 转基因茄子植株的获得 A：miR396b过表达植株的再生;B：miR396b反义抑制植株的再生。

Fig. 3 Regeneration of transgenic eggplant plants A：Regeneration of miR396b overexpression plants;B：Regeneration of miR396b antisense plants.

图4 茄子miR396b过表达植株（A）、反义抑制植株（B）的PCR鉴定和miR396b表达（C）的qRT-PCR分析 M：DL2000marker;WT：野生型植株;P：质粒对照;1305：转1305质粒植株;OE-1：miR396b 过表达植株;AE-1,AE-2,AE-3：miR396b反义抑制植株。下同。

Fig. 4 PCR identification of miR396b-overexpressing eggplants（A）and antisense eggplants（B）,and qRT-PCR analysis of miR396b（C） M：DL2000marker;WT：wild-type plants;P：positive plasmid control;1305：plants transformed with blank 1305 plasmid;OE-1：miR396b overexpression plants;AE-1,AE-2,AE-3：miR396b antisense plants. The same below.

图5 大丽轮枝菌侵染后转基因茄子的表型和病情指数

Fig. 5 Analysis of the phenotype and disease index of transgenic eggplants after Verticillium dahliae inoculation

图6 miR396b转基因植株中大丽轮枝菌繁殖量（ITS）荧光定量

Fig. 6 qRT-PCR analysis of fungal colonization of Verticillium dahliae internal transcribed spacer（ITS）transcript levels in transgenic eggplant of miR396b

图7 miR396b转基因茄子植株抗氧化物酶活性分析

Fig. 7 Analysis on the activities of antioxidant enzymes in miR396b transgenic eggplant

图8 茄子中miR396b靶基因SmGRF2在大丽轮枝菌（Verticillium dahliae）胁迫下的表达

Fig. 8 Expression analysis of miR396b target SmGRF2 under Verticillium dahliae stress

参考文献 23

[1]	Cao D, Wang J, Ju Z, Liu Q, Li S, Tian H. 2016. Regulations on growth and development in tomato cotyledon,flower and fruit via destruction of miR396 with short tandem target mimic. Plant Science, 247:1-12. doi: 10.1016/j.plantsci.2016.02.012 URL
[2]	Cuperus J T, Fahlgren N, Carrington J C. 2011. Evolution and functional diversification of miRNA genes. Plant Cell, 23 (2):431-442. doi: 10.1105/tpc.110.082784 URL
[3]	Chen L, Luan Y, Zhai J. 2015. Sp-miR396a-5p acts as a stress-responsive genes regulator by conferring tolerance to abiotic stresses and susceptibility to Phytophthora nicotianae infection in transgenic tobacco. Plant Cell Reports, 34 (12):2013-2025. doi: 10.1007/s00299-015-1847-0 pmid: 26242449
[4]	Häffner E, Karlovsky P, Splivallo R, Traczewska A, Diederichsen E. 2014. ERECTA,salicylic acid,abscisic acid,and jasmonic acid modulate quantitative disease resistance of Arabidopsis thaliana to Verticillium longisporum. BMC Plant Biology, 14 (1):85. doi: 10.1186/1471-2229-14-85 URL
[5]	Llave C. 2002. Cleavage of scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science, 297 (5589):2053. doi: 10.1126/science.1076311 URL
[6]	Li A L, Wen Z, Yang K, Wen X P. 2019. Conserved miR396b-GRF regulation is involved in abiotic stress responses in pitaya(Hylocereus polyrhizus). International Journal of Molecular Sciences, 20 (10):2501. doi: 10.3390/ijms20102501 URL
[7]	Li He-sheng. 2000. Principles and techniques of plant physiological and biochemical experiments. Beijing: Higher Education Press:164-165.
	李合生. 2000. 植物生理生化实验原理和技术. 北京: 高等教育出版社:164-165.
[8]	Liu S P, Zhu Y P, Xie C, Jue D W, Hong Y B, Chen M, Hubdar A K, Yang Q. 2012. Transgenic potato plants expressing StoVe1 exhibit enhanced resistance to Verticillium dahliae. Plant Mol Biol Rep, 30:1032-1039. doi: 10.1007/s11105-012-0413-y URL
[9]	Liu W, Zhou Y, Li X, Wang X, Dong Y, Wang N. 2017. Tissue-specific regulation of gma-miR396 family on coordinating development and low water availability responses. Frontiers in Plant Science, 8:1-14.
[10]	Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method. Methods, 25 (4):402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[11]	Jones-Rhoades M W, Bartel D P, Bonnie B. 2006. MicroRNAs and their regulatory roles in plants. Annual Review of Plant Biology, 57 (1):19-53. doi: 10.1146/annurev.arplant.57.032905.105218 URL
[12]	Kim J H, Kende H. 2004. A transcriptional coactivator,AtGIF1,is involved in regulating leaf growth and morphology in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 101 (36):13374-13379.
[13]	Kim J S, Mizoi J, Kidokoro S, Maruyama K, Nakajima J, Nakashima K, Mitsuda N, Takiguchi Y, Ohmetakagi M, Kondou Y. 2012. Arabidopsis GROWTH-REGULATING FACTOR7 functions as a transcriptional repressor of abscisic acid and osmotic stress esponsive genes,including DREB2A. Plant Cell, 24 (8):3393-3405. doi: 10.1105/tpc.112.100933 URL
[14]	Mittler R. 2002. Oxidative stress,antioxidants and stress tolerance. Trends Plant Sci, 7 (9):405-410. doi: 10.1016/s1360-1385(02)02312-9 pmid: 12234732
[15]	Mu X Y, Liu X R, Cai J H, Zhu W J, Wang Z, Yang Q, You X. 2018. MiR395 overexpression increases eggplant sensibility to Verticillium dahliae infection. Russian Journal of Plant Physiology, 65 (2):203-210. doi: 10.1134/S1021443718020164 URL
[16]	Pantelides I S, Tjamos S E, Paplomatas E J. 2010. Ethylene perception via ETR1 is required in Arabidopsis infection by Verticillium dahliae. Mol Plant Pathol, 11:191-202. doi: 10.1111/j.1364-3703.2009.00592.x pmid: 20447269
[17]	Soto-Suárez M, Baldrich P, Weigel D, Rubio-Somoza I, San S B. 2017. The Arabidopsis miR396 mediates pathogen-associated molecular pattern-triggered immune responses against fungal pathogens. Scientific Reports, 7 (1):1-14. doi: 10.1038/s41598-016-0028-x URL
[18]	Wang Jun-hong. 2020. Current status and countermeasures of eggplant verticillium wilt. China Fruit and Vegetables, 40 (3):98-100. (in Chinese)
	王俊红. 2020. 茄子黄萎病的防治现状及对策. 中国果菜, 40 (3):98-100.
[19]	Wei Yanhong, Liu Zhen, Li Ke, Meng Yuan, Wang Hui, Mao Jiangping, Ma Doudou, Li Shaohuan, Ma Juanjuan, Lu Xian, Zhang Dong. 2020. Genome-wide identification and expression analysis of miR396 family during adventitious root development in apple. Acta Horticulturae Sinica, 47 (7):1237-1252. (in Chinese)
	韦燕红, 刘桢, 李珂, 孟媛, 汪蕙, 毛江萍, 马豆豆, 李少欢, 马娟娟, 卢显, 张东. 2021. 苹果miR396家族鉴定及在不定根发育过程中的表达分析. 园艺学报, 47 (7):1237-1252.
[20]	Yang L, Jue W D, Li W, Zhang R, Chen M, Yang Q. 2013. Identification of miRNA from eggplant(Solanum melongena L.)by small RNA deep sequencing and their response to Verticillium dahliae infection. PloS ONE, 8 (8):e72840.
[21]	Yu Y, Sun F, Chen N, Sun G, Wang C Y. 2020. MiR396 regulatory network and its expression during grain development in wheat. Protoplasma, 258 (1):103-113. doi: 10.1007/s00709-020-01556-3 URL
[22]	Zhang Xian-zheng. 1994. Experimental technology of plant physiology. Shenyang: Liaoning Science and Technology Press:99-100. (in Chinese)
	张宪政. 1994. 植物生理学实验技术. 沈阳: 辽宁科学技术出版社:99-100.
[23]	Zou Qi. 2000. Plant physiology experiment guide. Beijing: China Agricultural Press:161-162. (in Chinese)
	邹琦. 2000. 植物生理学实验指导. 北京: 中国农业出版社:161-162.