苹果树腐烂病菌β-葡萄糖苷酶基因的克隆及表达分析

doi:10.7668/hbnxb.2017.06.012

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摘要为探究苹果树腐烂病菌β-葡萄糖苷酶基因VmGluI的序列特征及其在病菌侵染致病过程中的表达，以强致病力菌株LXS080601为材料，利用RT-PCR技术克隆基因cDNA序列，通过生物信息学软件对VmGluI编码蛋白的系统进化关系、理化性质和二级结构进行分析，采用实时荧光定量PCR技术测定VmGluI的表达量。结果显示，苹果树腐烂病菌VmGluI的开放阅读框为1 689 bp（GenBank登录号KY646110），编码562个氨基酸，蛋白分子量62.7 kDa。VmGluI编码的蛋白具有糖基水解酶1家族的保守功能域，其二级结构主要以α-螺旋和无规则卷曲为主，β-转角较少。VmGluI编码氨基酸序列与苹果和梨树腐烂病菌β-葡萄糖苷酶基因（KUI68239.1和KUI56905.1）同源性最高，分别为98.7%和87.8%，而与非植物病原真菌β-葡萄糖苷酶基因亲缘关系较远。接种苹果树腐烂病菌的富士枝条中，VmGluI显著上调表达，推测其在腐烂病菌侵染致病过程中起重要作用。离体枝条接种48 h后基因表达量开始显著升高，72 h时表达量最高为对照6.3倍；活体枝条接种12 h后，基因表达量已为对照的11.2倍，接种后48 h基因表达出现低谷，但96 h时表达量为对照的16.8倍，表明苹果树腐烂病菌在侵染离体和活体枝条过程中，VmGluI表达水平和时序变化存在明显差异。

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	李婷
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	王彩霞

关键词 ：苹果树腐烂病菌； β-葡萄糖苷酶基因；基因克隆；生物信息学分析；基因表达

Abstract：To explore the sequence characteristics of β-glucosidase gene VmGluI from Valsa mali var. mali (Vmm)and analyze its expression pattern in the process of pathogen infection,the cDNA sequence of VmGluI was amplified from the strong pathogenic strain LXS080601 by using RT-PCR technology.The phylogenetic relationship,physicochemical properties and secondary structure of the protein coded by VmGluI were analyzed by bioinformatics software.The expression levels of VmGluI in apple tiwgs(Malus domestica Borkh.Fuji) inoculated by Vmm were analyzed by Real-time quantitative PCR.The results showed that the cDNA sequence of VmGluI from Vmm was obtained and its GenBank accession number was KY646110.The open reading frame of 1 689 bp encoded 562 amino acid residues with a predicted molecular weight of 62.7 kDa.VmGluI shared characteristic conserved domains of glycosyl hydrolase family 1. α -helices and random coils were its main elements of secondary structure of VmGluI,and β-turns were less.Multiple sequence alignment and phylogenetic tree analysis showed that the encoded protein had the closest genetic relationship with β-glucosidase genes from Vmm (KUI68239.1) and V.mali var. pyri (KUI56905.1),and the similarities of amino acid sequences were 98.7% and 87.8%,respectively.However,the relationship between VmGluI and other β-glucosidase genes from non-pathogenic fungi was far away.The Real-time quantitative PCR results showed that VmGluI was significantly up-regulated in Fuji tiwgs after inoculation by Vmm.Thus,the authors speculated that VmGluI could paly an important role in the pathogenic process of Vmm.The expression level of VmGluI began to increase significantly in detached twigs at 48 h and the highest expression level was 6.3 times higher than that in the control at 72 h.However,the gene expression level increased significantly at 12 h in living twigs,which was 11.2 times higher than that in the control at 96 h.Although there was a low level of gene expression at 48 h,the amount of gene expression was 16.8 times higher than that in the control at 96 h.The results indicated that expression levels and the time course changes of VmGluI were obvious difference in detached and living twigs inoculated with Vmm.

Key words： Valsa mali var.mali ; VmGluI ; Gene cloning ; Bioinformatics analysis ; Gene expression

收稿日期: 2017-09-21

:	Q78
	S436.03

基金资助:国家自然科学基金项目（31272001；31371883）；现代农业产业技术体系建设专项资金（CARS-28）；国家农业行业公益性项目（201203034-02）；山东省“泰山学者”建设工程专项

通讯作者: 王彩霞(1977-),女,山东德州人,教授,博士,硕士生导师,主要从事果树病害研究。

作者简介: 李婷(1992-),女,山东莱芜人,在读硕士,主要从事果树病害研究。

引用本文:

李婷, 练森, 李保华, 梁文星, 王彩霞. 苹果树腐烂病菌β-葡萄糖苷酶基因的克隆及表达分析[J]. 华北农学报, 2017, 32(6): 78-84. doi: 10.7668/hbnxb.2017.06.012.
LI Ting, LIAN Sen, LI Baohua, LIANG Wenxing, WANG Caixia. Molecular Cloning and Expression Analysis of β-glucosidase Gene from Valsa mali var. mali. ACTA AGRICULTURAE BOREALI-SINICA, 2017, 32(6): 78-84. doi: 10.7668/hbnxb.2017.06.012.

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http://www.hbnxb.net/CN/10.7668/hbnxb.2017.06.012 或 http://www.hbnxb.net/CN/Y2017/V32/I6/78

[1] 陈策.苹果树腐烂病发生规律和防治研究[M].北京:中国农业科学技术出版社,2009.
[2] 李保华,王彩霞,董向丽.我国苹果主要病害研究进展与病害防治中的问题[J].植物保护,2013,39(5):46-54.
[3] 曹克强,国立耘,李保华,等.中国苹果树腐烂病发生和防治情况调查[J].植物保护,2009,35(2):114-117.
[4] 王彩霞,董向丽,张振芳,等.2011年烟台苹果产区腐烂病发病情况调查与原因分析[J].农业科学与技术:英文版,2014,38(1):83-86.
[5] 陈晓林,牛程旺,李保华,等.苹果树腐烂病菌产生细胞壁降解酶的种类及其活性分析[J].华北农学报,2012,27(2):207-212.
[6] Shi X,Yu D,Kong Q,et al.Optimizing prokaryotic expression of a xylanasegene from the apple pathogen Valsa mali var.mali[J].Advance Journal of Food Science&Technology,2015,9(9):701-705.
[7] 许春景,吴玉星,戴青青,等.苹果树腐烂病菌多聚半乳糖醛酸酶基因Vmpg7和Vmpg8 的功能[J].中国农业科学,2016,49(8):1489-1498.
[8] 臧睿.陕西苹果树腐烂病菌不同分离株生物学特性及致病性研究[D].杨凌:西北农林科技大学,2006.
[9] 史祥鹏.苹果树腐烂病菌木聚糖酶基因的克隆及功能分析[D].青岛:青岛农业大学,2016.
[10] Saloheimo M,Kujapanula J,Ylösmäki E,et al.Enzymatic properties and intracellular localization of the novel Trichoderma reesei β-Glucosidase BGLⅡ(CellA)[J].Applied & Environmental Microbiology,2002,68(9):4546-4553.
[11] Singhania R R,Patel A K,Sukumaran R K,et al.Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol production[J].Bioresource Technology,2013,127(1):500-507.
[12] Chen R Y,Baiya S,Sangkyu L,et al.Recombinant expression and characterization of the cytoplasmic rice β-glucosidase Os1BGlu4[J].PLoS One,2014,9(5):e96712.
[13] 陈斌,钱骅,闫淑珍,等.半夏内生真菌所产β-葡萄糖苷酶的酶学特性研究[J].中国野生植物资源,2016,35(2):7-9.
[14] Fang X,Shen Y,Zhao J,et al.Status and prospect of lignocellulosic bioethanol production in China[J].Bioresource Technology,2010,101(13):4814-4819.
[15] Jeng W Y,Wang N C,Lin M H,et al.Structural and functional analysis of three β-glucosidases from bacterium Clostridium cellulovorans,fungus Trichoderma reesei and termite Neotermes koshunensis[J].Journal of Structural Biology,2011,173(1):46-56.
[16] Větrovsky T,Baldrian P,Gabriel J.Extracellular enzymes of the white-rot fungus fomes fomentarius and purification of 1,4-β-glucosidase[J].Applied Biochemistry & Biotechnology,2013,169(1):100-109.
[17] 刘中美,谢梦圆,周哲敏.黑曲霉-葡萄糖苷酶的分离纯化及酶学性质[J].食品与生物技术学报,2015,34(11):1198-1202.
[18] 张大智,詹儒林,柳凤,等.杧果细菌性角斑病菌细胞壁降解酶的致病作用[J].果树学报,2016(5):585-593.
[19] 金勤,周国英,刘君昂,等.细胞壁降解酶在油茶炭疽病菌致病过程中的作用研究[J].植物保护,2017,43(3):97-102.
[20] 唐德芳,裴小琼,李晓璐,等.黑曲霉β-葡萄糖苷酶的筛选,克隆及表达[J].应用与环境生物学报,2009,15(3):423-426.
[21] Li L,Ji Q L,Hao J F,et al.Cloning and expression of β-Glucosidase gene from Bacillus pumilus[J].Applied Mechanics & Materials,2013,301:1271-1274.
[22] Waksman G.Molecular cloning of a beta-glucosidase-encoding gene from Sclerotinia sclerotiorum,by expression in Escherichia coli[J].Current Genetics,1989,15(4):295-297.
[23] Yin Z,Ke X,Huang D,et al.Validation of reference genes for gene expression analysis in Valsamali var.mali,using Real-time quantitative PCR[J].World Journal of Microbiology & Biotechnology,2013,29(9):1563-1571.
[24] Zhang Y,Shi X P,Li B H,et al.Salicylic acid confers enhanced resistance to Glomerella leaf spot in apple[J].Plant Physiology and Biochemistry,2016,106:64-72.
[25] 李玉婷,赵奇,房冰,等.禾谷缢管蚜热激蛋白Hsp90 基因的克隆和表达分析[J].植物保护学报,2017,44(1):16-23.
[26] Kenneth J,Livak T D.Analysis of relative gene expression data using Real-time quantitative PCR and the 2^-△△CT method[J].Method,2001,25(4):402-408.
[27] 房伟,方泽民,刘娟娟,等.新型海洋微生物β-葡萄糖苷酶基因的克隆,表达及重组酶性质[J].生物工程学报,2009,25(12):1914-1920.
[28] 陈辉,冯志勇,冯川,等.斑玉蕈β-葡萄糖苷酶基因的序列分析及皂苷对基因表达的影响[J].食用菌学报,2017,24(1):1-6.
[29] 陈相永,陈捷胤,肖红利,等.植物病原真菌寄生性与分泌蛋白组CAZymes的比较分析[J].植物病理学报,2014,44(2):163-172.
[30] 王彩霞,陈晓林,李保华.腐烂病菌侵染对苹果愈伤组织防御酶活性及丙二醛含量的影响[J].植物生理学报,2014(7):909-916.
[31] 雍道敬,李保华,王彩霞.苹果树腐烂病菌不同致病力菌株对苹果的诱导效应[J].植物生理学报,2014(6):810-816.
[32] Bhat S,Goodenough P W,Bhat M K,et al.Isolation of four major subunits from Clostridium thermocellum cellulosome and their synergism in the hydrolysis of crystalline cellulose[J].International Journal of Biological Macromolecules,1994,16(6):335-342.