球炭疽菌果胶酸裂解酶基因CcPL20552克隆及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-1075

摘要/Abstract

摘要：

目的基于RNA-Seq数据，果胶酸裂解酶候选效应蛋白基因CcPL20552为球炭疽菌致病过程中致病基因之一，且具有效应蛋白的特征。研究CcPL20552基因信号肽功能及其蛋白表达特点，为揭示其基因功能提供依据，也为揭示球炭疽菌的分子致病机制奠定基础。方法以球炭疽菌侵染的马铃薯茎秆cDNA为模板，克隆CcPL20552基因CDS编码区全长以及去除信号肽（-SP）序列，构建带有His标签的全长融合表达载体pET28a-PL20552和去信号肽融合表达载体pET28a-SP 20552，于大肠杆菌BL21（DE3）中通过自诱导和IPTG诱导表达，纯化蛋白后测定其浓度及活性。结果 CcPL20552基因编码区全长为720 bp，蛋白质预测分子量为24.48 kD，且包含一段由18个氨基酸组成的信号肽序列，蛋白主要以包涵体形式存在于裂解后的菌体沉淀中，通过变复性并利用His标签纯化出目的蛋白，相同诱导条件下全长蛋白表达量显著高于去除信号肽蛋白表达量，100 mmol/L浓度的咪唑洗脱液为最适洗脱浓度，全长和去信号肽蛋白浓度分别为224.25和174.02 μg/mL，活性分别为0.634 6和0.952 0 U/mL。结论 CcPL20552基因可通过自诱导和IPTG诱导表达，温度越低蛋白表达量越高，信号肽对该蛋白表达影响较大，且信号肽具有分泌功能。

关键词: 球炭疽菌, 果胶酸裂解酶基因, 克隆, 信号肽, 原核表达

Abstract:

Objective Based on RNA-Seq data, the candidate effector protein gene CcPL20552 of pectate lyase is one of the pathogenic genes in the pathogenic process of Colletotrichum coccodes, and it has the characteristics of effector proteins. The study provides a basis for revealing the gene function and protein expression characteristics of CcPL20552 by studying its signal peptide function and also lays the foundation for revealing the molecular pathogenic mechanism of C. coccodes. Method Using potato stem cDNA infected with C. coccodes as a template, the full-length CDS coding region and signal peptide (-SP) sequence of CcPL20552 gene were cloned. Full-length fusion expression vector pET28a-PL20552 with His tag and fusion expression vector pET28a-SP 20552 without signal peptide were constructed, and expressed in Escherichia coli BL21 (DE3) through self-induction and IPTG induction expression, then the concentration and activity of purified protein were measured. Result The full-length coding region of CcPL20552 gene is 720 bp, the predicted molecular weight of the protein is 24.48 kD and it contains a signal peptide sequence consisting of 18 amino acids. But proteins mainly exist in the form of inclusion bodies in the sediment of lysed bacterial cells, the target protein is purified through denaturation and His labeling. Under the same induction conditions, the expression of full-length protein was significantly higher than that of protein without signal peptide. The optimal elution concentration is 100 mmol/L imidazole eluent.The full-length and designal peptide protein concentrations were 224.25 and 174.02 μg/mL, respectively, and the activities were 0.634 6 and 0.952 0 U/mL. Conclusion The results indicate that the CcPL20552 gene can be expressed through self induction and IPTG induction and the lower the temperature, the higher the protein expression level. The signal peptide has a greater impact on the protein expression, and the signal peptide has a secretory function.

Key words: Colletotrichum coccodes, pectate lyase gene, clone, signal peptide, prokaryotic expression

王婷, 王一丹, 任姝锦, 马婷, 金梦军, 杨成德. 球炭疽菌果胶酸裂解酶基因CcPL20552克隆及表达分析[J]. 生物技术通报, 2025, 41(5): 290-299.

WANG Ting, WANG Yi-dan, REN Shu-jin, MA Ting, JIN Meng-jun, YANG Cheng-de. Cloning and Expression Analysis of Pectate Lyase Gene CcPL20552 from Colletotrichum coccodes[J]. Biotechnology Bulletin, 2025, 41(5): 290-299.

图/表 7

图1 克隆及菌落PCR验证M：Marker （DL=2000）； A：（1： PL20552； 2： SP 20552）；B： PL20552菌落PCR验证；C： SP 20552菌落PCR验证

Fig. 1 PCR verification for cloning and colonyM: Marker (DL=2000)). A:(1: PL20552. 2: SP 20552). B: PL20552 colony PCR validation.C: SP 20552 colony PCR validation

图2 信号肽序列导入酵母菌YTK12中菌落PCR验证A： pSUC2-SP20552菌落PCR验证； B： PSUC2菌落PCR验证； C：Avr1b菌落PCR验证

Fig. 2 PCR validation of colony after introduction of signal peptide sequence into yeast YTK12A: pSUC2-SP20552 colony PCR validation. B: PSUC2 colony PCR validation. C:Avr1b colony PCR validation

图3 TTC显色反应

Fig. 3 TTC color reaction

图4 目的基因和原核表达载体双酶切及菌落PCR验证A：目的基因和原核表达载体双酶切，M：Marker （DL=10 000）， 1： PL20552， 2： SP 20552， 3： pET28a， 4： pET28a未酶切；B： pET28a-PL20552菌落PCR验证；C： pET28a-SP 20552菌落PCR验证

Fig. 4 Dual enzyme digestion of target gene and prokaryotic expression vector and colony PCR validationA: Dual enzyme digestion of target gene and prokaryotic expression vector, M:Marker (DL=10 000), 1: PL20552, 2: SP 20552, 3: pET28a, 4: pET28a unenzymatically digested; B: pET28a-PL20552 colony PCR validation; C: pET28a-SP 20552 colony PCR validation

图5 重组蛋白SDS-PAGE分析A：自诱导破碎后上清和沉淀；M：蛋白marker； 1：pET28a诱导上清；2：pET28a-SP20552 16 ℃诱导沉淀；3：pET28a-SP20552 16 ℃诱导上清；4：pET28a-SP20552 37 ℃诱导沉淀；5：pET28a-SP20552 37 ℃诱导上清；6：pET28a-PL20552 16 ℃诱导沉淀；7：pET28a-PL20552 16 ℃诱导上清；8：pET28a-PL20552 37 ℃诱导沉淀；9：pET28a-PL20552 37 ℃诱导上清。B：28 ℃诱导破碎后上清和沉淀；M：蛋白marker；1：pET28a诱导上清；2：pET28a-SP20552 未诱导沉淀；3：pET28a-SP20552未诱导上清；4：pET28a-SP20552诱导沉淀；5：pET28a-SP20552诱导上清；6：pET28a-PL20552未诱导沉淀；7：pET28a-PL20552未诱导上清；8：pET28a-PL20552诱导沉淀；9：pET28a-PL20552诱导上清。C：37 ℃诱导破碎后上清和沉淀； M：蛋白marker； 1：pET28a诱导沉淀，2：pET28a诱导上清； 3：pET28a-SP20552未诱导沉淀，4：pET28a-SP20552未诱导上清， 5：pET28a-SP20552诱导沉淀，6：pET28a-SP20552诱导上清；7：pET28a-PL20552未诱导沉淀，8：pET28a-PL20552未诱导上清，9：pET28a-PL20552诱导沉淀，10：pET28a-PL20552诱导上清

Fig. 5 SDS-PAGE analysis of recombinant proteinA: Precipitation and supernatant after self induced fragmentation; M: protein marker;1:pET28a induced supernatant; 2: pET28a-SP20552 16 ℃ induced precipitation; 3: pET28aSP20552 16 ℃ induced supernatant; 4: pET28a-SP20552 37 ℃ induced precipitation; 5: pET28a-SP20552 37 ℃ induced supernatant; 6: pET28a-PL20552 16 ℃ induced precipitation; 7: pET28a-PL20552 16 ℃ induced supernatant; 8: pET28a-PL20552 37 ℃ induced precipitation; 9: pET28a-PL20552 37 ℃ induced supernatant. B: The supernatant and precipitate after induced fragmentation at 28 ℃; M: protein Marker; 1: pET28a induced supernatant; 2: pET28a-SP20552 uninduced precipitation; 3: pET28a-SP20552 uninduced supernatant; 4: pET28a-SP20552 induced precipitation; 5: pET28a-SP20552 induced supernatant; 6: pET28a-PL20552 uninduced precipitation; 7: pET28a-PL20552 uninduced supernatant; 8: pET28a-PL20552 induced precipitation; 9: pET28a-PL20552 induced supernatant. C: The supernatant and precipitate after induced fragmentation at 37 ℃; M: protein marker, 1: pET28a induced precipitation; 2: pET28a induced supernatant; 3: pET28a-SP20552 uninduced precipitation; 4: pET28a-SP20552 uninduced supernatant; 5: pET28a-SP20552 induced precipitation; 6: pET28a-SP20552 induced supernatant; 7: pET28a-PL20552 uninduced precipitation; 8: pET28a-PL20552 uninduced supernatant; 9: pET28a-PL20552 induced precipitation; 10: pET28a-PL20552 induced supernatant

图6 重组蛋白纯化后SDS-PAGE分析A：pET28a-PL20552重组蛋白纯化；B：pET28a-SP20552重组蛋白纯化，泳道1-8分别是洗脱1-8次纯化蛋白洗脱液，M：蛋白marker

Fig. 6 SDS-PAGE analysis of purified recombinant proteinA: Purification of recombinant protein pET28a-PL20552. B: Purification of recombinant protein pET28a-SP20552; 1-8: elution of 1-8 times purified protein eluate; M: protein marker

图7 BSA（A）和D-半乳糖醛酸（B）标准曲线

Fig. 7 Standard curve of BSA(A) and D-galacturonic acid(B)

参考文献 31

1	孙雪梅, 赵吉霞, 张文妹, 等.有机肥等氮替代化肥对马铃薯氮素吸收及产量的影响 [J].土壤通报, 2025, 56(1): 157-170.
	Sun XM, Zhao JX, Zhang WM, et al. The effect of organic fertilizer and other nitrogen substitutes for chemical fertilizers on nitrogen absorption and yield of potatoes [J]. Chinese Journal of Soil Science, 2025, 56(1): 157-170.
2	Wang ZJ, Liu H, Zeng FK, et al. Potato processing industry in China: current scenario, future trends and global impact [J]. Potato Res, 2023, 66(2): 543-562.
3	徐进, 朱杰华, 杨艳丽, 等. 中国马铃薯病虫害发生情况与农药使用现状 [J]. 中国农业科学, 2019, 52(16): 2800-2808.
	Xu J, Zhu JH, Yang YL, et al. Status of major diseases and insect pests of potato and pesticide usage in China [J]. Sci Agric Sin, 2019, 52(16): 2800-2808.
4	崔月贞, 杨小利, 杨成德, 等. 拮抗马铃薯晚疫病菌的高寒草地牧草内生细菌的鉴定及其生物功能测定 [J]. 植物保护学报, 2016, 43(5): 789-795.
	Cui YZ, Yang XL, Yang CD, et al. Identification and determination of biological functions of endophytic bacteria from alpine pasture against Phytophthora infestans [J]. J Plant Prot, 2016, 43(5): 789-795.
5	Yarmeeva M, Kutuzova I, Kurchaev M, et al. Colletotrichum species on cultivated Solanaceae crops in Russia [J]. Agriculture, 2023, 13(3): 511.
6	陈淑琴, 王生荣. 放线菌DX23对马铃薯炭疽病菌的拮抗作用及其鉴定 [J]. 甘肃农业大学学报, 2015, 50(2): 95-99.
	Chen SQ, Wang SR. Antagonism of actinomycete DX23 to Colletotrichum coccodes of potato and its identification [J]. J Gansu Agric Univ, 2015, 50(2): 95-99.
7	刘治会, 杨成德, 金梦军, 等. 枯草芽胞杆菌262XY2'固体发酵条件优化及对马铃薯炭疽病的防治效果 [J]. 中国生物防治学报, 2019, 35(4): 586-596.
	Liu ZH, Yang CD, Jin MJ, et al. Optimization of solid fermentation conditions of Bacillus subtilis 262XY2' and its control effect on Colletotrichum coccodes [J]. Chin J Biol Contr, 2019, 35(4): 586-596.
8	金梦军, 杨成德, 李统华, 等. 细胞壁降解酶在球刺盘孢侵染过程中的作用 [J]. 植物保护, 2024, 50(3): 88-98, 110.
	Jin MJ, Yang CD, Li TH, et al. The role of cell wall-degrading enzymes of Colletotrichum coccodes during its infection [J]. Plant Prot, 2024, 50(3): 88-98, 110.
9	Tian ZM, Wu YH, Guo HJ, et al. First report of potato black dot caused by Colletotrichum coccodes in Hebei Province of China [J]. Plant Dis, 2019, 103(10): 2669.
10	高馨梅, 邵晨曦, 梁英梅, 等. 山田胶锈菌效应蛋白GyHGSRE1的功能初探 [J/OL]. 南京林业大学学报: 自然科学版, 2024: 1-10. .
	Gao XM, Shao CX, Liang YM, et al. Preliminary study on the function of GyHGSRE 1, an effector protein of Puccinia Yamada [J/OL]. China Ind Econ, 2024: 1-10. .
11	Yuan MH, Ngou BPM, Ding PT, et al. PTI-ETI crosstalk: an integrative view of plant immunity [J]. Curr Opin Plant Biol, 2021, 62: 102030.
12	汤敬诚, 李睿, 刘文波, 等. Neopestalotiopsis thailandica全基因组分泌蛋白及其效应因子的预测分析 [J/OL]. 分子植物育种, 2024: 1-11. .
	Tang JC, Li R, Liu WB, et al. Prediction and analysis of secreted proteins and their effecting factors in Neopestalotiopsis thailandica whole genome [J/OL]. China Ind Econ, 2024: 1-11. .
13	吴佳椰露, 傅艺炜, 包崇来, 等. 茄腐镰孢菌分泌蛋白与效应子的预测分析 [J/OL]. 植物病理学报, 2024: 1-17.
	Wu JYL, Fu YW, Bao CL, et al. Prediction and analysis of secreted proteins and effectors of Fusarium solani [J/OL]. Acta Phytopathologica Sinica, 2024: 1-17.
14	尹潇潇, 张芷菡, 颜绣莲, 等. 多个稻曲病菌效应因子的信号肽验证和表达分析 [J]. 中国水稻科学, 2024, 38(3): 256-265.
	Yin XX, Zhang ZH, Yan XL, et al. Signal peptide validation and expression analysis of multiple effectors from Ustilaginoidea virens [J]. Chin J Rice Sci, 2024, 38(3): 256-265.
15	Owji H, Nezafat N, Negahdaripour M, et al. A comprehensive review of signal peptides: Structure, roles, and applications [J]. Eur J Cell Biol, 2018, 97(6): 422-441.
16	侯杨威, 王鑫源, 杨林林, 等. 地黄轮纹病病原菌的细胞壁降解酶活性测定及致病性分析 [J]. 北方园艺, 2022(19): 106-113.
	Hou YW, Wang XY, Yang LL, et al. Determination of cell wall-degrading enzyme activity and pathogenicity analysis of rotunda pathogens [J]. North Hortic, 2022(19): 106-113.
17	Ramezani Y, Taheri P, Mamarabadi M. Identification of Alternaria spp. associated with tomato early blight in Iran and investigating some of their virulence factors [J]. J Plant Pathol, 2019, 101(3): 647-659.
18	Jia YJ, Feng BZ, Sun WX, et al. Polygalacturonase, pectate lyase and pectin methylesterase activity in pathogenic strains of Phytophthora capsici incubated under different conditions [J]. J Phytopathol, 2009, 157(10): 585-591.
19	Meir S, Amsellem Z, Al-Ahmad H, et al. Transforming a NEP1 toxin gene into two Fusarium spp. to enhance mycoherbicide activity on Orobanche—failure and success [J]. Pest Manag Sci, 2009, 65(5): 588-595.
20	Ben-Daniel BH, Bar-Zvi D, Tsror Lahkim L. Pectate lyase affects pathogenicity in natural isolates of Colletotrichum coccodes and in pelA gene-disrupted and gene-overexpressing mutant lines [J]. Mol Plant Pathol, 2012, 13(2): 187-197.
21	Chen J, Luo H, Tao M, et al. Quantitation of nucleoprotein complexes by UV absorbance and Bradford assay [J]. Biophys Rep, 2021, 7(6): 429-436.
22	郑东影, 梅婕, 陈玮, 等. DNS法与菲林试剂法测定酿酒大曲糖化酶活力的比较分析 [J]. 酿酒, 2023, 50(5): 134-138.
	Zheng DY, Mei J, Chen W, et al. Comparison of the DNS method and the film reagent mmethod for determination of daqu glucoamylase activity [J]. Liquor Mak, 2023, 50(5): 134-138.
23	谢茂芳, 薛保国, 吴坤. 黑曲霉果胶裂解酶基因的克隆与原核表达 [J]. 河南农业科学, 2013, 42(1): 82-85.
	Xie MF, Xue BG, Wu K. Cloning and prokaryotic expression of pectin lyase gene from Aspergillus niger [J]. J Henan Agric Sci, 2013, 42(1): 82-85.
24	Zhao QX, Yuan S, Zhang YL, et al. Expression, purification and characterization of pectate lyase A from Aspergillus nidulans in Escherichia coli [J]. World J Microbiol Biotechnol, 2007, 23(8): 1057-1064.
25	赵合美, 朱原, 杨雪, 等. 高地芽孢杆菌FY1果胶酸裂解酶基因BaPel的克隆及表达分析 [J/OL]. 吉林农业大学学报, 2022: 1-7.
	Zhao HM, Zhu Y, Yang X, et al. Cloning and Expression Analysis of BaPel Gene for Pectin Acid lyase in High Altitude Bacillus FY1 [J/OL]. Journal of Jilin Agricultural University, 2024:1-7.
26	苗朝悦, 杜乐, 王佳琦, 等. 重组蛋白质在大肠杆菌体系中的可溶性表达策略 [J]. 中国生物工程杂志, 2023, 43(9): 33-45.
	Miao ZY, Du L, Wang JQ, et al. Soluble expression strategies for production of recombinant proteins in Escherichia coli [J]. China Biotechnol, 2023, 43(9): 33-45.
27	Pliego-Arreaga R, Regalado C, Amaro-Reyes A, et al. Lactose-induced expression of recombinant turnip peroxidase in Escherichia coli [J]. Rev Mex De Ing Quim, 2013, 12: 505-511.
28	王双, 李孟建, 黄凤兰, 等. 稻瘟菌角质酶MGG_09100的生物信息学分析及其原核表达 [J]. 中国生物制品学杂志, 2020(9): 1003-1008.
	Wang S, Li MJ, Huang FL, et al. Bioinformatics and prokaryotic expression of cutinase MGG_09100 of Magnaporthe oryzae [J]. Chin J Biol, 2020(9): 1003-1008.
29	张树军, 狄建军, 张国文. 大肠杆菌yfiF基因原核表达系统构建、表达条件优化及蛋白纯化 [J]. 生物技术, 2016, 26(3): 229-233.
	Zhang SJ, Di JJ, Zhang GW. Construction of the prokaryotic expression system of yfiF gene in E. coli, optimization of its induced expression condition and purification of the protein [J]. Biotechnology, 2016, 26(3): 229-233.
30	赵庆新, 王鑫, 张宇玲, 等. 番茄(Solanum lycopersicum)果胶酸裂解酶P56在大肠杆菌中的重组表达 [J]. 武汉植物学研究, 2007, 25(6): 539-543.
	Zhao QX, Wang X, Zhang YL, et al. Expression of P56 pectate lyase from Solanum lycopersicum in Escherichia coli [J]. Plant Sci J, 2007, 25(6): 539-543.
31	Grams N, Komar H, Jainchill D, et al. Comparative expression analysis of Phytophthora sojae polysaccharide lyase family 3 (pectate lyase) genes during infection of the soybean Glycine max [J]. Phytopathol Res, 2019, 1(1): 15.

[1]	刘鑫, 王嘉雯, 李进伟, 牟策, 杨盼盼, 明军, 徐雷锋. 兰州百合三个LdBBXs基因的克隆与表达分析[J]. 生物技术通报, 2025, 41(5): 186-196.
[2]	班秋艳, 赵鑫月, 迟文静, 黎俊生, 王琼, 夏瑶, 梁丽云, 贺巍, 李叶云, 赵广山. 茶树光敏色素互作因子CsPIF3a的克隆及其与光温逆境的响应[J]. 生物技术通报, 2025, 41(4): 256-265.
[3]	彭婷, 林颖, 谭圆圆, 饶英, 黄覃, 张文娥, 汪波, 田瑞丰, 刘国锋. 多星韭AwANSs基因的克隆与表达分析[J]. 生物技术通报, 2025, 41(3): 230-239.
[4]	李欣芃, 张武汉, 张莉, 舒服, 何强, 郭杨, 邓华凤, 王悦, 孙平勇. γ射线诱变创制水稻突变体及其分子鉴定[J]. 生物技术通报, 2025, 41(3): 35-43.
[5]	许圆梦, 毛娇, 王梦瑶, 王数, 任江陵, 刘宇涵, 刘思辰, 乔治军, 王瑞云, 曹晓宁. 糜子PmDEP1和PmEP3基因的克隆与表达特征分析[J]. 生物技术通报, 2025, 41(2): 150-162.
[6]	焦小雨, 吴琼, 刘丹丹, 孙明慧, 阮旭, 王雷刚, 王文杰. 茶树CsWAK8克隆及其在响应冷胁迫过程中的功能分析[J]. 生物技术通报, 2025, 41(2): 210-220.
[7]	向春繁, 李勒松, 王娟, 梁艳丽, 杨生超, 栗孟飞, 赵艳. 当归肉桂醇脱氢酶AsCAD功能鉴定及表达分析[J]. 生物技术通报, 2025, 41(2): 295-308.
[8]	李明, 刘祥宇, 王益娜, 和四梅, 沙本才. 紫金龙异紫堇定生物合成相关6-OMT基因克隆与功能表征[J]. 生物技术通报, 2025, 41(2): 309-320.
[9]	乔岩, 杨芳, 任盼荣, 祁伟亮, 安沛沛, 李茜, 李丹, 肖俊飞. 马铃薯野生种烯酰水合酶超家族基因ScDHNS的克隆与功能分析[J]. 生物技术通报, 2024, 40(9): 92-103.
[10]	庞梦真, 徐汉琴, 刘海燕, 宋娟, 王佳涵, 孙丽娜, 姬佩梅, 尹泽芝, 胡又川, 赵晓萌, 梁闪闪, 张泗举, 栾维江. 水稻黄化早抽穗突变体 hz1 的基因鉴定及功能分析[J]. 生物技术通报, 2024, 40(7): 125-136.
[11]	孙慧琼, 张春来, 王锡亮, 徐宏申, 窦苗苗, 杨博慧, 柴文婷, 赵珊珊, 姜晓东. 藜麦FLS基因家族的鉴定、表达及DNA变异分析[J]. 生物技术通报, 2024, 40(7): 172-182.
[12]	沈真辉, 曹瑶, 杨林雷, 罗祥英, 子灵山, 陆青青, 李荣春. 金耳和毛韧革菌麦角硫因生物合成基因的克隆及生物信息学分析[J]. 生物技术通报, 2024, 40(7): 259-272.
[13]	黄丹, 姜山, 彭涛. 褐角苔FfCYP98基因克隆及其功能分析[J]. 生物技术通报, 2024, 40(7): 273-284.
[14]	王玉书, 赵琳琳, 赵爽, 胡琦, 白慧霞, 王欢, 曹业萍, 范震宇. 大白菜BrCYP83B1基因的克隆及表达分析[J]. 生物技术通报, 2024, 40(6): 152-160.
[15]	郝思怡, 张君珂, 王斌, 曲朋燕, 李瑞得, 程春振. 香蕉ELF3的克隆与表达分析[J]. 生物技术通报, 2024, 40(5): 131-140.