枣疯病植原体胸苷激酶基因的克隆、序列分析与原核表达

doi:10.3969/j.issn.1004-1524.20221392

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (8): 1763-1772.DOI: 10.3969/j.issn.1004-1524.20221392

枣疯病植原体胸苷激酶基因的克隆、序列分析与原核表达

宋传生(), 康晓飞, 樊庆忠, 王俊刚^*(), 石雪, 张子汝, 谭青青, 曾小娇, 刘芳, 李英赛, 侯常跃

菏泽学院农业与生物工程学院,山东菏泽 274015

收稿日期:2022-09-28 出版日期:2023-08-25 发布日期:2023-08-29
作者简介:宋传生(1984—),男,山东滕州人,博士,副教授,研究方向为园艺植物病理学。E-mail:244799824@qq.com
通讯作者: *王俊刚,E-mail:hzuwjg@163.com
基金资助:
山东省自然科学基金(ZR2019PC039);国家自然科学基金(31800545);菏泽学院博士基金(XY16BS35);菏泽学院培育项目(XY18PY04);菏泽学院培育项目(XY18PY15)

Cloning, sequence analysis, prokaryotic expression of thymidine kinase from jujube witches’-broom phytoplasma

SONG Chuansheng(), KANG Xiaofei, FAN Qingzhong, WANG Jungang^*(), SHI Xue, ZHANG Ziru, TAN Qingqing, ZENG Xiaojiao, LIU Fang, LI Yingsai, HOU Changyue

College of Agricultural and Biological Engineering, Heze University, Heze 274015, Shandong, China

Received:2022-09-28 Online:2023-08-25 Published:2023-08-29

摘要/Abstract

摘要：

枣疯病(jujube witches’-broom, JWB)俗称枣树的“癌症”,是由枣疯病植原体导致的侵染性病害,给我国的枣栽培与生产造成了严重危害,对枣疯病植原体增殖基因的研究有助于枣疯病的有效防治。为获得枣疯病植原体胸苷激酶基因(tdk),利用PCR方法扩增该基因并测序,结果表明,该基因的开放阅读框长度为576 bp,编码191个氨基酸,其蛋白质分子量为21.76 ku。序列分析和预测表明,该蛋白质为植原体细胞质中性质相对稳定的亲水蛋白质。遗传距离和进化树分析显示,植原体tdk基因比其16S rDNA基因的保守程度低,tdk基因和16S rDNA基因序列的进化树高度相似,表明tdk基因适于植原体分子分类。为获得枣疯病植原体TDK蛋白,成功构建了原核表达载体pET-28a-JWB-Heze-tdk;在诱导条件为0.1 mmol·L^-1 IPTG、20 ℃、140 r·min^-1时,该原核表达载体在Escherichia coli BL21(DE3)菌株中可表达出大量可溶性的N端融合了6×His标签的JWB-Heze TDK蛋白;利用Ni-IDA琼脂糖树脂纯化出了可溶性的JWB-Heze TDK融合蛋白。研究结果为JWB-Heze TDK体外酶活性测定和抗体制备奠定了基础。

关键词: 枣疯病, 植原体, 胸苷激酶, 基因克隆, 原核表达, 蛋白质纯化

Abstract:

Jujube witches’-broom (JWB), commonly known as the “cancer” of jujube trees, is an infectious disease caused by the JWB phytoplasma, which had caused serious harm to the cultivation and production of jujube in China. The study on the proliferation gene of JWB phytoplasma is helpful to the effective prevention and control of the JWB disease. To obtain the thymidine kinase gene (tdk) of JWB phytoplasma, it was amplified by PCR and then sequenced. The results showed that it contained a complete open reading frame which was composed of 576 nucleotides and encoded a protein of 191 amino acid residues with a molecular mass of 21.76 ku. The genetic distance matrices based on tdk and 16S rDNA gene sequences indicated that the tdk gene in phytoplasma was more variable than its 16S rDNA gene. Also, the phylogenetic trees constructed respectively using the tdk and 16S rDNA genes of the same phytoplasmas were highly similar. So, the tdk gene was suitable for molecular classification of phytoplasma. To obtain the TDK protein of JWB phytoplasma, the prokaryotic expression vector of pET-28a-JWB-Heze-tdk was successfully constructed. A large amount of soluble recombinant protein with 6×His tag fused to N-terminus of JWB-Heze TDK was expressed in Escherichia coli BL21 (DE3) bacteria when induced using 0.1 mmol·L^-1 IPTG at 20 ℃ and 140 r·min^-1. Then, the soluble JWB-Heze TDK fusion protein was purified with Ni-IDA agarose resins. This study laid a foundation for the enzyme activity assays in vitro and antibody preparation of the JWB-Heze TDK.

Key words: jujube witches’-broom, phytoplasma, thymidine kinase, gene cloning, prokaryotic expression, protein purification

中图分类号:

S436.629

宋传生, 康晓飞, 樊庆忠, 王俊刚, 石雪, 张子汝, 谭青青, 曾小娇, 刘芳, 李英赛, 侯常跃. 枣疯病植原体胸苷激酶基因的克隆、序列分析与原核表达[J]. 浙江农业学报, 2023, 35(8): 1763-1772.

SONG Chuansheng, KANG Xiaofei, FAN Qingzhong, WANG Jungang, SHI Xue, ZHANG Ziru, TAN Qingqing, ZENG Xiaojiao, LIU Fang, LI Yingsai, HOU Changyue. Cloning, sequence analysis, prokaryotic expression of thymidine kinase from jujube witches’-broom phytoplasma[J]. Acta Agriculturae Zhejiangensis, 2023, 35(8): 1763-1772.

图/表 7

图1 预测的JWB-Heze TDK蛋白质三维结构

Fig.1 Predicted three-dimensional structure of JWB-Heze TDK protein

图2 不同物种胸苷激酶氨基酸序列比对图 1~12,不同16Sr组的植原体。

Fig.2 Sequence comparison of amino acids of thymidine kinase from different species 1-12, Phytoplasmas from different 16Sr groups.

图3 基于植原体tdk和16S rDNA序列构建的进化树 A,基于植原体tdk序列构建的进化树;B,基于植原体16S rDNA序列构建的进化树。▲所注为本研究中克隆的JWB-Heze tdk;进化树构建方法为NJ法,bootstrap重复次数为500。

Fig.3 Phylogenetic trees based on phytoplasma tdk sequence and 16S rDNA sequence A, Phylogenetic tree based on the tdk sequences of Phytoplasmas; B, Phylogenetic tree based on the 16S rDNA sequences of phytoplasmas. ▲ shows the JWB-Heze tdk cloned in this study; NJ method was used to construct the phylogenetic tree and the number of bootstrap repeats was 500.

图4 JWB-Heze TDK融合蛋白诱导表达结果 M,蛋白质分子量标准;1~3,pET-28a-JWB-Heze-tdk载体;4~6,pET-28a空载体。1、4,未经IPTG诱导;2、5,IPTG浓度为1.0 mmol·L-1;3、6,IPTG浓度为0.1 mmol·L-1。2、3,泳道中最粗的条带为目标蛋白质;箭头所指为目标蛋白质的位置。

Fig.4 Expression of the JWB-Heze TDK fusion protein M, Protein marker; 1-3, pET-28a-JWB-Heze-tdk vector; 4-6, pET-28a empty vector. Lanes 1 and 4, not induced by IPTG; Lanes 2 and 5, induced by 1.0 mmol·L-1 IPTG; 3 and 6, induced by 0.1 mmol·L-1 IPTG. The thickest band in lanes 2 and 3 were the target proteins; The arrow indicated the location of the target protein.

图5 0.1 mmol·L-1 IPTG、28 ℃、140 r·min-1条件下诱导表达的JWB-Heze TDK融合蛋白的纯化结果 M,蛋白质分子量标准;SF,菌体裂解液上清液;FT,过柱流穿液;E1~E9,不同浓度咪唑的蛋白质洗脱液洗脱的蛋白质,蛋白质洗脱液中咪唑的浓度分别为10、20、50、60、100、150、200、250、400 mmol·L-1;箭头所指为目标蛋白质的位置。图7同。

Fig.5 Purification of JWB-Heze TDK fusion protein induced by 0.1 mmol·L-1 IPTG at 28 ℃ and 140 r·min-1 M, Protein marker; SF, Cell lysate supernatant; FT, Flow-through liquid; E1-E9, Proteins eluted by eluates containing 10, 20, 50, 60, 100, 150, 200, 250 and 400 mmol·L-1 imidazole respectively; The arrow indicated the location of the target protein. The same as in figure 7.

图6 JWB-Heze TDK融合蛋白质包涵体的鉴定和裂解结果 M,蛋白质分子量标准;1,未经处理的菌体裂解液沉淀;2、3,分别为菌体裂解液沉淀经蒸馏水洗涤后离心获得的上清液和沉淀;4、5,分别为菌体裂解液沉淀在20 ℃ 8 mol·L-1尿素溶液中处理后离心获取的上清液和沉淀;6、7,分别为菌体裂解液沉淀在50 ℃ 8 mol·L-1尿素溶液中处理后离心获得的上清液和沉淀;箭头所指为目标蛋白质的位置。

Fig.6 Identification and lysis of inclusion bodies of the JWB-Heze TDK fusion protein M, Protein marker; 1, Bacterial lysate precipitation without treatment; 2 and 3, Obtained from the distilled water used for washing the bacterial lysate precipitation; 4 and 5, Obtained from the 8 mol·L-1 urea solution used for dissolving the bacterial lysate precipitation at 20 ℃;6 and 7, Obtained from the 8 mol·L-1 urea solution used for dissolving the bacterial lysate precipitation at 50 ℃. The arrow indicated the location of the target protein.

图7 0.1 mmol·L-1 IPTG、20 ℃、140 r·min-1条件下诱导表达的JWB-Heze TDK融合蛋白的纯化结果

Fig.7 Purification of JWB-Heze TDK fusion protein induced by 0.1 mmol·L-1 IPTG at 20 ℃ and 140 r·min-1

参考文献 30

[1]	孙浩元. 中国枣树优良品种资源的保存、繁殖技术及毛叶枣引种研究[D]. 北京: 北京林业大学, 2001.
	SUN H Y. Study on preservation and propagation techniques of excellent jujube variety resources in China and introduction of Zizyphus mauritiana[D]. Beijing: Beijing Forestry University, 2001. (in Chinese with English abstract)
[2]	肖婧. 枣树种质资源收集与‘岱健枣’综合特性评价[D]. 泰安: 山东农业大学, 2015.
	XIAO J. Collection of jujube germplasm resources and evaluation of comprehensive characteristics of ‘Daijian Jujube’[D]. Taian: Shandong Agricultural University, 2015. (in Chinese with English abstract)
[3]	刘孟军, 赵锦, 周俊义. 枣疯病[M]. 北京: 中国农业出版社, 2010.
[4]	田国忠. 枣疯病的预防和治疗策略研究[J]. 林业科技通讯, 1998(2): 14-16.
	TIAN G Z. Study on prevention and treatment strategies of jujube witches’ broom[J]. Forest Science and Technology, 1998(2): 14-16. (in Chinese)
[5]	李继东, 陈立川, 王会鱼, 等. 枣疯病发生机制与防治研究进展[J]. 河南农业大学学报, 2021, 55(1): 1-7.
	LI J D, CHEN L C, WANG H Y, et al. Research progress on the pathogenic mechanism and control of jujube witches’ broom disease[J]. Journal of Henan Agricultural University, 2021, 55(1): 1-7. (in Chinese with English abstract)
[6]	The IRPCM Phytoplasma/Spiroplasma Working Team-Phytoplasma Taxonomy Group. ‘Candidatus Phytoplasma’, a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects[J]. International Journal of Systematic and Evolutionary Microbiology, 2004, 54(Pt 4): 1243-1255.
[7]	RAZIN S, YOGEV D, NAOT Y. Molecular biology and pathogenicity of mycoplasmas[J]. Microbiology and Molecular Biology Reviews, 1998, 62(4): 1094-1156.
[8]	穆雪, 赵洋, 李春艳, 等. 国内外樱桃植原体(Phytoplasma)病害研究进展[J]. 果树学报, 2019, 36(12): 1754-1762.
	MU X, ZHAO Y, LI C Y, et al. International advances of the research on the phytoplasma diseases in cherry[J]. Journal of Fruit Science, 2019, 36(12): 1754-1762. (in Chinese with English abstract)
[9]	任争光, 王合, 林彩丽, 等. 实时荧光定量PCR(SYBR Green I)检测不同抗枣疯病枣树品种嫁接接穗中的植原体浓度[J]. 植物病理学报, 2015, 45(5): 520-529.
	REN Z G, WANG H, LIN C L, et al. A real-time(SYBR Green I) PCR assay for detection and quantification of jujube witches’-broom phytoplasma in the grafted jujube cultivar scions with different resistance[J]. Acta Phytopathologica Sinica, 2015, 45(5): 520-529. (in Chinese with English abstract)
[10]	KUBE M, MITROVIC J, DUDUK B, et al. Current view on phytoplasma genomes and encoded metabolism[J]. The Scientific World Journal, 2012: 185942.
[11]	WINTERSBERGER E. Regulation and biological function of thymidine kinase[J]. Biochemical Society Transactions, 1997, 25(1): 303-308.
[12]	AUFDERKLAMM S, TODENHÖFER T, GAKIS G, et al. Thymidine kinase and cancer monitoring[J]. Cancer Letters, 2012, 316(1): 6-10.
[13]	GASPARRI F, WANG N N, SKOG S, et al. Thymidine kinase 1 expression defines an activated G1 state of the cell cycle as revealed with site-specific antibodies and ArrayScan^TM assays[J]. European Journal of Cell Biology, 2009, 88(12): 779-785.
[14]	ULLAH H M, ROBERTSON D, FITES R C. A gene for thymidine kinase in plants[J]. Plant Physiology, 1999, 119: 1567-1568.
[15]	HE Q, ZOU L, ZHANG P A, et al. The clinical significance of thymidine kinase 1 measurement in serum of breast cancer patients using anti-TK1 antibody[J]. The International Journal of Biological Markers, 2000, 15(2): 139-146.
[16]	NING S F, WEI W E, LI J L, et al. Clinical significance and diagnostic capacity of serum TK1, CEA, CA 19-9 and CA 72-4 levels in gastric and colorectal cancer patients[J]. Journal of Cancer, 2018, 9(3): 494-501.
[17]	WANG Y, JIANG X R, DONG S L, et al. Serum TK1 is a more reliable marker than CEA and AFP for cancer screening in a study of 56, 286 people[J]. Cancer Biomarkers, 2016, 16(4): 529-536.
[18]	DEL RE M, BERTOLINI I, CRUCITTA S, et al. Overexpression of TK1 and CDK9 in plasma-derived exosomes is associated with clinical resistance to CDK4/6 inhibitors in metastatic breast cancer patients[J]. Breast Cancer Research and Treatment, 2019, 178(1): 57-62.
[19]	ZUO S P, WANG H X, LI L, et al. Thymidine kinase 1 drives skin cutaneous melanoma malignant progression and metabolic reprogramming[J]. Frontiers in Oncology, 2022, 12: 802807.
[20]	WANG J, SONG L Q, JIAO Q Q, et al. Comparative genome analysis of jujube witches’-broom phytoplasma, an obligate pathogen that causes jujube witches’-broom disease[J]. BMC Genomics, 2018, 19(1): 1-12.
[21]	CLAUSEN A R, GIRANDON L, ALI A, et al. Two thymidine kinases and one multi-substrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana[J]. FEBS Journal, 2012, 279(20): 3889-3897.
[22]	LEE I M, HAMMOND R W, DAVIS R E, et al. Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasmalike organisms[J]. Phytopathology, 1993, 83(8): 834.
[23]	宋传生, 胡佳续, 林彩丽, 等. 泡桐丛枝植原体河北平山和江西吉安株系胸苷酸激酶基因多态性分析[J]. 林业科学, 2014, 50(8): 108-118.
	SONG C S, HU J X, LIN C L, et al. Thymidylate kinase gene polymorphism of two strains of Paulownia witches’-broom Phytoplasma[J]. Scientia Silyae Sinicae, 2014, 50(8): 108-118. (in Chinese with English abstract)
[24]	MARTINI M, LEE I M, BOTTNER K D, et al. Ribosomal protein gene-based phylogeny for finer differentiation and classification of phytoplasmas[J]. International Journal of Systematic and Evolutionary Microbiology, 2007, 57(9): 2037-2051.
[25]	UPADHYAY A K, MURMU A, SINGH A, et al. Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in Escherichia coli[J]. PLoS One, 2012, 7(3): e33951.
[26]	RAMÓN A, SEÑORALE-POSE M, MARÍN M. Inclusion bodies: not that bad...[J]. Frontiers in Microbiology, 2014, 5: 56.
[27]	STRANDBERG L, ENFORS S O. Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli[J]. Applied and Environmental Microbiology, 1991, 57(6): 1669-1674.
[28]	陈来同, 茹炳根. 在大肠杆菌表达体系中以包涵体形式存在的真核生物蛋白质的分离、复性和二硫键的形成[J]. 中国生化药物杂志, 1997, 17(1): 46-49.
	CHEN L T, RU B G. Separation, renaturation and disulfide bond formation of eukaryotic protein in the form of inclusion bodies in Escherichia coli expression system[J]. Chinese Journal of Biochemical and Pharmaceuticals, 1997, 17(1): 46-49. (in Chinese)
[29]	朱希强, 袁勤生. EC-SOD在大肠杆菌中的可溶性表达[J]. 食品与药品, 2005, 7(4): 31-34.
	ZHU X Q, YUAN Q S. The resolution expression of EC-SOD in supernatant by E. coli[J]. Food and Drug, 2005, 7(4): 31-34. (in Chinese with English abstract)
[30]	王海彬. 肿瘤坏死因子相关凋亡诱导配体(TRAIL)的临床前抗肿瘤作用研究[D]. 杭州: 浙江大学, 2014.
	WANG H B. Study on preclinical anti-tumor effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)[D]. Hangzhou: Zhejiang University, 2014. (in Chinese with English abstract)

编辑推荐

Metrics

阅读次数

全文

1335

HTML			PDF

最新录用	在线预览	正式出版	最新录用	在线预览	正式出版
0	0	229	0	0	1106

来源	本网站	其他网站

次数	986	349
比例	74%	26%

摘要

315

最新录用	在线预览	正式出版

0	0	315

	来源	本网站

	次数	315
	比例	100%

枣疯病植原体胸苷激酶基因的克隆、序列分析与原核表达

Cloning, sequence analysis, prokaryotic expression of thymidine kinase from jujube witches’-broom phytoplasma

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 30

相关文章 15

编辑推荐

Metrics

本文评价

[1]	刘光瑞, 宗渊, 李云, 曹东, 刘宝龙, 包雪梅, 李建民. 当归转录因子AsMYB44的克隆与功能研究[J]. 浙江农业学报, 2023, 35(6): 1253-1264.
[2]	刘琪, 曹影丽, 魏宁波, 杨侃侃, 梁月巧, 宋祥军, 邵颖, 涂健, 祁克宗. 鸡DDX41基因克隆表达、细胞定位及其在禽腺病毒4型感染调控天然免疫中的作用[J]. 浙江农业学报, 2023, 35(5): 1028-1036.
[3]	姚彦林, 马骊, 刘丽君, 蒲媛媛, 李学才, 王旺田, 方彦, 孙万仓, 武军艳. 白菜型油菜开花调控基因BrFT的生物信息学特性和表达分析[J]. 浙江农业学报, 2023, 35(5): 992-1000.
[4]	燕存尧, 贾凯, 闫会转, 高杰. 芜菁BrrLOX7基因克隆、表达及生物信息学分析[J]. 浙江农业学报, 2023, 35(4): 831-840.
[5]	郭春倩, 田洁. 大蒜己糖激酶基因AsHXK2的克隆及其参与根际促生菌缓解干旱胁迫的表达分析[J]. 浙江农业学报, 2022, 34(9): 1925-1934.
[6]	刘凯, 谢楠, 郭炜, 马恒甲. 三角鲂MHCⅠα基因全长cDNA克隆与生物信息学分析[J]. 浙江农业学报, 2022, 34(6): 1162-1174.
[7]	洪森荣, 向琼钰, 谢颖, 熊晨露, 徐晨慧, 徐璐珂, 陈荣华, 蔡红. 怀玉山三叶青烟草病毒增殖蛋白1基因克隆、亚细胞定位和组织表达分析[J]. 浙江农业学报, 2022, 34(6): 1193-1204.
[8]	樊有存, 张红岩, 杨旭升, 韩芊, 刘玉皎, 武学霞. 蚕豆耐盐相关基因VfHKT1;1的克隆、生物信息学分析及表达特性[J]. 浙江农业学报, 2022, 34(4): 756-765.
[9]	杨卫军, 董艳蕾, 吴秋芳, 张美玲, 韩丽滨, 张元臣. 棉蚜ATP合成酶基因AgoATPb的克隆与表达[J]. 浙江农业学报, 2022, 34(2): 329-336.
[10]	唐国亮, 张玉宝, 王若愚, 王亚军, 赵霞, 苏学思, 金卫杰. 魔芋花叶病毒衣壳蛋白的原核表达和多克隆抗体制备[J]. 浙江农业学报, 2022, 34(11): 2471-2481.
[11]	朱寅初, 王宏宇, 云涛, 华炯钢, 叶伟成, 倪征, 陈柳, 张存. 浙江地区鹅星状病毒分离鉴定及其衣壳蛋白多克隆抗体的制备[J]. 浙江农业学报, 2022, 34(10): 2149-2159.
[12]	赵秀平, 王双, 闫星伊, 段强, 张帅, 陈永胜, 李国瑞. 稻瘟病菌MGG-01005的表达纯化与生物信息学分析[J]. 浙江农业学报, 2021, 33(3): 470-478.
[13]	何佳琦, 翟莹, 张军, 邱爽, 李铭杨, 赵艳, 张梅娟, 马天意. 大豆转录因子GmDof1.5的克隆与非生物胁迫诱导表达[J]. 浙江农业学报, 2021, 33(1): 1-7.
[14]	苏学思, 张玉宝, 王若愚, 王亚军, 唐国亮, 金卫杰. 车前草花叶病毒衣壳蛋白的原核表达和多克隆抗体制备[J]. 浙江农业学报, 2021, 33(1): 104-111.
[15]	刘金玉, 黄鹰. 粟酒裂殖酵母SpTrz2蛋白全长和N端的原核表达与多克隆抗体制备[J]. 浙江农业学报, 2021, 33(1): 34-42.