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

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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

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