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

doi:10.3969/j.issn.1004-1524.20221392

Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (8): 1763-1772.DOI: 10.3969/j.issn.1004-1524.20221392

• Horticultural Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S436.629

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.

Figures/Tables 7

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

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

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.

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.

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.

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.

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

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