番茄WRKY转录因子<i>SlWRKY75</i>的克隆、表达及功能分析

doi:10.7668/hbnxb.20194162

摘要/Abstract

摘要：

WRKY转录因子是植物中特有的转录因子家族之一,研究证实WRKY转录因子在植物的生长发育及对生物与非生物胁迫响应中具有重要的调控作用。为揭示番茄WRKY基因的功能,基于前期转录组数据,以番茄高抗青枯病Hm 2-2(R)与高感青枯病BY 1-2(S)2个自交系株系为试验材料,克隆得到1个番茄WRKY转录因子SlWRKY75基因(Solyc05g015850.3)。通过生物信息学分析、氨基酸序列多重比对、系统发育树构建、实时荧光定量PCR(qRT-PCR)和病毒诱导的基因沉默(VIGS)等技术方法对其基因及编码蛋白的结构、表达模式与功能进行分析。结果表明,该基因的cDNA全长为653 bp,其最大开放阅读框为519 bp,编码172个氨基酸,其蛋白相对分子量为19.878 51 ku,理论等电点为9.32,属于亲水性非分泌蛋白,且不存在跨膜结构,同时,该蛋白具有1个高度保守的WRKY结构域和一个CX₄CX₂₃HXH锌指基序,且同属于第Ⅱ类家族。系统发育树分析表明,SlWRKY75与潘那利番茄SpWRKY75亲缘关系最近,并与其他茄科聚为1组,而与橡胶树HbWRKY75、陆地棉GhWRKY75等的亲缘关系较远,在系统发育树上处于不同的分支。qRT-PCR结果表明,SlWRKY75基因的表达具有组织特异性,并可被青枯菌、水杨酸和茉莉酸所诱导。利用VIGS技术得出,沉默SlWRKY75降低了植株对青枯病的抗性,表明SlWRKY75正调控番茄对青枯病的抗性。通过以上研究结果得出,SlWRKY75基因在番茄调控青枯病抗性响应过程中扮演着重要的角色。

关键词: 番茄青枯病, WRKY转录因子, 基因克隆, 表达分析, 病毒诱导的基因沉默(VIGS)

Abstract:

WRKY transcription factor is a family of transcription factors unique to plants.Studies have demonstrated that WRKY transcription factor played an important role in plant growth and development and in plant response to biological and abiotic stress.In order to reveal the function of tomato WRKY gene,two inbred lines of tomato with high resistance to bacterial wilt Hm 2-2(R)and high susceptibility to bacterial wilt BY 1-2(S)were used as experimental materials based on the preliminary transcriptome data,and a WRKY transcription factor SlWRKY75 gene(Solyc05g015850.3)was cloned.The structure,expression pattern and function of the gene and its encoded protein were analyzed by bioinformatics analysis,multiple alignment of amino acid sequences,phylogenetic tree construction,Real-time Quantitative PCR(qRT-PCR)and virus induced gene silencing(VIGS).The results showed that the full length of the cDNA of this gene was 653 bp,its maximum open reading frame was 519 bp,encoding 172 amino acids,the relative molecular weight of the protein was 19.878 51 ku,the theoretical isoelectric point was 9.32.The protein belonged to the hydrophilic non-secreted protein,and there was no transmembrane structure.Meanwhile,the protein had a highly conserved WRKY domain and a CX₄CX₂₃HXH zinc finger motif,which belonged to the Class Ⅱ family.Phylogenetic tree analysis showed that SlWRKY75 was closely related to Solanum pennellii SpWRKY75 and clustered into a group with other Solanaceae,while it was far related to Hevea brasiliensis HbWRKY75 and Gossypium hirsutum GhWRKY75 and was in different branches in the phylogenetic tree.The results of qRT-PCR analysis showed that the expression of SlWRKY75 gene were tissue-specific and could be induced by Ralstonia solanacearum,salicylic acid and jasmonic acid.VIGS result showed that silencing SlWRKY75 reduced plant resistance to bacterial wilt,indicating that SlWRKY75 positively regulated tomato resistance to bacterial wilt.These results suggested that SlWRKY75 gene played an important role in regulating tomato resistance to bacterial wilt.

Key words: Tomato bacterial wilt, WRKY transcription factor, Gene cloning, Expression analysis, Virus induced gene silencing(VIGS)

陈娜, 詹文文, 刘兴雨, 石磊鑫, 李若楠, 谢镕, 却志群. 番茄WRKY转录因子SlWRKY75的克隆、表达及功能分析[J]. 华北农学报, 2023, 38(6): 1-10. doi: 10.7668/hbnxb.20194162.

CHEN Na, ZHAN Wenwen, LIU Xingyu, SHI Leixin, LI Ruonan, XIE Rong, QUE Zhiqun. Cloning,Expression,and Functional Analysis of WRKY Transcription Factor SlWRKY75 in Tomato[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(6): 1-10. doi: 10.7668/hbnxb.20194162.

http://www.hbnxb.net/CN/Y2023/V38/I6/1

图/表 10

图1 番茄SlWRKY75基因 A:M.Marker DL2000;1.SlWRKY75基因。B:M.Marker DL2000;5—7.阳性克隆;1—4,8.阴性克隆。

Fig.1 Tomato SlWRKY75 gene A:M.Marker DL2000;1.SlWRKY75 gene.B:M.Marker DL2000;5—7.Positive clone;1—4,8.Negative clone.

图2 SlWRKY75 cDNA及预测的开放阅读框

Fig.2 Sequences of SlWRKY75 cDNA and predicted ORF

图3 SlWRKY75 的保守区

Fig.3 Conservative domain of SlWRKY75

图4 SlWRKY75蛋白的亲疏水性分析

Fig.4 Hydrophilic analysis of SlWRKY75 protein

图5 SlWRKY75蛋白的信号肽和跨膜结构分析 A.SlWRKY75蛋白的信号肽预测;B.SlWRKY75蛋白的跨膜结构预测。

Fig.5 Signal peptide and transmembrane structure analysis of SlWRKY75 protein A.Signal peptide prediction of SlWRKY75 protein;B.Transmembrane structure prediction of SlWRKY75 protein.

图6 SlWRKY75蛋白的二级结构和三级结构预测 A.SlWRKY75蛋白的二级结构预测;B.SlWRKY75蛋白的三级结构预测。

Fig.6 Secondary structure and tertiary structure prediction of SlWRKY75 protein A.The secondary structure prediction of SlWRKY75 protein;B.The tertiary structure prediction of SlWRKY75 protein.

图7 番茄SlWRKY75蛋白与其他物种同源蛋白多重序列比对方框.高度保守的WRKYGQK七肽序列;线条.WRKY结构域;三角.锌指基序。

Fig.7 Multiple alignment of homologous protein for the SlWRKY75 in tomato and other species The box.The highly conserved WRKYGQK heptapeptide sequence;The line.The WRKY domain;The triangle.The zinc finger motif.

图8 SlWRKY75同源蛋白的系统进化树

Fig.8 Phylogenetic tree of SlWRKY75 homologous protein

图9 番茄SlWRKY75基因表达分析 n=3,不同的小写字母代表显著差异(P<0.05)。图10同。

Fig.9 Expression analysis of tomato SlWRKY75 gene n=3,the different lowercase letters represent significant differences(P<0.05).The same as Fig.10.

图10 SlWRKY75基因正调控番茄对青枯病的抗性 A.TRV介导的SlWRKY75基因沉默番茄植株中SlWRKY75相对表达量;B.TRV介导的SlWRKY75基因沉默番茄植株叶片的表型特征。CK.对照组,接种清水;TRV∷Empty.对照组,接种病毒空载体;TRV∷SlWRKY75.VIGS 处理组,转染SlWRKY75重组病毒载体。

Fig.10 SlWRKY75 gene positively regulates tomato resistance to bacterial wilt A.SlWRKY75 relative expression in tomato plants with SlWRKY75 silencing mediated by TRV;B.Phynotypic characterization of tomato leaf with SlWRKY75 silencing mediated by TRV.CK.Control group,trans-infected with water;TRV∷Empty.Control group,trans-infected with empty virus vector;TRV∷SlWRKY75.Experimental group,trans-infected with SlWRKY75 recombinant virus vector.

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番茄WRKY转录因子SlWRKY75的克隆、表达及功能分析

Cloning,Expression,and Functional Analysis of WRKY Transcription Factor SlWRKY75 in Tomato

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