番茄SlMAPK6基因克隆及其表达特性分析

Cloning and expression analysis of SlMAPK6 gene in tomato

【目的】克隆番茄促分裂原活化蛋白激酶(MAPK)基因SlMAPK6,并分析其在不同非生物胁迫及信号物质处理下的表达模式,为深入探究SlMAPK6基因在番茄逆境响应中调控机制提供理论参考。【方法】采用同源克隆技术从矮番茄中克隆SlMAPK6基因cDNA全长序列,并进行生物信息学分析,采用实时荧光定量PCR检测矮番茄幼苗在干旱(20%PEG-6000)、盐(300 mmol/L NaCl)及信号物质[0.50 mmol/L水杨酸(SA)和0.10 mmol/L 2,1,3-苯并噻二唑(BTH)]处理下SlMAPK6基因的表达模式。【结果】克隆获得SlMAPK6基因cDNA全长序列为1360 bp,开放阅读框(ORF)为1131 bp,编码376个氨基酸残基,其编码蛋白理论等电点(pI)6.76,不稳定指数44.36,脂肪系数90.00,为不稳定的强脂溶性蛋白,亲水性平均值(GRAVY)-0.396,亲水性氨基酸均匀分布在整个肽链中,故该蛋白为亲水性蛋白;亚细胞定位于细胞核及胞浆质中,无跨膜区,含有36个磷酸化位点,其中丝氨酸(Ser)磷酸位点19个、苏氨酸(Thr)磷酸位点9个。SlMAPK6蛋白二级结构中,α-螺旋占38.03%,延伸链占10.37%,无规则卷曲占51.60%。SlMAPK6蛋白与马铃薯StMAPK4、风铃辣椒CbMAPK4和中华辣椒CcMAPK4蛋白同源性均为99.47%。SlMAPK6和SlMAPK5蛋白的亲缘关系最近,与拟南芥MAPK家族B亚族成员AtMAPK4、AtMPK5、AtMPK11、AtMPK12和AtMPK13蛋白的亲缘关系也较近。经SA、BTH、NaCl和PEG-6000处理后,SlMAPK6基因均出现抑制表达,说明SlMAPK6基因负向调控番茄植株的逆境响应。【结论】SlMAPK6基因可能参与负调控番茄植株的抗病和抗逆胁迫机制。SlMAPK6蛋白通过Ser或Thr磷酸化识别细胞信号的传导功能,在矮番茄逆境胁迫响应中发挥重要作用。

【Objective】To clone the tomato mitogen-activated protein kinase(MAPK)gene SlMAPK6,to analyze its expression level under different abiotic stresses and signal substances,which provided theoretical reference for exploration of the regulatory mechanism of the SlMAPK6 gene in tomato stress response.【Method】Using homologous cloning technology to clone the full-length cDNA sequence of SlMAPK6 gene from dwarf tomato,conducting bioinformatics analysis,and real-time fluorescent quantitative PCR was used to detect the expression pattern of SlMAPK6 gene under the drought(20%PEG-6000),salt(300 mmol/L NaCl),signal substances[0.50 mmol/L salicylic acid(SA)and 0.10 mmol/L 2,1,3-benzothiadiazole(BTH)]treatments.【Result】The full-length cDNA of SlMAPK6 was 1360 bp and contained an open reading frame(ORF)of 1131 bp that encoded 376 amino acids residues,the theoretical isoelectric point(pI)was 6.76,instability index 44.36,and the fat coefficient 90.00,it was an unstable strong fat-soluble protein.The average hydrophilicity(GRAVY)was-0.396,hydrophilic amino acids were evenly distributed in the entire peptide chain,so the protein was a hydrophilic protein,subcellularly located in the nucleus and cytoplasm,without a transmembrane region.It contained 36 phosphorylation sites,including 19 serine(Ser)phosphate sites and 9 threonine(Thr)phosphate sites.In the secondary structure of SlMAPK6 protein,α-helix accounted for 38.03%,extended chain accounted for 10.37%,and random coil accounted for 51.60%.The homology of SlMAPK6 protein with potato StMAPK4,capsicum CbMAPK4 and Chinese capsicum CcMAPK4 were all 99.47%.SlMAPK6 and SlMAPK5 had the closest relationship,and it was also closely related to members of the arabidopsis MAPK family B subfamily:AtMAPK4,AtMPK5,AtMPK11,AtMPK12 and AtMPK13 proteins.After SA,BTH,NaCl and PEG-6000 treatments,the expression of SlMAPK6 gene was suppressed,indicating that SlMAPK6 gene negatively regulated the stress response of tomato plants.【Conclusion】The Sl-MAPK6 gene may be involved in negatively regulating the disease resistance and stress resistance of tomato plants.The Sl-MAPK6 protein recognizes the transduction function of cell signals through Ser or Thr phosphorylation,and plays an important role in the response of dwarf tomato to stress.