梁思桃, 高雁茹, 刘广清, 黄瑞基, 刘强, 任振新, 乔清华. 2022: 番茄抗病品种吉美08 SlDFR基因的克隆与表达. 南方农业学报, 53(3): 813-820. DOI: 10.3969/j.issn.2095-1191.2022.03.023
引用本文: 梁思桃, 高雁茹, 刘广清, 黄瑞基, 刘强, 任振新, 乔清华. 2022: 番茄抗病品种吉美08 SlDFR基因的克隆与表达. 南方农业学报, 53(3): 813-820. DOI: 10.3969/j.issn.2095-1191.2022.03.023
LIANG Si-tao, GAO Yan-ru, LIU Guang-qing, HUANG Rui-ji, LIU Qiang, REN Zhen-xin, QIAO Qing-hua. 2022: Cloning and expression analysis of SlDFR gene in diseaseresistant tomato varieties Jimei 08. Journal of Southern Agriculture, 53(3): 813-820. DOI: 10.3969/j.issn.2095-1191.2022.03.023
Citation: LIANG Si-tao, GAO Yan-ru, LIU Guang-qing, HUANG Rui-ji, LIU Qiang, REN Zhen-xin, QIAO Qing-hua. 2022: Cloning and expression analysis of SlDFR gene in diseaseresistant tomato varieties Jimei 08. Journal of Southern Agriculture, 53(3): 813-820. DOI: 10.3969/j.issn.2095-1191.2022.03.023

番茄抗病品种吉美08 SlDFR基因的克隆与表达

Cloning and expression analysis of SlDFR gene in diseaseresistant tomato varieties Jimei 08

  • 摘要: 【目的】克隆番茄(Solanum lycopersicum)抗病品种吉美08二氢黄酮醇4-还原酶(dihydroflavonol 4-reductase,DFR)基因,并分析枯萎病菌尖孢镰刀菌番茄专化型(Fol4287)诱导下其在番茄不同组织中的表达模式,为深入研究SlDFR基因在番茄抗病过程中的调控机制提供理论依据。【方法】采用同源克隆技术从番茄中克隆SlDFR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(qRT-PCR)检测番茄根、茎、叶中SlDFR基因在枯萎病菌诱导下的表达模式。【结果】从番茄抗病栽培品种吉美08中克隆得到SlDFR基因,cDNA序列全长1659 bp,包含1个1149 bp的开放阅读框(ORF),编码379个氨基酸。SlDFR蛋白的相对分子质量为42.43 kD,等电点(pI)为6.08,是一个亲水的、稳定的酸性蛋白,亚细胞定位在高尔基体上,无信号肽和跨膜结构域,有38个磷酸化位点,其中,丝氨酸18个,苏氨酸15个,酪氨酸5个。SlDFR蛋白的二级结构中α-螺旋占37.73%,延伸链占13.98%,无规则卷曲占40.69%。系统发育进化树分析结果表明,SlDFR蛋白序列与同属茄科马铃薯(Solanum pennellii)亲缘关系较近,其次为黑果枸杞(Lycium ruthenicum)。qRT-PCR分析结果显示,SlDFR基因在番茄叶中表达量最高,在根中表达量最低。SlDFR基因在番茄根、茎、叶中表达量受番茄枯萎病菌诱导,均呈现不同程度上调,其中,在根中表达量变化最大,在叶中表达量变化较小,推测SlDFR基因的表达量与番茄枯萎病菌胁迫响应有关,且番茄根部类黄酮的合成为重要胁迫响应途径。【结论】SlDFR基因表达量受Fol4287的诱导,可能参与番茄枯萎病胁迫响应过程,且对番茄枯萎病菌的响应在番茄根部更强烈,并通过调控番茄根部黄酮类物质的合成提高根系分泌物的抑菌活性从而增强番茄对枯萎病的抗性。

     

    Abstract: 【Objective】To clone the tomato dihydroflavonol 4-reductase(DFR)gene of the resistant varieties Jimei 08 and to analyze its expression in different tissues of tomato induced by Fusarium wilt,so as to provide theoretical reference for exploration of the regulation mechanism of SlDFR gene in the process of tomato disease resistance.【Method】The fulllength cDNA sequence of SlDFR gene from tomato was cloned through homologous cloning technology,and bioinformatics analysis was conducted,and real-time fluorescent quantitative PCR was used to detect the expression pattern of SlDFR gene in tomato roots,stems and leaves induced by Fusarium wilt.【Result】The full-length cDNA of SlDFR was 1659 bp and cDNA contained an open reading frame(ORF)of 1149 bp that encoded 379 amino acids. SIDFR protein had a molecular weight of 42.43 kD and its theoretical isoelectric point(pI)was 6.08,indicating that it was a stable hydrophilic one. And its subcellular localization was on Golgi apparatus without signal peptide and transmembrane domain,with 38 phosphorylation sites,including 18 serine(Ser)phosphate sites,15 threonine(Thr)phosphate sites and 5 tyrosine(Tyr) phosphate sites. In the secondary structure of SlDFR protein,α-helix accounted for 37.73%,extended chain accounted for 13.98%,and random coil accounted for 40.69%. Phylogenetic analysis showed that tomato DFR was closely related to Solanum pennelli followed by Lycium ruthenicum. The results of real-time fluorescence quantitative PCR showed that the expression of SlDFR gene was the highest in leaves,and lowest in roots. After SlDFR gene expression being induced by Fusarium wilt,the expression of SlDFR gene was up-regulated in varying degrees. The change of expression was the largest in roots and the smallest in leaves,indicating that SlDFR gene was related to the stress response of tomato Fusarium wilt,and the synthesis of flavonoids in roots was an important stress response pathway.【Conclusion】After SlDFR gene expression being induced by Fol4287,SlDFR gene could participate in the stress response of tomato Fusarium wilt,and the response to Fusarium wilt is stronger in the root,and probably the gene regulates flavonoid synthesis in the root to enhance antibacterial activity of root exudates,thus increasing the resistance of tomato to Fusarium wilt.

     

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