陆地棉衰老相关基因GhSAG101的克隆及抗病功能分析

Cloning and disease-resistant function of a senescence-associated gene GhSAG101 in Gossypium hirsutum

【目的】挖掘棉花抗病相关基因,为创制抗病棉新品种提供理论依据和候选基因。【方法】以陆地棉(Gossypium hirsutum)TM-1为研究对象,克隆了棉花衰老相关基因GhSAG101(senescence-associated gene 101)。通过实时荧光定量聚合酶链式反应分析GhSAG101在棉花幼苗不同组织中的表达量,以及在黄萎病菌诱导下表达量的变化;通过蛋白序列分析预测了GhSAG101的保守位点和活性位点;利用病毒诱导的基因沉默技术沉默棉花中GhSAG101,以空载体作为对照,通过棉花整株接种和离体叶片接种黄萎病菌研究GhSAG101沉默对黄萎病抗性的影响。【结果】GhSAG101基因编码区长度为1764 bp,编码587个氨基酸残基的蛋白质,包含水解酶家族、EDS1(enhanced disease susceptibility 1)家族的保守结构域和亲核位点。GhSAG101在根中表达量最高,茎中次之,在叶片中表达量最低,黄萎病菌侵染中后期GhSAG101在根中被诱导上调表达。在棉花中沉默GhSAG101基因降低了黄萎病菌在寄主体内的扩展速度,减缓了病害发生过程。【结论】GhSAG101负调控棉花对黄萎病的抗性,可作为棉花抗病育种的候选基因。

[Objective]Screening and study of cotton disease resistance genes will provide theoretical basis and genetic resources for cotton disease resistance breeding.[Method]The senescence associated gene 101(GhSAG101)was cloned from Gossypium hirsutum L.TM-1.The expression levels of GhSAG101 in different organs of cotton seedlings,as well as in cotton roots under Verticillium dahliae inoculation were analyzed by real time quantitative polymerase chain reaction(qRT-PCR).The conserved domains and active sites of GhSAG101 were predicted through protein sequence analysis.Knockdown lines of GbSAG101 generated through virus induced gene silencing(VIGS)system were employed for the examination of Verticillium dahliae resistance by inoculation of the whole plant and detached leaves,and the empty vector transferred plants were used as the negative control.[Result]The coding region of GhSAG101 gene is 1764 bp in length,encodes a protein with 587 amino acid residues that includes a nucleophilic elbow and the conserved domains belonging to the abhydrolase superfamily and enhanced disease susceptibility 1(EDS1)family.The highest expression level of GhSAG101 was found in roots,followed by stems,and the lowest expression level was found in leaves.GhSAG101 expression was induced by V.dahliae infection in roots at the middle and late infection stage.Suppression of GhSAG101 expression in cotton reduced the spread of V.dahliae and retarded the disease process.[Conclusion]GhSAG101 negatively regulates cotton resistance to V.dahliae and can be used as a candidate gene in the breeding of cotton with improved disease resistance.