大丽轮枝菌转录因子VDAG_04814基因功能探究  

Functional Exploration of Transcription Factor VDAG_04814 Gene in Verticillium dahliae

作　　者：贾鑫宇 东保柱 杨继峰 周洪友[1,2] JIA Xinyu;DONG Baozhu;YANG Jifeng;ZHOU Hongyou(College of Horticulture and Plant Protection,Inner Mongolia Agricultural University,Hohhot 010010,China;Key Laboratory of Biopesticide Creation and Resource Utilization for Autonomous Region Higher Education Institutions(Under Cultivation),Hohhot 010010,China;Inner Mongolia Hinggan League Institute of Agriculture and Animal Husbandry Sciences,Ulanhot 137400,China)

机构地区：[1]内蒙古农业大学园艺与植物保护学院,内蒙古呼和浩特010010 [2]生物农药创制与资源利用自治区高等学校重点实验室(培育中),内蒙古呼和浩特010010 [3]内蒙古自治区兴安盟农牧科学研究所,内蒙古乌兰浩特137400

出　　处：《华北农学报》2024年第6期202-209,共8页Acta Agriculturae Boreali-Sinica

基　　金：内蒙古自然科学基金项目(2022QN03001);中央引导地方科技发展资金项目(2023ZY0014);内蒙古自治区重点研发项目(2023YFHH0087)。

摘　　要：为明确Zn(Ⅱ)2Cys6转录因子基因VDAG_04814对大丽轮枝菌的生长发育及致病过程中的作用。利用聚乙二醇介导的同源重组构建VDAG_04814基因敲除突变体。将野生型菌株和突变体菌株分别接种至添加过氧化氢、氯化钠、氯化钾、山梨醇、十二烷基硫酸钠、刚果红的PDA培养基以及铺有无菌玻璃纸的培养基,分析其抗氧化胁迫、盐胁迫、渗透胁迫、细胞壁细胞膜完整性胁迫的水平和菌株穿透能力;测定其致病力,检测马铃薯植株中真菌生物量。经潮霉素筛选和PCR验证,正确的敲除转化子可扩增到上下游各1500 bp及敲除片段序列全长4500 bp的DNA条带。ΔVDAG_04814菌株生长速度缓慢、黑色素形成能力降低;在添加过氧化氢、氯化钠、氯化钾的氧化胁迫和盐胁迫培养基上,ΔVDAG_04814相较于野生型菌株受到的抑制率更高;在添加山梨醇的渗透胁迫培养基上,ΔVDAG_04814的生长抑制率显著低于野生型菌株;在添加十二烷基硫酸钠、刚果红的细胞壁细胞膜完整性胁迫培养基中生长并没有受到抑制;在铺有无菌玻璃纸培养基上,ΔVDAG_04814没有菌落长出,而野生型菌株有正常形态的菌落长出;致病力测定表明,ΔVDAG_04814菌株的致萎程度较野生型菌株显著下降,致萎指数为47.22~55.56。综合上述结果,VDAG_04814基因能够调控大丽轮枝菌的生长发育、抗胁迫能力、穿透能力以及对马铃薯的致病力,可为马铃薯黄萎病的防控提供新靶点。In order to clarify the role of the Zn(Ⅱ)2Cys6 transcription factor gene VDAG_04814 in the growth,development and pathogenicity of the Verticillium dahliae.It constructed a VDAG_04814 gene knockout mutant using homologous recombination mediated by polyethylene glycol.Wild-type and mutant strains were inoculated separately onto PDA media supplemented with hydrogen peroxide,sodium chloride,potassium chloride,sorbitol,sodium dodecyl sulfate,Congo red,as well as onto media overlaid with sterile cellophane,to analyze their levels of resistance to oxidative stress,salt stress,osmotic stress,stress on cell wall and plasma membrane integrity,and strain penetration ability.Their pathogenicity was assayed,and the fungal biomass in potato plants was detected.After hygromycin selection and PCR validation,the correct knockout transformants were able to amplify DNA bands of 1500 bp upstream and downstream,respectively,as well as the full-length 4500 bp knockout fragment sequence.The results demonstrated that the growth rate and melanin formation ability of theΔVDAG_04814 mutants were significantly reduced.On media subjected to oxidative stress and salt stress with the addition of hydrogen peroxide,sodium chloride,and potassium chloride,ΔVDAG_04814 showed a higher inhibition rate compared to the wild-type.On osmotic stress media with sorbitol,the growth inhibition rate ofΔVDAG_04814 was significantly lower than the wild type.No growth inhibition was observed forΔVDAG_04814 on media subjected to cell wall and membrane integrity stress with the addition of sodium dodecyl sulfate and Congo red.On media overlaid with sterile cellophane,no colonies grew forΔVDAG_04814,whereas the wild-type strain produced normal colonies.Pathogenicity tests indicated that the wilting index ofΔVDAG_04814 was significantly reduced compared to the wild-type,with wilting index ranging from 47.22 to 55.56.It has been demonstrated that VDAG_04814 can regulate the growth,development,stress resistance,penetration ability and pathogenicity of V.dahli

关 键 词：大丽轮枝菌 转录因子 基因功能验证 侵染能力 

分 类 号：Q965.9[生物学—昆虫学]