香蕉枯萎病菌Dicer-like基因的功能分析  

Functional Analysis of Dicer-like Genes in Fusarium oxysporum f.sp.cubense Race 4

作　　者：张欣 漆艳香[1] 曾凡云[1] 王艳玮 谢培兰 谢艺贤[1] 彭军[1] ZHANG Xin;QI Yanxiang;ZENG Fanyun;WANG Yanwei;XIE Peilan;XIE Yixian;PENG Jun(Key Laboratory of Integrated Pest Management on Tropical Crops,Ministry of Agriculture and Rural Affairs,Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests,Environment and Plant Protection Institute,Chinese Academy of Tropical Agricultural Sciences,Haikou 571101,China)

机构地区：[1]中国热带农业科学院环境与植物保护研究所,农业农村部热带作物有害生物综合治理重点实验室,海南省热带农业有害生物监测与控制重点实验室,海口571101

出　　处：《园艺学报》2023年第2期279-294,共16页Acta Horticulturae Sinica

基　　金：海南省自然科学基金项目(322RC756);中国热带农业科学院环境与植物保护研究所自主选题项目(hzsjy2019004);中国热带农业科学院基本科研业务费专项资金项目(1630042018010);海南省基础与应用基础研究计划(自然科学领域)高层次人才项目(2019RC269);国家现代农业产业技术体系建设专项资金项目(CARS-31-07)。

摘　　要：香蕉枯萎病病原菌尖孢镰刀菌古巴专化型(Fusarium oxysporum f.sp.cubense)4号生理小种(Foc4)含有两个进化上高度保守的Dicer-like基因FocDCL1和FocDCL2。为了探究该基因的功能及其在RNAi中的作用机制,利用同源重组的方法获得ΔFocDCL1、ΔFocDCL2和ΔFocDCL1/2基因敲除突变体。表型分析显示,与Foc4野生型相比,突变体菌丝的营养生长无显著差异,但产孢量下降;ΔFocDCL2突变体在非生物胁迫刚果红处理后菌落明显变小且气生菌丝增多;ΔFocDCL2和ΔFocDCL1/2的致病力下降。miRNA深度测序数据显示,与Foc4野生型相比,ΔFocDCL1、ΔFocDCL2和ΔFocDCL1/2敲除突变体的miRNA长度分布和5′–端首位碱基出现频率都发生了改变,都能产生自身特异miRNA。DCL功能存在交叉和冗余,miRNA可以通过依赖独立的、交叉的或者联合的DCL发生;此外,鉴定到不依赖DCL形成的miRNA。这些结果表明FocDCL在产孢量、非生物胁迫、致病力以及小RNA发生中发挥作用。The fungal pathogen Fusarium oxysporum f.sp.cubense(Foc)is an important soil-borne pathogenic fungus which causes Banana Fusarium wilt.The Foc race 4(Foc 4)attacking almost all Cavendish cultivars is currently noteworthy in global banana production.In order to explore the function of DCL-like genes in Foc4,the gene-knockout mutantsΔFocDCL1,ΔFocDCL2 andΔFocDCL1/2 were obtained by homologous recombination technique.The phenotype analysis showed the mutants showed no significant differences on vegetative growth but significant reduced in conidia production compared to the Foc4.TheΔFocDCL2 mutant increased sensitive to Congo red,and the mutant significantly reduced the size of the colony and increased the number of aerial hyphae after Congo red treatment.Next,the pathogenicity test showed that virulence ofΔFocDCL2 andΔFocDCL1/2 were significantly reduced compared with Foc4,respectively.Furthermore,miRNA deep sequencing data showed the length distribution of the total reads,and the frequency of the 5′first nucleotide bias of the mutants were markedly changed compared with Foc4,respectively.The miRNA could be generated depending on individual,alternative or joint DCL manners,indicating the FocDCL with overlapped and redundant function in small RNA biogenesis.In addition,DCLs-independent miRNA also be identified in the sequencing data.These results indicated that FocDCL function involved in the stress response,conidia,pathogenicity and generation of miRNAs in Foc4.

关 键 词：香蕉 枯萎病 Fusarium oxysporum f.sp.cubense Dicer-like基因 致病力测试 miRNA 

分 类 号：S668.1[农业科学—果树学]