尖孢镰刀菌古巴专化型4号生理小种特有SIX基因的功能分析

Fusarium oxysporum f. sp. Cubense (Foc), causal agent of Fusarium wilt of banana, is among the most destructive pathogens of banana and plantain. Race 4 isolates affect banana cultivars that are susceptible to races 1 and 2, as well as the commercially significant Cavendish cultivars. Recent researchs with F. oxysporum f. sp. lycopersici (Fol) have elucidated the role of SIX-genes (secreted in xylem),which encode several specific and small and cysteine-rich proteins, in pathogenicity in the Fol tomato pathosystem. Foc,which infectied the vascular as Fol isolates,may switch on similar genes to determine its ability in host xylem colonization.The Foc4 specific SIX genes may determine that Foc4 can infect wider banana cultivars than Foc1.We searched across the Foc1 and Foc4 assemblies to identify the ortholog counterparts of SIX genes (SIX1-SIX8). Our analysis revealed that there are different type or copies of SIX genes in Foc1 and Foc4,and there are specific SIX genes in Foc4.And Meldrum infered that SIX7 and SIX8 genes may play a role in cultivar specificity in Foc.So we will use hybridisation analysis and PCR to testify the difference between the two races by using different geological isolates and international standard strains, then we will knockout and complement the Foc4 specific SIX gene by transformation and check the deletion mutants' and complementary isolats' pathogenicity to different hosts to investigate whether it affect Foc4 host diversity selection.The results help explore the mechanism for host diversity selection of Foc4 from the molecular level,and will help do research about interaction between Foc and xylem of banana in the future, and also can provide basis for banana Fusarium wilt resistant breeding.

引起香蕉枯萎病的尖孢镰刀菌古巴专化型4号生理小种（Foc4）比1号生理小种（Foc1）侵染的宿主范围更广，几乎危害所有香蕉品种。近来对尖孢镰刀菌番茄专化型（Fol）的相关研究揭示其入侵番茄后在木质部分泌SIX蛋白启动致病力。Foc亦可能启动相似基因决定其在寄主木质部的定植能力，Foc4中特有的SIX基因可能为其比Foc1侵染的香蕉品种更广的决定因子。项目组比较已完成的Foc1与Foc4 的基因组序列，发现Foc4中存在特有的SIX基因。为此，本项目拟采用DNA杂交与PCR方法分析2个小种中的SIX基因，确定Foc4特有的SIX基因，进而开展该基因的敲除与回复，检测基因缺失突变体与回补菌株对不同香蕉品种的致病力，明确Foc4特有的SIX基因在寄主差异性选择中发挥的作用。研究结果有利于从分子水平上探讨Foc4寄主差异性选择的机制，为开展Foc4与香蕉在木质部内的互作及抗病育种工作提供基础。

尖孢镰刀菌在与寄主的相互作用中分泌几个特定的富含半胱氨酸的小分子量蛋白进入木质部中启动致病力，被称为SIX (secreted in xylem)蛋白，为明确其在寄主选择中的作用，比较分析了尖孢镰刀菌古巴专化型中SIX1、SIX2、SIX4、SIX6、SIX7、SIX8同源基因序列，并通过同源重组转化方法敲除其中4号生理小种中的SIX2、SIX6基因后分析了其在寄主差异性选择中的功能。.应用PCR方法扩增分析56株尖孢镰刀菌古巴专化型与18株其它专化型及非致病型尖孢镰刀菌菌与其它种或属共21株菌株中的SIX1、SIX2、SIX4、SIX6、SIX7、SIX8基因。非致病性尖孢镰刀菌与其它镰刀属种或其它属的菌株DNA中均无上述SIX基因目的条带；SIX1基因的2个引物均能从供试的Foc菌株及部分其它专化型菌株DNA中扩增出目的条带；仅从供试的Foc4菌株基因组DNA中扩增出SIX2基因序列，且灵敏度达5 pg/25μL，并可用于检测感病的球茎组织；SIX4基因引物仅能从供试的番茄专化型与部分甘蓝专化型菌株DNA中扩增出目的条带；SIX6引物仅能从供试Foc1、Foc2、Foc4菌株DNA中扩增出目的条带；SIX7基因引物序列仅能从供试的亚热带4号生理小种(ST4)中扩增出663 bp的目的条带；SIX8基因引物能从所有供试的致病尖孢镰刀菌中DNA扩增出目的条带。.将Foc4的FoSIX2 和FoSIX6基因左右两端DNA片段分别克隆连接到pCT74载体中以获得同源重组DNA片段，通过原生质体转化，把GFP和hph转入Foc4中，采用4对特异性引物对检测筛选阳性转化子，获得FoSIX2 和FoSIX6两个基因的敲除转化子。通过对ΔFoSIX2和ΔFoSIX6突变体的生物学特征观察， FoSIX2 和FoSIX6基因缺失使菌落形态呈现不规则状，菌丝在显微镜下生长速率减慢，菌丝稀疏，生长速率减慢，产孢率降低，菌丝异核率增加。ΔFoSIX2和ΔFoSIX6突变体都能穿透玻璃纸生长，孢子在香蕉苗的幼嫩根部附着量减少，孢子入侵数目降低。ΔFoSIX2突变菌株基本上丧失了对巴西蕉的致病力，而对粉蕉仍有较强的致病能力；ΔFoSIX6突变菌株则对粉蕉苗、巴西香蕉苗盆栽致病力均呈极显著下降。通过KOV21质粒将敲出基因进行回复突变得到回复突变体，而回复突变体表型和致病力均可恢复至野生型。