利用寄主诱导的基因沉默技术验证大丽轮枝菌糖代谢相关基因的致病力

【目的】筛选大丽轮枝菌(Verticillium dahliae)糖代谢相关基因，证实其与大丽轮枝菌的生长发育和致病性相关,为防治由大丽轮枝菌引起的棉花黄萎病提供理论基础。【方法】利用寄主诱导的基因沉默（host-induced gene silencing，HIGS）技术对大丽轮枝菌糖代谢相关基因进行筛选。将单个或多个含靶标基因的病毒载体注射本氏烟草瞬时表达基因，7-10 d后八氢番茄红素脱氢酶PDS会出现明显的烟草白化症状，蘸根法接种大丽轮枝菌V.d991，对应的大丽轮枝菌靶标基因会被干扰，接菌14 d后，观察转基因烟草表型，统计烟草病情指数，同时利用荧光定量PCR技术检测转基因烟草中大丽轮枝菌V.d991的生物量和靶标基因VDPDF-1、VDEG-1、VDMAN-1和VDPD-1的表达量。【结果】HIGS方法快速筛选获得大丽轮枝菌4个糖代谢相关基因，分别为VDPDF-1、VDEG-1、VDMAN-1和VDPD-1，相比空载体对照，转化这4个靶标基因的烟草病情指数分别降低了35%、30%、20%和10%，混合注射VDEG-1/VDPDF-1、VDPD-1/VDPDF-1/VDMAN-1、VDPD-1/VDMAN-1、VDPD-1/VDPDF-1和VDEG-1/VDPD-1的转基因烟草病情指数分别降低了45%、45%、40%、40%和35%，除VDPD-1转基因烟草外，其余转基因烟草与注射空载体的阴性对照相比病情指数都有显著降低。接种后大丽轮枝菌在寄主体内的生物量和干扰后靶基因表达量也有不同程度降低，混合基因注射比单基因注射干扰效果降低更明显,其中生物量降低最显著的是混合注射的EG/PDF(VDEG-1/VDPDF-1)和EG/PD(VDEG-1/VDPD-1)，生物量的降低可达0.94,干扰大丽轮枝菌的靶标基因后，表达量降低最明显的是混合注射PD/PDF(VDPD-1/VDPDF-1)中VDPDF-1，降低了0.91。【结论】HIGS方法快速筛选到大丽轮枝菌糖代谢相关的4个基因VDPDF-1、VDEG-1、VDMAN-1和VDPD-1，当其被干扰后，转基因烟草的病情指数降低，抗病性增强，而且混合注射多个基因载体比单基因注射效果更为明显，证实这些糖代谢相关的基因与大丽轮枝菌的致病性相关。

Verification of Verticillium dahliae Pathogenicity of Glycometabolism Related Genes by Using Host-Induced Gene Silencing Method

【Objective】 The objective of this study is to screen glycometabolism related genes from Verticillium dahliae, confirm the relationship between those genes and the growth and pathogenicity of V. dahliae, and to provide a theoretical basis for the control of plant Verticillium wilt. 【Method】 The glycometabolism related genes of V. dahliae were screened using host-induced gene silencing (HIGS) method. The Tobacco rattle virus vectors containing target genes were injected into tobacco for transient expression of those genes, then V.d991 was inoculatedto those transgenic tobacco using the root-dip approach, PDS would be whitened 7-10 days later, and their disease indexes were counted after 14 days. The fluorescence quantitative PCR was used to detect the amount of V.d991 biomass and the expression of VDPDF-1, VDEG-1, VDMAN-1and VDPD-1. 【Result】 Four glycometabolism related genes from V. dahliae (VDPDF-1, VDEG-1, VDMAN-1 and VDPD-1) were selected because of the great change of their disease indexes. Transgenic tobacco disease indexes of these four genes were reduced by 35%, 30%, 20%, and 10%, respectively, compared with the negative control. After injected with the mixture of VDEG-1/VDPDF-1, VDPD-1/VDPDF-1/ VDMAN-1, VDPD-1/VDMAN-1, VDPD-1/VDPDF-1,and VDEG-1/VDPD-1, transgenic tobacco disease indexes were reduced by 45%, 45%, 40%, 40%, and 35%. In addition to VDPD-1 transgenic tobacco, the transgenic tobacco disease indexes had significant differences compared with the control. After inoculation withV.d991, the V.d991 biomass and expression of target gene were decreased, mixed gene injection had better interference effect than the single gene. The most obvious biomass reduction wereEG/PDF (VDEG-1/VDPDF-1)and EG/PD (VDEG-1/VDPD-1), reached to 0.94, and the most significant interference effect was VDPDF-1, from PD/PDF (VDPD-1/VDPDF-1)，reduced by 0.91. 【Conclusion】 Four genes were selected from V. dahliae by HIGS method. When these genes were interfered, those plants had high resistance to disease, mixed injection of multiple gene vectors were more significant than the single gene. These genes were confirmed with the glycometabolism in V.d991, probably related with V. dahliae pathogenicity.