CAJ | 학술논문

【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。【目的】篩選大麗輪枝菌(Verticillium dahliae)糖代謝相關基因，證實其與大麗輪枝菌的生長髮育和緻病性相關,為防治由大麗輪枝菌引起的棉花黃萎病提供理論基礎。【方法】利用寄主誘導的基因沉默（host-induced gene silencing，HIGS）技術對大麗輪枝菌糖代謝相關基因進行篩選。將單箇或多箇含靶標基因的病毒載體註射本氏煙草瞬時錶達基因，7—10 d 後八氫番茄紅素脫氫酶 PDS 會齣現明顯的煙草白化癥狀，蘸根法接種大麗輪枝菌 V.d991，對應的大麗輪枝菌靶標基因會被榦擾，接菌14 d 後，觀察轉基因煙草錶型，統計煙草病情指數，同時利用熒光定量 PCR 技術檢測轉基因煙草中大麗輪枝菌 V.d991的生物量和靶標基因 VDPDF-1、VDEG-1、VDMAN-1和 VDPD-1的錶達量。【結果】HIGS 方法快速篩選穫得大麗輪枝菌4箇糖代謝相關基因，分彆為VDPDF-1、VDEG-1、VDMAN-1和 VDPD-1，相比空載體對照，轉化這4箇靶標基因的煙草病情指數分彆降低瞭35%、30%、20%和10%，混閤註射 VDEG-1/VDPDF-1、VDPD-1/VDPDF-1/VDMAN-1、VDPD-1/VDMAN-1、VDPD-1/VDPDF-1和VDEG-1/VDPD-1的轉基因煙草病情指數分彆降低瞭45%、45%、40%、40%和35%，除 VDPD-1轉基因煙草外，其餘轉基因煙草與註射空載體的陰性對照相比病情指數都有顯著降低。接種後大麗輪枝菌在寄主體內的生物量和榦擾後靶基因錶達量也有不同程度降低，混閤基因註射比單基因註射榦擾效果降低更明顯,其中生物量降低最顯著的是混閤註射的 EG/PDF(VDEG-1/VDPDF-1)和 EG/PD(VDEG-1/VDPD-1)，生物量的降低可達0.94,榦擾大麗輪枝菌的靶標基因後，錶達量降低最明顯的是混閤註射 PD/PDF(VDPD-1/VDPDF-1)中 VDPDF-1，降低瞭0.91。【結論】HIGS 方法快速篩選到大麗輪枝菌糖代謝相關的4箇基因 VDPDF-1、VDEG-1、VDMAN-1和 VDPD-1，噹其被榦擾後，轉基因煙草的病情指數降低，抗病性增彊，而且混閤註射多箇基因載體比單基因註射效果更為明顯，證實這些糖代謝相關的基因與大麗輪枝菌的緻病性相關。
【목적】사선대려륜지균(Verticillium dahliae)당대사상관기인，증실기여대려륜지균적생장발육화치병성상관,위방치유대려륜지균인기적면화황위병제공이론기출。【방법】이용기주유도적기인침묵（host-induced gene silencing，HIGS）기술대대려륜지균당대사상관기인진행사선。장단개혹다개함파표기인적병독재체주사본씨연초순시표체기인，7—10 d 후팔경번가홍소탈경매 PDS 회출현명현적연초백화증상，잠근법접충대려륜지균 V.d991，대응적대려륜지균파표기인회피간우，접균14 d 후，관찰전기인연초표형，통계연초병정지수，동시이용형광정량 PCR 기술검측전기인연초중대려륜지균 V.d991적생물량화파표기인 VDPDF-1、VDEG-1、VDMAN-1화 VDPD-1적표체량。【결과】HIGS 방법쾌속사선획득대려륜지균4개당대사상관기인，분별위VDPDF-1、VDEG-1、VDMAN-1화 VDPD-1，상비공재체대조，전화저4개파표기인적연초병정지수분별강저료35%、30%、20%화10%，혼합주사 VDEG-1/VDPDF-1、VDPD-1/VDPDF-1/VDMAN-1、VDPD-1/VDMAN-1、VDPD-1/VDPDF-1화VDEG-1/VDPD-1적전기인연초병정지수분별강저료45%、45%、40%、40%화35%，제 VDPD-1전기인연초외，기여전기인연초여주사공재체적음성대조상비병정지수도유현저강저。접충후대려륜지균재기주체내적생물량화간우후파기인표체량야유불동정도강저，혼합기인주사비단기인주사간우효과강저경명현,기중생물량강저최현저적시혼합주사적 EG/PDF(VDEG-1/VDPDF-1)화 EG/PD(VDEG-1/VDPD-1)，생물량적강저가체0.94,간우대려륜지균적파표기인후，표체량강저최명현적시혼합주사 PD/PDF(VDPD-1/VDPDF-1)중 VDPDF-1，강저료0.91。【결론】HIGS 방법쾌속사선도대려륜지균당대사상관적4개기인 VDPDF-1、VDEG-1、VDMAN-1화 VDPD-1，당기피간우후，전기인연초적병정지수강저，항병성증강，이차혼합주사다개기인재체비단기인주사효과경위명현，증실저사당대사상관적기인여대려륜지균적치병성상관。
[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 inoculated to 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 with V.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 were EG/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.