西瓜枯萎病菌T—DNA插入轉化子庫的構建及其質量評價

裴月令　曾凡云　彭軍　龍海波　郭建榮

摘 要 為研究西瓜枯萎病菌的致病分子機理，開展了病原菌的轉化子庫構建工作。采用攜帶潮霉素抗性pTFCM雙元載體的農桿菌AGL-1介導的ATMT轉化方法，獲得菌株FON-01的轉化子，通過表型觀察篩選突變體。結果表明：當乙酰丁香酮（AS）濃度為200 μmol/L、農桿菌OD600為0.4、分生孢子濃度為1×106個/mL時，于28 ℃共培養48 h轉化效率高達（663.33±24.95）個/106個孢子。構建了總數為3 832個的轉化子庫并對其進行了質量評價，從2 201個轉化子中篩選出菌落形態、生長速度及產孢量等方面有所變異的突變體73個。病原菌轉化子庫的構建為進一步開展致病分子機理及相關基因克隆等方面研究奠定基礎。

關鍵詞 西瓜枯萎病菌；ATMT；轉化子庫

中圖分類號 S432.1 文獻標識碼 A

Generation of a Transformant Library of Fusarium oxysporum.

f. sp. niveum by ATMT and Its Quality Assessment

PEI Yueling，ZENG Fanyun，PENG Jun，LONG Haibo，GUO Jianrong﹡

Environment and Plant Protection Institute，CATAS/Key Laboratory of Integrated Pest Management on

Tropical Crops，Ministry of Agriculture/Hainan Key Laboratory for Monitoring and Control of Tropical

Agricultural Pests，Haikou，Hainan 571101， China

Abstract In order to study the pathogenic molecular mechanism of Fusarium oxysporum. f. sp. Niveum，the transformant library was conducted. Agrobacterium strainAGL-1 containing the binary vector pTFCM was used for the transformation of strain FON-01，and the mutants were screened by penotype observation. Transformation efficiency was（663.33±24.95），when the acetosyringone concentration was 200 μmol/L，OD600 of AGL-1 was 0.4，conidia concehntration was 106/mL，co-culture time was 48 h and co-culture temperature was 28 ℃. 3 832 transformants were obtained and quality assessment to the library was finished. 73 mutants with variation of colony shape，growth speed and sporulation ability were screened from 2 201 transformants. The construction of transformants library would provide the foundation for further study of pathogenic molecular mechanism and related genescloning.

Key words Fusarium oxysporum. f. sp. niveum；ATMT；Transformant library

doi 10.3969/j.issn.1000-2561.2015.01.013

西瓜[Citrullus lanatus（Thunb.）Matsum & Nakai]是中國重要的水果之一，在中國農業經濟中占有重要地位。中國是西瓜生產和消費大國，種植面積和產量均占世界一半以上（FAO）。與其它瓜菜類作物相比，種植西瓜的經濟效益非常高，已經成為增加農戶經濟收入的重要途徑。由尖孢鐮刀菌西瓜專化型[Fusarium oxyxporum Schl. f. sp. niveum（E. F. Smith）Snyder et Hansen]引起的西瓜枯萎病是一種世界性土傳病害，是中國西瓜種植業中的重要病害，生產上防治困難，可造成30%以上的產量損失[1]。

隨著農業經濟的發展，中國的西瓜種植業也呈現設施化、基地化、專業化的發展趨勢，重茬現象非常普遍，為西瓜枯萎病的發生、發展和流行創造了極為有利的條件，導致該病嚴重發生。近年來，該病在中國各個西瓜種植區均有發生，一般病株率10%～20%，重者達80%～90%[2]，已經成為制約西瓜產業發展的主要因素之一。……