农杆菌介导的获取疏绵状嗜热丝孢菌插入突变体体系的建立_英文_.docxVIP

农杆菌介导的获取疏绵状嗜热丝抱菌插入突变体体系的建立—英文— Research Paper Acta Microbiologica Sinica 微生物学报 52 ( 12 ) : 1449 ) 145 7 ; 4 December201 2 ISSN 0001 )620 9 ; CN 11 )199 5 / Q http: / / journals, im, ac, cn / actamicrocn Agrobacterium tumefaciens-mediatedtransformation as a tool for insertional mutagenesis in thermophilic fungus Thermomyces lanuginosus # # *Hua Han, Xiaoxue Xu, Yanjie Peng, Dehui Kong, DuochuanL i Department Eonf vronmenta Boogy, Shandong Agrcutura Unversty, TaJ an 271018 ,Chnailililliiii Abstract: , Objective, To establish a stable transformation system of ththee rmophilic fungusT hermomyces lanuginosus for ts nsertona mutageness, , ehods, Agrobacterum tumefacens-medated transformaton (ATMT ) was apped to iiiliMtiiiiliestablish transformation system oTf , lanuginosus, Southernb lotting of hph gene acnld on ing of transforming DNA ( T- DNA) flanking sequences were used tteorm dinee insert number ansdit e of T-DNA in the fungal genome, respectively, , Results, A reliable transformation methodi s established for T, lanuginosus, Specifically, pre-germinating spores of T, lan uginosus used ate o- cultivated period was a rperequisite, T, lanuginosus germinating spores co-cultivated with Agrobacterium tumefaciens at 28 ? for 48 h achieved the highest transformation efficiency, Addition of A cetosyringone ( AS) during preculture of A, tumefaciens and co-cultivation of T, lanuginosus germinating spores with A, tumefaciens was sseentially required, and the besrte suits were obtained with AS at thee oncentration of 500 M , S outhe rnb lotting u analysis showed that jmoriaty of transforman(t s 79 ? 2 % ) contained a single insertion of T -DNA, Thermal asymmetric interlaced PCR ( TAIL-PCR) analysis showed randoimns ertion of T-DNA in the fungal genome, Using the rtansformation system, somes table phenotypic mutants oTf, lanuginosus wereo btained, ,Conclusion, We repo, rtfor thef irst time, a simple and eiffeient method fotrra nsforming T, lanuginosus by using ATMT, This approa