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Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic fungus Penicillium digitatum 被引量:10
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作者 Ji-ye WANG Hong-ye LI 《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》 SCIE CAS CSCD 2008年第10期823-828,共6页
Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformat... Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 106 conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alter-natively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics. 展开更多
关键词 Penicillium digitatum agrobacterium tumefaciens-mediated transformation atmt Hygromycin B resistance gene lnsertional mutagenesis
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Establishment of Agrobacterium tumefaciens-Mediated Transformation System for Rice Sheath Blight Pathogen Rhizoctonia solani AG-1 IA 被引量:3
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作者 YANG Ying-qing YANG Mei +3 位作者 LI Ming-hai LI Yong HE Xiao-xia ZHOU Er-xun 《Rice science》 SCIE 2011年第4期297-303,共7页
To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transf... To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transformation.The conditions for transformation of isolate GD118 were optimized in five aspects,i.e.pre-induction time,co-culture time,acetosyringone(AS) concentration at the co-culture phase,co-culture temperature and pH value of induction solid medium(ISM) at the co-culture phase.Finally,a system of Agrobacterium tumefaciens-mediated transformation(ATMT) for R.solani AG-1 IA was established successfully.The optimal conditions for this ATMT system were as follows:the concentration of hygromycin B at 30 μg/mL for transformant screening,8 h of pre-induction,20 h of co-culture,200 μmol/L of AS in ISM,co-culture at 25 ℃ and pH 5.6 to 5.8 of ISM at the co-culture phase.The transformants still displayed high resistance to hygromycin B after subculture for five generations.A total of 10 randomly selected transformants were used for PCR verification using the specific primers designed for the hph gene,and the results revealed that an expected band of 500 bp was amplified from all of the 10 transformants.Moreover,PCR amplification for these 10 transformants was carried out using specific primers designed for the Vir gene of A.tumefaciens,with four strains of A.tumefaciens as positive controls for eliminating the false-positive caused by the contamination of A.tumefaciens.An expected band of 730 bp was amplified from the four strains of A.tumefaciens,whereas no corresponding DNA band could be amplified from the 10 transformants.The results of the two PCR amplifications clearly showed that T-DNA was indeed inserted into the genome of target isolate GD118. 展开更多
关键词 rice sheath blight Rhizoctonia solani agrobacterium tumefaciens-mediated transformation t-dna insertional mutagenesis METHODOLOGY
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Establishment of Agrobacterium tumefaciens-mediated Genetic Transformation System for Aspergillus awamori
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作者 Feng CHEN Kun WANG +3 位作者 Chao YIN Deming LI Nan REN Junxing LI 《Agricultural Biotechnology》 CAS 2012年第3期44-48,51,共6页
[ Objective ] This study aimed to establish Agrobacterium tumefaciens-mediated genetic transformation system for Aspergillus awamori and investigate the feasibility of expressing heterologous proteins in A. awamori. [... [ Objective ] This study aimed to establish Agrobacterium tumefaciens-mediated genetic transformation system for Aspergillus awamori and investigate the feasibility of expressing heterologous proteins in A. awamori. [ Method] Appropriate A. awamori host strains were determined according to the secretory protein profile. Selectable marker was selected for genetic transformation by drug sensitivity analysis. The established A. awamori genetic transformation system was used for transformation and expression analysis of Rhizomucor miehei lipase (RML). The feasibility of using A. awamori to express heterologous proteins was investigated by identification of transformants and property analysis. [ Result~ Based on the analysis of secretory protein profile, A. awamori strains CBS115.52 and CICC2257 were determined as the host strains for heterologous protein expression ; drug sensitivity analysis shows that hygromycin B resistance gene ( HygBr ) is an effective ge- netic seleetable marker; by using Agrobacterium tumefaciens-mediatcd transformation (ATMT) method, the plasmid pHGW-amdS containing HygBr was successfully transformed into A. awamori strain CBS115.52 to establish the genetic transformation system ofA. awamorl with HygBr as selectable marker. RML was transformed into A. awamori and its expression was validated by substrate hydrolysis test, SDS-PAGE and Western blot. [ Conclusion] This study demonstrates that the genetic transformation system of A. awamori mediated by Agrobacterium tumefaciens has potential feasibility for expression of heterologous proteins. 展开更多
关键词 Aspergillus awamoH agrobacterium tumefaciens-mediated transformation atmt Hygromycin B resistant gene (HygBr) Rhizomucor miehei lipase (RML)
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Induction of chromosomal inversion by integration of T-DNA in the rice genome 被引量:7
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作者 Chuanfeng Zhu,Jiahe Wu,Chaozu He State Key Laboratory of Plant Genomics,Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China 《Journal of Genetics and Genomics》 SCIE CAS CSCD 2010年第3期189-196,共8页
Transfer DNA (T-DNA) of Agrobacterium tumefaciens integration in the plant genome may lead to rearrangements of host plant chromosomal fragments, including inversions. However, there is very little information conce... Transfer DNA (T-DNA) of Agrobacterium tumefaciens integration in the plant genome may lead to rearrangements of host plant chromosomal fragments, including inversions. However, there is very little information concerning the inversion. The present study re- ports a transgenic rice line selected from a T-DNA tagged population, which displays a semi-dwarf phenotype. Molecular analysis of this mutant indicated an insertion of two tandem copies of T-DNA into a locus on the rice genome in a head to tail mode. This insertion of T-DNA resulted in the inversion of a 4.9 Mb chromosomal segment. Results of sequence analysis suggest that the chromosomal inversion resulted from the insertion of T-DNA with the help of sequence microhomology between insertion region of T-DNA and target sequence of the host plant. 展开更多
关键词 agrobacterium tumefaciens-mediated transformation atmt t-dna chromosomal inversion
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