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
[ 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
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
基金the National Natural Science Foundation of China (No. 30571236)the Modern Agriculture (Citrus) Technology System (MATS) of Chinathe Science and Technology Department of Zhejiang Province, China (No. 2007C22007)
文摘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.
文摘[ 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.
基金supported by the National High Technology Development Program of China (No.2008AA10Z104)China Postdoctoral Science Foundation (No.20060600519)
文摘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.
基金Supported by the National Natural Science Foundation of China(30870006)by the Chinese Marine Renewable Energy Special Foundation(SDME2011SW01)by the National Programs for High Technology Research and Development of China(2012AA10180402)~~
