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- 저자 : Angenon, (검색결과 3 건)
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Genomic stability in Nicotiana plants upon silencing of the mismatch repair gene MSH2
Marcke, Inke Van,Angenon, Geert 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.4
The Mismatch Repair (MMR) system is a highly conserved pathway for the maintenance of genomic stability in many organisms. In plants, this is particularly important because of the lack of a reserved germline. Suppression of MMR leads to an accumulation of random mutations in the genome over successive generations, and thus maximizes genetic diversity. MMR deficiency has been shown to be a useful technique in plant breeding, complementary to chemical or physical mutagenesis. We have developed an artificial microRNA (amiRNA) targeting the MSH2 gene, which is generally applicable in Solanaceae. Two amiRNA precursors were inserted in a transformation vector, under the control of the CaMV 35S promoter and the meiosis active AtDMC1 promoter, respectively. Introduction of this amiRNA construct in Nicotiana tabacum and N. plumbaginifolia reduced the MSH2 transcript levels to 20-30 %. Morphological and developmental abnormalities and plants with white sectors on the first pair of leaves or on the cotyledons (referred to as 'chimeric albinos') appeared in the transformed Nicotiana lines at higher frequencies than in the control lines. Also, some plants which show an increased tolerance for the herbicide chlorsulfuron were found. However, the mutant phenotypes were not transmitted to subsequent generations. We conclude that the designed amiRNA was capable of suppressing the MSH2 activity, which caused the occurrence of somatic mutations. Apparently, the silencing of MSH2 was not strong enough in the germline to cause inheritable mutations.
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Genomic stability in Nicotiana plants upon silencing of the mismatch repair gene MSH2
Inke Van Marcke,Geert Angenon 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.4
The Mismatch Repair (MMR) system is a highly conserved pathway for the maintenance of genomic stability in many organisms. In plants, this is particularly important because of the lack of a reserved germline. Suppression of MMR leads to an accumulation of random mutations in the genome over successive generations, and thus maximizes genetic diversity. MMR deficiency has been shown to be a useful technique in plant breeding, complementary to chemical or physical mutagenesis. We have developed an artificial microRNA (amiRNA) targeting the MSH2 gene, which is generally applicable in Solanaceae. Two amiRNA precursors were inserted in a transformation vector, under the control of the CaMV 35S promoter and the meiosis active AtDMC1 promoter, respectively. Introduction of this amiRNA construct in Nicotiana tabacum and N. plumbaginifolia reduced the MSH2 transcript levels to 20–30 %. Morphological anddevelopmental abnormalities and plants with white sectors on the first pair of leaves or on the cotyledons (referred to as ‘chimeric albinos’) appeared in the transformed Nicotiana lines at higher frequencies than in the control lines. Also, some plants which show an increased tolerance for the herbicide chlorsulfuron were found. However, themutant phenotypes were not transmitted to subsequent generations. We conclude that the designed amiRNA was capable of suppressing the MSH2 activity, which causedthe occurrence of somatic mutations. Apparently, the silencing of MSH2 was not strong enough in the germline to cause inheritable mutations.
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Agrobacterium tumefaciens-mediated genetic transformation of Digitalis purpurea L.
Naivy Pe´rez-Alonso,Elio Jime´nez,Borys Chong-Pe´rez,Alina Capote,Anabel Pe´rez,Yovanny Izquierdo,Geert Angenon 한국식물생명공학회 2014 Plant biotechnology reports Vol.8 No.5
Genetic transformation is a tool of specialinterest for developing new biotechnological strategies forthe production of bio-active compounds such as cardenolides,which are exclusively obtained from plants. To date,Digitalis plants are the main economically viable source ofcardenolides for the pharmaceutical industry. This studydescribes the development of efficient plant regenerationand Agrobacterium-mediated genetic transformation protocolsfor Digitalis purpurea L. First, a plant regenerationprocedure starting from leaf segments of in vitro-cultivatedplants was established and the minimal inhibitory concentrationof G-418 (geneticin) for callus induction wasdetermined. Both leaf segments and callus tissue weresensitive to G-418 70 mg l-1. Afterwards, two Agrobacteriumstrains were used to test their T-DNA transferability on D. purpurea leaf tissues, EHA105 andC58C1RifR (pMP90), both harboring the binary vectorpTJK136. Strain C58C1RifR (pMP90) yielded a highernumber of transformed plants than EHA105. Successfultransformation was confirmed by histochemical b-glucuronidase(GUS) assays of the putative transgenic tissuesand PCR analyses using b-glucuronidase (uidA)- andneomycin phosphotransferase II (nptII)-specific primers. Southern blot hybridization confirmed the stable integrationof the nptII gene in the transgenic plants. In total, 518independent transgenic lines were regenerated with anaverage of 6.91 transgenic lines per initial leaf segmentinfected with A. tumefaciens strain C58C1RifR (pMP90). To date, only a few studies have been published on thegenetic transformation of Digitalis species. The protocolsfor plant regeneration and genetic transformation describedin this paper will contribute to functional studies for abetter understanding of cardenolide biosynthetic pathwaysand the metabolic engineering of cardenolides to develophigh-yielding improved genotypes.
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