Genetic Transformation in Rhizopus arrhizus of Antisense VeFAD2 Gene via Agrobacterium tumefaciens-mediated Transformation and Its Influencing Factors

LIU Ming-jing; LI Ji-yuan; FAN Zheng-qi; TIAN Min; FAN Miao-hua

Agrobacteriurn tumefaciens-mediated transformation (AtMT) method was used to study the genetic transformation in Rhizopus arrhizus of antisense expression vector pBI121-fad2 of VeFAD2 gene. We studied the key factors influencing transformation, such as A.tumefaciens strains,bacterial cell volume initially used, co-cultivation time, effective period and concentration of AS. The results shows that the ideal A.tumefaciens strain is AGL-1, the optimum bacterial cell volume initially used is 50-100 μL and co-culture 24 hours will get the most transformants. At co-culture period, AS induction is indispensable. It is beneficial to improve transformation ratio by adding to 200 μmol·L-1 AS at preincubate period and increasing AS concentration to 400-600 μmol·L-1 at co-culture period. It determined that antisense VeFAD2 gene has been integrated into the genome of R. arrhizus by PCR-Southern detection.

1. Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang 311400, Zhejiang, China

Abstract: Agrobacteriurn tumefaciens-mediated transformation (AtMT) method was used to study the genetic transformation in Rhizopus arrhizus of antisense expression vector pBI121-fad2 of VeFAD2 gene. We studied the key factors influencing transformation, such as A.tumefaciens strains,bacterial cell volume initially used, co-cultivation time, effective period and concentration of AS. The results shows that the ideal A.tumefaciens strain is AGL-1, the optimum bacterial cell volume initially used is 50-100 μL and co-culture 24 hours will get the most transformants. At co-culture period, AS induction is indispensable. It is beneficial to improve transformation ratio by adding to 200 μmol·L-1 AS at preincubate period and increasing AS concentration to 400-600 μmol·L-1 at co-culture period. It determined that antisense VeFAD2 gene has been integrated into the genome of R. arrhizus by PCR-Southern detection.

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Reference (19)

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Genetic Transformation in Rhizopus arrhizus of Antisense VeFAD2 Gene via Agrobacterium tumefaciens-mediated Transformation and Its Influencing Factors

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通讯作者: 陈斌, bchen63@163.com

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