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Method for using antibiotic-free selection marker in barbadosnut gene transformation

A screening marker, antibiotic-free technology, used in biochemical equipment and methods, applications, genetic engineering, etc., to achieve the effects of high biosafety, simple method, and wide application value

Active Publication Date: 2013-01-30
CENTRAL SOUTH UNIVERSITY OF FORESTRY AND TECHNOLOGY
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Problems solved by technology

However, it has not been reported yet in the genetic transformation of Jatropha curcas

Method used

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  • Method for using antibiotic-free selection marker in barbadosnut gene transformation
  • Method for using antibiotic-free selection marker in barbadosnut gene transformation
  • Method for using antibiotic-free selection marker in barbadosnut gene transformation

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Embodiment 1

[0030] 1. Agrobacterium-mediated transformation of Jatropha curcas without antibiotics

[0031] Sterilize young leaves from the greenhouse and cut into 9mm 2 , the abaxial surface of the leaf was placed in the medium for pre-cultivation for 14 days, and then transformed. Construct the binary vector pC1304PMI, which includes the PMI gene (ie, 6-phosphate-mannose isomerase gene) as a screening gene and the GUS gene as a reporter gene, (pC1304PMI vector was constructed according to the literature method, reference: PMI gene as a selection marker Construction of plant expression vectors and its application in transgenic citrus (Journal of Agricultural Biotechnology 2008,16(5):858-864.). The vector was introduced into Agrobacterium tumefaciens GV3101 (purchased from Beijing Bei Nachuanglian Institute of Biotechnology) by electroporation, and a single colony of the transformed bacteria was inoculated into a culture medium containing 50 mg / L kalamycin and 50 mg / L rifampicin. in liq...

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Abstract

The invention discloses a method for using antibiotic-free selection marker in barbadosnut gene transformation. The method constructs a mannose antibiotic-free selection marker carrier, transfers a phosphomannose isomerase (PMI gene) and a reporter gene (GUS gene) into a barbadosnut cell by mediation by agrobacterium, uses mannose as a selective agent in place of an antibiotic, screens the transformed receptor cell, and detects the transgentic plants by polymerase chain reaction (PCR) to see if the transferred gene is expressed in the cell. Based on the technical scheme provided by the invention, a bud with uncertain resistance is successfully obtained by transforming a barbadosnut leave tray, the genetic transformation rate of cells screened by mannose antibiotic-free selection marker is over 10% which is two times that of cells screened by antibiotics. Besides the high transformation rate, the method also has the characteristics of convenience in detection, high biosafety of transgenic plants, and no potential adverse effect of antibiotics on the environment.

Description

technical field [0001] The invention belongs to the technical field of plant transgenesis, and in particular relates to an application method of an antibiotic-free screening marker in Jatropha curcas gene transformation. Background technique [0002] Jatropha curcas is one of the most researched energy plants in the world that can produce biodiesel. Carrying out genetic transformation can greatly promote the research on the genetic breeding of Jatropha curcas. In view of the difficulty of gene transformation of Jatropha curcas, there are not many reports on this aspect at home and abroad at present, and the selection marker system used for the transformation of Jatropha curcas in existing research is to use hygromycin, karimycin and other antibiotics or herbicides. Agent resistance screening. This type of drug has a greater impact on plant transformation, and will affect the growth and regeneration of transformed cells, especially for woody plants such as Jatropha curcas. T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 陈介南卢孟柱刘伯斌王琼
Owner CENTRAL SOUTH UNIVERSITY OF FORESTRY AND TECHNOLOGY
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