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Ammopiptanthus mongolicus cold-resistant gene AmEBP1

A technology of holly and gene, which is applied in the field of cold-resistant gene AmEBP1 of holly, which can solve the problems of cold-resistant gene cloning and transformation that have not been reported successfully.

Inactive Publication Date: 2010-11-24
TAISHAN RES INST OF FORESTRY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there have been no successful reports on the cloning and transformation of cold resistance genes in woody plants

Method used

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  • Ammopiptanthus mongolicus cold-resistant gene AmEBP1
  • Ammopiptanthus mongolicus cold-resistant gene AmEBP1
  • Ammopiptanthus mongolicus cold-resistant gene AmEBP1

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0010] (1) The in vitro regeneration system of woody plants is used as the transformation material, and genetic transformation is carried out by means of Agrobacterium dipping, pollen tube passage, gene gun and other methods.

[0011] (2) Conversion operation steps

[0012] 1. Agrobacterium transformation method

[0013] (1) The AmEBP1 gene expression vector was transformed into Agrobacterium tumefaciens LBA4404 by the three-parent hybridization method, and used as Agrobacterium infecting transformed plants.

[0014] (2) Bacterial liquid preparation

[0015] 1) Use an inoculation loop to scrape the surface of the frozen culture of Agrobacterium strains containing recombinant plasmids, streak on the above-mentioned YEP plate containing the corresponding antibiotics, and place it in a constant temperature incubator at 28°C for 2 days, until colonies grow out on the plate It can be used for liquid culture. Plates with colonies were stored in a 4°C refrigerator. After one mont...

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Abstract

The invention discloses an ammopiptanthus mongolicus cold-resistant gene AmEBP1, and relates to a national secondary endangered and protected evergreen broad-leaved woody plant derived from the tertiary period in the desert regions of northwest China. The molecular cloning of the gene comprises the following steps of: obtaining a low temperature-induced and up regulation-expressed EST fragment through low temperature induction and differential expression analysis; and obtaining a full-length cDNA sequence of the gene through RACE amplification, and registering in a gene bank with the registration number of DQ519359. The AmEBP1 contains a whole ORF region with 1,188 nucleotides at the overall length, and the nucleotides code 395 amino acids and a terminator codon (TGA); a eukaryotic expression vector pCAMBIA2300-AmEBR1 is used to transform arabidopsis thaliana, and the obtained transgenic plant has improved cold resistance; and the ammopiptanthus mongolicus cold-resistant gene AmEBP1 is the first international woody plant cold-resistant gene which is cloned and passes genetic transformation verification.

Description

1. Technical field [0001] The present invention relates to a national second-class endangered plant originating from the ancient tertiary period in the desert area of ​​northwest my country, and it is also the molecule of the cold resistance gene AmEBP1 of the evergreen broad-leaved woody plant holly that has been preserved so far in the desert area of ​​central Asia. Cloning and genetic transformation. Through low temperature induction and differential expression analysis, the EST fragment of the antifreeze gene of A. chinensis was obtained by low temperature induction and expression, and then the full-length cDNA sequence of the antifreeze gene AmEBP1 of A. chinensis was obtained by RACE amplification, which was registered in the gene bank , registered as DQ519359. AmEBP1 contains a complete ORF region, the full length of ORF is 1188 nucleotides, encoding 395 amino acids and a stop codon (TGA). Subsequently, the eukaryotic expression vector pCAMBIA2300-AmEBP1 of the cold re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C12N15/54C12Q1/68C12N15/11
Inventor 冯殿齐王斌刘静曹鹏秀翁曼丽
Owner TAISHAN RES INST OF FORESTRY
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