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Omega 3 fatty acid desaturase gene of halophyte, expression vector thereof, vegetable cell and plant transformed therewith

A technology of desaturase and halophytes, applied in angiosperms/flowering plants, cells modified by introducing foreign genetic material, botanical equipment and methods, etc., can solve the problem that there is no ω3 fatty acid desaturase gene, etc.

Inactive Publication Date: 2005-03-16
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No ω3 fatty acid desaturase gene has been reported from halophytes

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Construction of Suaeda salsa cDNA library and isolation and sequencing of cDNA clones

[0028] The invention uses the aerial parts of Suaeda salsa seedlings as materials to construct a cDNA library. After the Suaeda salsa seedlings growing for 4 weeks were treated with 400mM NaCl solution for 48 hours, the total RNA of its aerial parts was extracted with RNAgent Kit (Promega), its mRNA was isolated with MESSAGEMAKER Kit (GIBCO BRL), and cDNA Synthesis Kit ( The Oligo-dT primer with the XhoI cloning site provided by Stratagene) synthesizes the first strand of cDNA, and then synthesizes the second strand. After the 5' end of the cDNA is digested by RNaseH, it is flattened or filled in with pfu DNA polymerase, and the The adapter of the EcoRI cloning site was connected, and then loaded between the EcoRI and XhoI cloning sites of the phage λ-ZAP expression vector to construct a phage library. After the library was packaged with the packaging extract of GigapackII...

Embodiment 2

[0029] Example 2 Isolation and identification of full-length cDNA clone of Suaeda salsa plastid ω3 fatty acid desaturase

[0030] Using BLAST homology analysis, cDNAs encoding ω3 fatty acid desaturase from Suaeda salsa plastid were isolated. BLAST is a way of performing homology searches in BLAST "nr" databases (including all non-redundant GenBank CDS, sequences from the protein database with three-dimensional structure, SWISS-PROT protein sequence database, EMBL and DDBJ databases). BLASTX (Gish and States, Nature Genetics, 1993, 3: 266-272) is a method provided by NCBI (National Center of Biology Information) to compare six translated sequences of nucleotide sequences with protein databases. P value (probability) is the degree of homology between the searched cDNA sequence and a certain sequence in the database. The larger the P value, the greater the homology between the two. According to BLASTX comparison, the sequence has the highest homology with tobacco plastid ω3 fatt...

Embodiment 3

[0033] Example 3 Construction of plant overexpression vector and its expression in plants

[0034] The plant expression vector involved in the present invention is pCAMBIAL3301, in order to make the foreign gene forwardly inserted into the pCAMBIAL3301 carrier, the gene fragment is inserted into the intermediate vector pRT101 (Topfer et al, Nucleic Acids Res, 1987,15: 5890), then use the PstI site on pRT101 to double-cut the exogenous fragment, insert it into the corresponding site of the pCAMBIAL3301 vector, connect and react overnight at 16°C, and the ligated product is verified by digestion with the corresponding restriction endonuclease. Transform the pCAMBIAL3301 recombinant plasmid into Agrobacterium strain GV3101, culture the Agrobacterium cells overnight in YEB medium (containing 60mg / ml rifampicin) at 28°C, draw a plate on an LB plate containing 60mg / ml rifampicin, and lyse with alkali Method extraction plasmid verification.

[0035] Arabidopsis thaliana cv. Columbia...

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PUM

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Abstract

The invention provides a halophytes omega 3 fatty acid desaturase gene and its expression vector and the said gene transformed cell and plant. The said gene expressed excessively in plants can increase the ª‡-linolenic acid content of leaf, can increase the cold-resistant, salt-tolerant and antimycotic ability, and can promote nutritious value of vegetable. That antisense suppression of the said gene expression in vivo plant can reduce ª‡-linolenic acid content, and can promote the plant high temperature resistance. The invention relates to chimeric gene construction with eukaryon expression promotor, and chimeric gene transformed host cell and recombination and transformation method. At same time, the invention provides a method for cultivating transgenic plant in which the gene is expressed excessively or antisense suppressed.

Description

technical field [0001] The invention belongs to the field of plant molecular biology and is related to plant fatty acid metabolism. Further speaking, the present invention relates to the full-length cDNA sequence of Suaeda salsa plastid ω3 fatty acid desaturase (ω3 fatty acid desaturase) and its encoded amino acid sequence, including the overexpression and antisense inhibition of the cDNA sequence Expression vectors, plants transformed with the sequences and methods for producing transgenic plants. Background technique [0002] Polyunsaturated fatty acids (PUFAs) are important components of membrane lipids and precursors of signaling molecules such as prostaglandins and prothrombin. Fatty acid desaturases are a class of enzymes involved in the synthesis of PUFAs, which catalyze the insertion of double bonds at specific positions in the carbon and hydrogen chains (Los and Murata, Biochim Biophys Acta, 1998, 1394: 3-15). ω3 fatty acid desaturase is a membrane-bound enzyme th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H5/00C12N5/10C12N9/00C12N15/52C12N15/63C12N15/82
Inventor 张学杰张慧李法曾赵彦修
Owner SHANDONG NORMAL UNIV
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