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Chorispora bungeana endoplasmic reticulum type omega-3 fatty acid desaturation enzyme gene and application thereof

A technology of alpine mustard and desaturase, applied in the field of alpine mustard endoplasmic reticulum type ω-3 fatty acid desaturase gene, to achieve the effects of strong response, increased trienoic acid content, and high stress resistance

Active Publication Date: 2014-04-23
NORTHWEST INST OF ECO ENVIRONMENT & RESOURCES CAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report of cloning the endoplasmic reticulum type omega-3 fatty acid desaturase gene from the alpine mustard, so the endoplasmic reticulum type omega-3 fatty acid desaturase gene is identified from the alpine mustard clone, and the It is transferred to tobacco, which has important practical significance for the improvement of tobacco resistance

Method used

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  • Chorispora bungeana endoplasmic reticulum type omega-3 fatty acid desaturation enzyme gene and application thereof
  • Chorispora bungeana endoplasmic reticulum type omega-3 fatty acid desaturation enzyme gene and application thereof
  • Chorispora bungeana endoplasmic reticulum type omega-3 fatty acid desaturation enzyme gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Alpine mustard endoplasmic reticulum type ω-3 fatty acid desaturase gene, the amino acid sequence of the endoplasmic reticulum type ω-3 fatty acid desaturase encoded by the gene is as shown in the sequence table from the first position of SEQ ID NO.2 to Shown at position 386, its nucleotide sequence is shown at position 1 to 1161 of SEQ ID NO.1 in the sequence listing.

[0032] 1. Alpine ionic mustard CbFAD3 Gene cloning.

[0033] 1. Alpine ion mustard CbFAD3 Gene Conserved Sequence Amplification:

[0034] 1.1. Use Omega's Plant RNA Extraction Kit to extract total RNA from the callus of Japonica japonica.

[0035] 1.2. Use Takara's PrimeScript? 1st Strand cDNA synthesis kit for reverse transcription to obtain cDNA.

[0036] 1.3. The following degenerate primers were designed according to the gene sequence retrieved from the NCBI (National Center for Biotechnology Information) database:

[0037] Sense Primer: 5'-GGGCGGCKATTCCTAAGCAYT-3'(Degeneracy=4)

[0...

Embodiment 2

[0084] Example 2 Alpine mustard CbFAD3 Eukaryotic Expression of Genes in Saccharomyces cerevisiae INVScl

[0085] 1. Construction of eukaryotic expression vector pYES2- CbFAD3

[0086] 1.1. Design primers according to the sequence of the coding region as follows:

[0087] Z3-UP: 5'-GGATCCACCATGGTTGTTGCAATGGACCA-3'

[0088] Z3-DN: 5'-CGAGCTCTAATTGATTTTAGATTTGTCAG-3'

[0089] 1.2. Alpine mustard CbFAD3 Gene Amplification:

[0090] Reaction system (50 μl): cDNA 2.5 μl, TaKaRa LA Taq (5 U / μl) 0.5 μl, 10×LA Taq Buffer II (Mg2+ free) 5 μl, MgCl2 (25 mM) 5 μl, dNTP Mix (2.5 mM)8 μl, CbFAD3s (10μM) 1μl, CbFAD3a (10μM) 1μl, ddH 2 O 27 μl.

[0091] Reaction conditions: 94°C, 3min

[0092] (94°C, 1min; 56°C, 15s; 63°C, 15s; 72°C, 1min) 30 cycles

[0093] 72℃, 10min

[0094] 1.3. Alpine mustard CbFAD3 The gene recovery method is the same as 1.4.1 in Example 1.

[0095] 1.4. Enzyme digestion and ligation of CbFAD3 gene and expression vector pYES2 plasmid:

[0096] (1) The re...

Embodiment 3

[0115] Example 3 Transforming Alpine mustard CbFAD3 Acquisition of Genetic Tobacco

[0116] 1. Construction of eukaryotic expression vector pBI121- CbFAD3

[0117] 1.1. Design primers according to the sequence of the coding region as follows:

[0118] ZF3: 5'-GCTCTAGAATGGTTGTTGCAATGGACCA-3'

[0119] ZF3': 5'-CGAGCTCCTAATTGATTTTAGATTTGTCAG-3'

[0120] 1.2. Alpine mustard CbFAD3 Gene Amplification:

[0121] Reaction system is the same as 1.2 in embodiment 2

[0122] Reaction conditions: 94°C, 3min

[0123] (94°C, 1min; 60°C, 30s; 72°C, 1min) 30 cycles

[0124] 72℃, 10min

[0125] 1.3. Alpine mustard mustard CbFAD3 The gene recovery method is the same as 1.4.1 in Example 1.

[0126] 1.4. Enzyme digestion and ligation of CbFAD3 gene and expression vector pBI121 plasmid:

[0127] (1) The recovered product in 1.2 and the expression vector pBI121 plasmid (preserved in our laboratory) were double-digested with restriction endonucleases XbaI and SacI from Takara Company re...

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Abstract

The invention relates to a chorispora bungeana endoplasmic reticulum type omega-3 fatty acid desaturation enzyme gene ((i)CbFAD3( / i)) and an application thereof. The gene is selected from chorispora bungeana and an amino acid sequence of an endoplasmic reticulum type omega-3 fatty acid desaturation enzyme encoded by the gene is represented from a 1st site to a 386th site in a sequence table SEQ ID NO.2; a nucleotide sequence of the endoplasmic reticulum type omega-3 fatty acid desaturation enzyme is represented from a 1st site to a 1161st site in a sequence table SEQ ID NO.1. Compared with wild tobaccos, tobaccos with the trans-chorispora bungeana (i)CbFAD3( / i) gene have high cold resistance, drought resistance and salt resistance; the chorispora bungeana endoplasmic reticulum type omega-3 fatty acid desaturation enzyme gene ((i)CbFAD3( / i)) has the important effect of improving the stress resistance of the tobaccos.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the alpine mustard endoplasmic reticulum type omega-3 fatty acid desaturase gene and application thereof. Background technique [0002] Low temperature, drought and salinity are common abiotic stresses in nature and are important factors affecting crop yield and quality. Breeding new crop varieties that can resist cold, drought and salt stress at the same time is of great significance for improving agricultural productivity, water resources and land use efficiency. Tobacco is an important economic crop, but many high-quality tobaccos are not highly resistant to stress, which not only limits their planting areas, but also makes them vulnerable to sudden climate disasters. Therefore, identifying new stress resistance genes and improving the comprehensive stress resistance of tobacco through genetic engineering technology will be an important way to improve tobacco. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/82
Inventor 石玉兰安黎哲
Owner NORTHWEST INST OF ECO ENVIRONMENT & RESOURCES CAS
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