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Peanut lysophosphatidic acid acyltransferase gene and application thereof

A technology of lysophosphatidic acid and acyltransferase, which is applied in the fields of molecular biology and biology, and can solve problems such as the limitation of acyl CoA selection specificity

Inactive Publication Date: 2014-04-16
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] A large number of studies have shown that G3PAT and DAGAT from different sources have less selectivity for acyl-CoA, while LPAAT has strong substrate selectivity, and the Sn- Position 2 is highly limited by LPAAT selection specificity for acyl-CoAs

Method used

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  • Peanut lysophosphatidic acid acyltransferase gene and application thereof
  • Peanut lysophosphatidic acid acyltransferase gene and application thereof
  • Peanut lysophosphatidic acid acyltransferase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] peanut AhLPAAT1 Gene cloning and recombination vector pGEM-T- AhLPAAT1 build

[0064] (1) Extraction of total RNA from peanut seeds

[0065] 1) Weigh 0.1g of peanut seeds, grind them into powder in liquid nitrogen, and quickly transfer them to a 1.5ml centrifuge tube;

[0066] 2) Add 1ml Trizol extract to the centrifuge tube, mix well, and place at room temperature for 5 minutes;

[0067] 3) Add 0.2ml of chloroform to the centrifuge tube, shake vigorously for 15s, place at room temperature for 3min, centrifuge at 12000rpm for 15min at 4°C, carefully absorb 200μl of the upper aqueous phase and add to another centrifuge tube;

[0068] 4) Repeat 3) until most of the protein is removed, place in isopropanol (0.8 times the volume) at room temperature for 10 minutes, then centrifuge at 12000g for 10 minutes at 4°C;

[0069] 5) Discard the supernatant, add 1ml of 75% ethanol, centrifuge at 7,500g at 4°C for 5min; discard the supernatant, add 1ml of absolute ethanol, ce...

Embodiment 2

[0089] Construction of Plant Overexpression Vectors

[0090] (1) Overexpression vector pBI121- AhLPAAT1 Recombinant vector and pBIOle17.8- AhLPAAT1 Construction of recombinant vector

[0091] Using pBI121 vector with 35S promoter and pOlesin17.8- GUS vector for the construction of plant overexpression vector, pOlesin17.8- GUS The vector was constructed by replacing the 35S promoter of the pBI121 vector with the peanut Olesin17.8 promoter. Based on the MCS of the pBI121 vector Sma I and Sac I restriction site and AhLPAAT1 A pair of primers were designed for the sequences at both ends of the cDNA, plus restriction enzyme sites and protective bases (the underlined sequence is the enzyme site):

[0092] Forward primer: 5'-TCC CCCGGG ATGACTACCACTGGGACACTCAAG-3'

[0093] Reverse primer: 5'-C GAGCTC TCATTTTTCTCCAAACGCCGTAGC-3'

[0094] The PCR reaction system is: 0.5 μl pGEM-T- AhLPAAT1 Intermediate vector plasmid, 1 μl dNTP Mixture (10mM), 2 μl forward primer (...

Embodiment 3

[0099] Genetic Transformation of Arabidopsis

[0100] (1) Pre-treatment of Arabidopsis transformation: Arabidopsis plants can be used for transformation when the main fur grows to 5-6cm and begins to bloom and form 1-2 siliques. The grown siliques need to be cut off before transformation.

[0101] (2) Preparation of dipping medium: The dipping medium used to soak Arabidopsis inflorescences is 1 / 2MS medium containing 5% sucrose, pH=5.8 (adjusted by KOH), and autoclaved. When used, add 0.02%-0.05% surfactant Silwet L-77.

[0102] (3) Agrobacterium preparation and Arabidopsis transformation:

[0103] 1) Activation of strains: Put the preserved transformed Agrobacterium tumefaciens EHA105 strains on YEB solid medium containing kanamycin (Km, 100 mg / L) and rifampicin (Rif, 30 mg / L) Activate the upper line, and pick a single colony for PCR detection;

[0104] 2) Pick the activated monoclonal positive Agrobacterium containing the target gene into 5ml of fresh YEB medium containing...

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Abstract

The invention relates to a peanut lysophosphatidic acid acyltransferase gene AhLPAAT1 and application thereof, belonging to the fields of molecular biology and biotechnology. The gene cDNA nucleotide sequence of the AhLPAAT1 is disclosed as SEQ ID NO:1, and the amino acid sequence of the coding protein is disclosed as SEQ ID NO:2. The peanut lysophosphatidic acid acyltransferase gene has the actions of changing the fatty acid content in plant tissues and especially seeds and enhancing the proportion of polyunsaturated fatty acids, and has wide application prospects in the fields of plant species breeding and agricultural production. The invention also relates to a transgenic plant with AhLPAAT1 gene. The invention also relates to a method for changing the fatty acid content in plant seeds and component proportion by using the AhLPAAT1 gene.

Description

technical field [0001] The invention relates to the fields of molecular biology and biotechnology, and more specifically relates to a peanut lysophosphatidic acid acyltransferase gene and its application. Background technique [0002] Oil is one of the main storage substances of oil crop seeds, and the oil content and composition of seeds determine the economic value of oil crops. Vegetable fatty acids are the main components of vegetable oils and are also an important part of biofilms. The composition of fatty acids has an important impact on the nutritional value of vegetable oils, food processing applications and industrial applications. The composition of fatty acids in biofilms also affects the plant membrane system. Stress resistance characteristics (Huang Bingyan et al., Henan Agricultural Sciences, 2009, 9:75-78). In plant seeds, fatty acids are mainly present as triacylglycerides (Somerville C and Browse J, Science, 1991, 252:80–87; Voelker T and Kinney AJ, Annu ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/84C12N1/21A01H5/00
Inventor 黎茵黄上志刘琛颜瑞卿
Owner SUN YAT SEN UNIV
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