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Gene PfDGAT2 relate to plant fatty acid metabolism, expression vector, construction method and application thereof

A plant fatty acid and expression vector technology, applied in plant gene improvement, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of unclear genes and their functions, and achieve short transformation time and high transformation efficiency. Effect

Active Publication Date: 2019-01-25
SHANXI AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The problem with the existing technology is that the genes involved in TAG biosynthesis and their functions in the mechanism of high-level accumulation of α-linolenic acid in the seeds of perilla and other plants are still unclear

Method used

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  • Gene PfDGAT2 relate to plant fatty acid metabolism, expression vector, construction method and application thereof
  • Gene PfDGAT2 relate to plant fatty acid metabolism, expression vector, construction method and application thereof
  • Gene PfDGAT2 relate to plant fatty acid metabolism, expression vector, construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034]The Escherichia coli strain DH5α in Example 1 was provided by the Key Laboratory of Crop Genetics and Breeding of Shanxi Agricultural University, the pMD18-T cloning vector was purchased from Beijing Quanshijin Biotechnology Co., Ltd., and the reagents used were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. All primers were synthesized by General Biosystems (Anhui) Co., Ltd. Perilla material: the preferred variety Jinsu No. 1, provided by the Cotton Research Institute of Shanxi Academy of Agricultural Sciences.

[0035] The present invention provides a gene PfDGAT2 related to plant fatty acid metabolism. The nucleotide sequence of the gene PfDGAT2 is shown in SEQ ID NO.11, which consists of 1249 bases.

[0036] The gene PfDGAT2 was prepared according to the following steps:

[0037] S1, extracting total RNA from perilla leaves and synthesizing the first strand of cDNA

[0038] Quick-freeze the fresh perilla leaves with liquid nitrogen, grind them in...

Embodiment 2

[0053] The Escherichia coli competent strain used in Example 2 is DH5α, the wild-type strain of Saccharomyces cerevisiae is INVSc1, the mutant strain is H1246α, and the yeast expression vector pYES2.0 is provided by the Key Laboratory of Crop Genetics and Breeding of Shanxi Agricultural University . The reagents used are Pfu DNA Polymerase (Tiangen Biochemical Technology Co., Ltd.), Agar Gel DNA Recovery Kit (Aidlab Co.), Plasmid Extraction Kit (from OMEGA Co.), Enzyme Reagent and Enzyme Ligation Reagent (Takara Co.), Yeast Transformation Kit (Coolaber Company), T4DNA Ligase (NEBiolabs); Ampicillin (Amp) (Solaibao Biological Company), LB medium (Qingdao Rishui Biology), YPD medium (Qingdao Pentax Biotechnology Co., Ltd.), SC -Ura medium (Coolaber company), galactose (D-(+)-Galactose) (Coolaber company), bacteriological agar powder (Qingdao Nisaixinhe Biotechnology Co., Ltd.). All primers were synthesized by General Biosystems (Anhui) Co., Ltd.

[0054] Based on the same inve...

Embodiment 3

[0086] Example 3 PfDGAT2 Gene Function Verification

[0087] Test materials: INVSc1 transfected with control plasmid pYES2.0 yeast, H1246α transfected with control plasmid pYES2.0 yeast, H1246α transfected with pYES2.0-PfDGAT2 yeast.

[0088] (1) Oil Thin Layer Chromatography (TLC)

[0089] The transgenic yeast was cultured in SC-Ura inhibition medium at 250r / min at 30°C overnight, and the OD was measured by ultraviolet spectrophotometer 600 Collect the bacteria by centrifugation at 0.4~0.5,13,000rpm, resuspend to OD with SC-Ura induction medium 600 Continue culturing at 0.4, 250r / min at 30°C, collect the transgenic yeast liquid induced by expression for 36 hours by centrifugation, vacuum freeze-dry and grind into powder for later use. The transgenic yeasts were INVSc1-transferred control plasmid pYES2.0 yeast, H1246α-transferred control plasmid pYES2.0 yeast and H1246α-transferred pYES2.0-PfDGAT2 yeast.

[0090] Weigh 20 mg of freeze-dried sample into a finger-shaped glass...

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Abstract

The invention belongs to the field of molecular biology, in particular to a gene PfDGAT2 related to plant fatty acid metabolism, an expression vector, a construction method and an application thereof.The nucleotide sequence of the gene PfDGAT2 is shown in SEQ ID NO:11, and the gene PfDGAT2 is related to fatty acid metabolic pathway in oil crop seeds such as perilla frutescens and the like, so that the gene PfDGAT2 can select alpha-linolenic acid molecule and transfer it to TAG molecule.

Description

technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to a gene PfDGAT2 related to plant fatty acid metabolism, an expression vector, a construction method and application. Background technique [0002] Perilla frutescens (L.) Britt. is an annual herbaceous plant of the Lamiaceae Perilla genus with strong adaptability. It is one of the first batch of 60 traditional Chinese medicine plants promulgated by the Ministry of Health of my country, which are both food and medicine. The oil yield of perilla seeds is as high as 45% to 55%, and it is rich in unsaturated fatty acids, of which the content of α-linolenic acid is more than 60%. α-Linolenic Acid (ALA), also known as n-3 (omega-3) polyunsaturated fatty acid, is an omega-3 fatty acid that is extremely important to human health. It participates in the synthesis of phospholipids in the human body Synthesis and metabolism, studies have found that α-linolenic acid has a high pro...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/81C12N15/66A01H5/00
CPCC12N9/1029C12N15/8247C12Y203/01158
Inventor 王计平周雅莉任文燕李润植安茜段露露郝月茹
Owner SHANXI AGRI UNIV