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Preparation of glyphosate resistance enhanced EPSPS (5-enolpyruvyl shikimate-3-phosphate synthase) mutant of Vitis vinifera and application of EPSPS mutant of Vitis vinifera

A technology of EPSP synthase and grape, which is applied in the fields of application, botany equipment and methods, biochemical equipment and methods, etc., and can solve the problems of non-transgenic crop cultivation and so on

Active Publication Date: 2014-12-24
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, wild-type EPSPS genes derived from plants are often very sensitive to glyphosate, so they cannot be directly used in the cultivation of transgenic crops

Method used

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  • Preparation of glyphosate resistance enhanced EPSPS (5-enolpyruvyl shikimate-3-phosphate synthase) mutant of Vitis vinifera and application of EPSPS mutant of Vitis vinifera
  • Preparation of glyphosate resistance enhanced EPSPS (5-enolpyruvyl shikimate-3-phosphate synthase) mutant of Vitis vinifera and application of EPSPS mutant of Vitis vinifera
  • Preparation of glyphosate resistance enhanced EPSPS (5-enolpyruvyl shikimate-3-phosphate synthase) mutant of Vitis vinifera and application of EPSPS mutant of Vitis vinifera

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 DNA molecular rearrangement (DNA Shuffling) of EPSP synthase gene

[0020] 1.1 Glyphosate herbicide resistance genes derived from grapes VvEPSPPS Synthesis

[0021] The applicant entered 5-enolpyruvylshikimate-3-phosphate synthase and Vitis vinifera on the NCBI website (http: / / www.ncbi.nlm.nih.gov), and obtained the coded grape 5-enolpyruvyl-shikimate with the serial number NM_001281247 - The mRNA sequence of the 3-phosphate synthase gene. Design a pair of PCR primers according to this sequence (VvEPSPSZ: 5'-AAGGATCCATGGCCTCTGTCGCCACTAAG

[0022] -3' and VvEPSPSF: 5'-AAGAGCTCTCAATGTTTGGTAAAACGCTGG-3'), using the Kyoho grape cDNA library preserved in this experiment (Northwest Botanical Journal, 2009, 29:1723-1729) as a template, the grape EPSP synthase was amplified by RT-PCR Gene( Vv EPSPS) full-length cDNA sequence. Reaction system: 1μl cDNA + 4μl 2.5mmol / L dNTPs + 25μl Buffer + KOD Plus (Toyobo Japan) polymerase 1U + 1μl VvEPSPSZ + 1μl VvEPSPSF +ddH ...

Embodiment 2

[0032] Example 2 Screening and sequence analysis of high glyphosate-resistant EPSP synthase mutants

[0033] 2.1 Screening of high glyphosate-resistant EPSP synthase mutants

[0034] The above-mentioned recovered and rearranged EPSP synthase gene fragment was digested with BamH I and Sac I, and then constructed into the prokaryotic expression vector pG251 (CN1338515) between the promoter and the t1t2 terminator, which carries the ampicillin resistance gene. Transform Escherichia coli strain DH5α by electroporation to obtain mutant expression library with a capacity of 10 8 , and then use a large number of plasmid extraction kits (Omega, USA) for plasmid extraction.

[0035] Take 1 μl of a large amount of extracted plasmid and transfer it into Escherichia coli ER2799 (NEB Company) and spread it on an M9 plate containing 200mM glyphosate for 48 hours. It was found that a colony grew well. The plasmid extracted from the clone (pVvEPSPS mutant ) into Escherichia coli ER2799 (NE...

Embodiment 3

[0038] Example 3 In vitro glyphosate resistance test

[0039] The wild type obtained in embodiment 1 VvEPSPPS After the gene was digested with BamH I and Sac I, it was constructed into the prokaryotic expression vector pG251 to obtain the plasmid pVvEPSPS. Transform the pVvEPSPS plasmid and the pVvEPSPS obtained in Example 2 respectively with Escherichia coli ER2799 bacterial strain mutant Plasmids were spread on M9 plates and cultured for 48 hours, and the respective transformants were picked out with toothpicks and inoculated in LB liquid medium for culture until the concentration reached 5 × 10 3 cells / μL, take 2μL of the respective cell fluids and 1 / 5 and 1 / 25 diluted cell fluids and spot them on M9 plates containing different concentrations of glyphosate (0, 50mM, 200mM), and observe after 48 hours of culture 50mM glyphosate has basically inhibited wild-type VvEPSPPS cell growth. However, when the concentration of glyphosate was 200mM, the mutant VvEPSPS mutant C...

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Abstract

The invention discloses a glyphosate resistance enhanced EPSP (5-enolpyruvyl shikimate-3-phosphate synthase) synthase multi-site mutant which is derived from Vitis vinifera and prepared by adopting a DNA shuffling technology, as well as a coding gene and application of the mutant. The mutant comprises 7 mutation sites, wherein the amino acid sequence of the mutant is as shown in the SEQ ID No.1, and the base sequence coded by the mutant is as shown in the SEQ ID No.2. Tests indicate that the EPSP synthase multi-site mutant derived from Vitis vinifera and the gene coded by the mutant have relatively high glyphosate resistance and relatively strong PEP affinity, and the characteristics of the mutant and the gene provide possibility to the gene for culturing anti-glyphosate transgenic crops.

Description

technical field [0001] The invention belongs to the field of microorganisms, and relates to a Vitis vinifera ) 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase / EPSPS) multi-site mutant preparation and its coding gene and application, specifically involving a method that utilizes DNA molecular rearrangement (DNA Shuffling) and function The complementation method is used to transform the EPSP synthase gene from grapes to obtain multi-site mutants, and then the encoded gene is used in glyphosate resistance research, rice transformation, and transgenic crop cultivation. Background technique [0002] The shikimate pathway is an important pathway for the synthesis of aromatic amino acids in plants and microorganisms. EPSP synthase is a key enzyme in the shikimate pathway, catalyzing 3-phosphoshikimate (S3P) and phosphoenolpyruvate (PEP) to generate 5-enolpyruvyl-shikimate-3-phosphate synthase (5-enolpyruvyl shikimate-3-phosphate synthase, EPSPS). The herbicide glyphos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/82
CPCC12N9/1092C12N15/8275C12Y205/01019
Inventor 田永生许晶姚泉洪彭日荷赵伟付晓燕王丽娟韩红娟王波
Owner SHANGHAI ACAD OF AGRI SCI
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