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EPSP synthase AroA-Ra multisite mutant of rahnella aquatilis

A technology of EPSP synthase, Lahenia aquatica, applied in the field of AroA-Ra multi-site mutants of EPSP synthase gene of Lahenia aquatica, can solve the problem of reduced affinity, insufficient enzyme activity and difficult to cope with the reaction needs and other issues to achieve the effect of strong affinity and high glyphosate resistance

Inactive Publication Date: 2012-07-11
SHANGHAI ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Many EPSP synthases that have no resistance or low resistance, reduce the affinity with glyphosate by mutating their amino acids to produce glyphosate resistance, and also reduce the affinity with PEP Sex, so that the enzyme activity is insufficient, it is difficult to meet the needs of the reaction

Method used

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  • EPSP synthase AroA-Ra multisite mutant of rahnella aquatilis
  • EPSP synthase AroA-Ra multisite mutant of rahnella aquatilis
  • EPSP synthase AroA-Ra multisite mutant of rahnella aquatilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0020] 11 Synthesis of glyphosate herbicide resistance gene AroA-Ra from Lahnella aquatica

[0021] By gene synthesis method (Nucleic Acids Research, 2004, 32, e98) synthetic EPSP synthase gene AroA-Ra of Lahnella aquatica, used primer is as follows:

[0022] 1. AraII-1:

[0023] GTGGAATCCC TGACATTACA ACCCGTTGCG CTGGTTAACG GCAGCATCAA TTTACCTGGC

[0024] 2. AraII-2:

[0025] AAAAGCAGCC AGTAAAAGTG CGCGGTTAGA AACACTTTTT GAGCCAGGTA AATTGATGCT

[0026] 3. AraII-3:

[0027] CTTTTACTGG CTGCTTTTGC ACAGGGTACT ACCCGCCTGA CTAACCTGCT CGACAGCGAT

[0028] 4. AraII-4:

[0029] GACACCCAGT TGAGTGAGTG CTGTCAACAT ATGACGCACA TCATCGCTGT CGAGCAGGTT

[0030] 5. AraII-5:

[0031] CTCACTCAAC TGGGTGTCAC GCATCGTTTA TCTGCATCCC GCACCGAGTG TGAAATCGAT

[0032] 6. AraII-6:

[0033] AAACAGTTCC AGACCTTTAG CATTGGAAAA AGCCGTGCCC AGACCATCGA TTTCACACTC

[0034] 7. AraII-7:

[0035] AAAGGTCTGG AACTGTTTCT TGGGAATGCA GGAA...

Embodiment 2

[0094] Example 2 Screening of high glyphosate-resistant EPSP synthase

[0095]The EPSP synthase gene fragment recovered and rearranged above, that is, the EPSP synthase gene AroA-Ra was double-digested with BamH I and SacI, and then constructed between the prokaryotic expression vector pG251 promoter and t1t2 terminator, and the vector contained ampicillin Penicillin 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. 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 100 mM glyphosate for 48 hours. It was found that three colonies grew well. These three colonies were inoculated on M9 plates containing 100mM and 200mM glyphosate, respectively, and it was found that only one clone could grow on the M9 plate...

Embodiment 3

[0096] Example 3 EPSP expression of high glyphosate resistance

[0097] 3.1 Sequence analysis of DNA fragments highly tolerant to glyphosate

[0098] DNA sequencing was performed on the full sequence of the highly glyphosate-tolerant plasmid pmAroA-Ra screened in Example 2 by step-by-step sequencing. The analysis results showed that the highly glyphosate-resistant mutant contained the following 1 to 6 mutation sites on the same molecule: Mutation 1 (C408E): the cysteine ​​at position 408 in the amino acid sequence of EPSP synthase was replaced Glutamic acid; mutation 2 (Q271K): glutamine at position 271 is replaced by lysine; mutation 3 (H128R): histidine at position 128 is replaced by arginine; mutation 4 (P101S) : Proline at position 101 is replaced by serine; mutation 5 (T97A): threonine at position 97 is replaced by alanine; mutation 6 (G96A): glycine at position 96 is replaced by Alanine (refer to the sequence listing SEQ ID No 1, 2).

[0099] Using the EPSP synthase g...

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Abstract

The invention relates to an EPSP synthase AroA-Ra multisite mutant of a rahnella aquatilis, particularly to the multisite mutant improved from the gene coded as 5- enol acetone shikimic acid-3-EPSP synthase of the rahnella aquatilis through DNA molecular rearrangement method and function complementation method, and the resistance of the improved multisite mutant to the glyphosate is greatly improved. The nucleotide sequence of the multisite mutant is as indicated by SEQ ID No1, the encoded amino acid sequence is as indicated by the SEQ ID No2, and the mutant has 6 different mutational sites under the same molecule. The multisite mutant not only retains the high affinity of the original EPSP synthase toward the PEP, but also reduces the affinity of the EPSP synthase toward the glyphosate herbicide, so that the glyphosate resistance of the mutant is greatly improved.

Description

technical field [0001] The invention belongs to the field of microorganisms, and relates to a multi-site mutant of the EPSP synthase gene AroA-Ra of Lahnella aquatica, in particular to a method of using DNA molecular rearrangement and functional complementation to synthesize the mutant from Lahnella aquatica The modified multi-site mutant of the gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), the modified multi-site mutant has a greatly improved resistance to glyphosate . Background technique [0002] The widespread use of glyphosate and its non-selective characteristics have prompted the cloning of glyphosate-resistant genes and the cultivation of glyphosate-resistant transgenic crops to become research hotspots. my country, as a major producer and exporter of glyphosate, currently does not have 5-enolpyruvylshikimate-3-phosphate synthase [5-enolpyruvylshikimate-3-phosphate synthase] with independent intellectual property rights, which is suitabl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/82
Inventor 彭日荷姚泉洪熊爱生付晓燕田永生赵伟金晓芬韩红娟陈晨
Owner SHANGHAI ACAD OF AGRI SCI
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