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Gene site-directed mutation method and glyphosate-resistant gene obtained by said method and its expression vector and trans foring factor

A technology of gene encoding and gene fragment, applied in the fields of recombinant bacterial 5-enolpyruvate shikimate-3-phosphate synthase gene, expression vector and transformant, can solve the problem of limited effect, affecting enzyme catalytic activity, zymogen Problems such as decreased substrate affinity

Inactive Publication Date: 2005-11-09
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the three-dimensional structure of the enzyme and the role of amino acid positions are not completely clear, the effect of using site-directed mutagenesis is very limited
The problem encountered by the previous site-directed mutagenesis method is that the affinity of the mutated enzyme to glyphosate is reduced and the affinity to the zymogen substrate is also reduced, thus affecting the catalytic activity of the enzyme

Method used

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  • Gene site-directed mutation method and glyphosate-resistant gene obtained by said method and its expression vector and trans foring factor
  • Gene site-directed mutation method and glyphosate-resistant gene obtained by said method and its expression vector and trans foring factor
  • Gene site-directed mutation method and glyphosate-resistant gene obtained by said method and its expression vector and trans foring factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Acquisition of mutant EPSP synthetase gene aroAM142 and its expression vector pET-aroA-M142

[0040] Using the above-mentioned gene site-directed mutagenesis method, the position 124-126 of the aroAM1 gene sequence was changed from ACC to ATG, and the 42nd amino acid residue of the expressed EPSP synthetase protein was replaced by Thr to Met.

[0041] The principle of site-directed mutagenesis figure 2 shown. figure 2Among them, primer1, primer2 and primer3 represent primer A, primer B and primer C, respectively; ACC represents the original sequence on the source template, ATG represents the sequence after mutation; NcoI and NdeI represent restriction enzyme sites, respectively.

[0042] The specific operation method is as follows:

[0043] Directly use the plasmid pET-aroA-M1 as the source template.

[0044] Since the aroAM1 gene is about 20 bp away from the 124-126 nucleotide sequence of the 42nd amino acid residue encoding the enzyme protein, there is...

Embodiment 2

[0058] Example 2: Acquisition of transformants BL21 (aroAM1), BL21 (EcaroA) and BL21 (aroAM142)

[0059] The plasmids pET-aroA-M1, pET-EcaroA (see CN1358858A patent application) and pET-aroA-M142 were respectively transformed into Escherichia coli BL21(DE3) by standard calcium chloride transformation method, and the obtained transformants were respectively named BL21(aroAM1) , BL21 (EcaroA) and BL21 (aroA-M142).

Embodiment 3

[0060] Example 3: Glyphosate resistance test of transformants BL21 (aroAM1), BL21 (EcaroA) and BL21 (aroAM142)

[0061] Transformants BL21 (aroAM1), BL21 (aroAM142) and BL21 (EcaroA) were inoculated into M9 liquid medium containing 1 mM IPTG, 50 μg / ml ampicillin, and 30 mM and 60 mM glyphosate, respectively, at 200 rpm and 37 ° C in air. Bath conditioned culture. Measure the OD of the culture medium every 2 hours 600 , record its growth, and the measurement results are as follows: Figure 5 , Figure 6 shown. It can be seen from the growth curve in the figure that the transformant carrying the mutant gene can still survive on the M9 basic medium containing 60mM glyphosate, and BL21 (aroAM142) grows faster than BL21 (aroAM1) and has a longer lag phase. short, showing increased resistance to glyphosate ( Figure 6 ); the growth of the transformant BL21 (EcaroA) carrying the wild-type gene on the M9 basic medium containing 30mM glyphosate has been severely inhibited ( Figu...

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Abstract

The invention refers to a gene-pointing mutation method, reformed aroA gene and carrier and transformer containing said gene. The method selects DNA, sequence that the distance between the point to be mutated and the monozyme pitch in point is 10-20 bases as source mould, according to sequence character designs the corresponding one 5'-end solicitatino and two 3'-end solicitation, and adopts two-step PCR expanding reaction to obtain pad-in point mutation gene segment. The invention makes further improvement on the gene aroA-M1 to obtain EPSP in order to compound enzyme gene aroA-M142 which makes further improvement on enzyme character.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a gene site-directed mutagenesis method and the recombinant bacterial 5-enol pyruvate shikimate-3-phosphate (EPSP) synthetase gene (aroA) obtained by the method, and containing the Gene expression vectors and transformants. Background technique [0002] Glyphosate is the most widely used broad-spectrum herbicide. It is non-toxic to humans and animals, it is difficult for weeds to develop resistance to it, and it has low soil residues. The market potential is huge. [0003] 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (E.C.2.5.1.19), catalyzes the penultimate reaction of the shikimate metabolic pathway in plants and microorganisms, making PEP (phosphoenolpyruvate ) reacts with 3-phosphoshikimate to generate EPSP (5-enolpyruvylshikimate-3-phosphate) and inorganic phosphorus. Since glyphosate has a structure similar to PEP, it can fo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N15/19C12N15/52C12P19/34
Inventor 何鸣聂燕芳徐培林
Owner SUN YAT SEN UNIV
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