Glyphosate resistant EPSP synthetase GmEPSPS, and coding gene and application thereof
A coding and coding sequence technology, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problem of low expression efficiency
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Embodiment 1
[0073]Embodiment 1, the modification synthesis of GmEPSPS gene
[0074] According to the mutant gene of 5-enolpyruvate-3-phosphate synthase (EPSPS) derived from Pseudomonas fluorescens, the original gene ORF sequence 5 was deleted Two base sequences of 147 bp at the 'end and 531 bp at the 3' end, leaving only a base sequence of 654 bp in the middle core domain sequence, which is spliced with a base sequence of 591 bp from the EPSPS gene of Allium scallops; Under the condition that the amino acid sequence composition of the EPSP synthetase protein is generally unchanged, base substitutions are performed with plant-favored codons, and a modified DNA sequence is initially obtained; AT-rich sequences and AT-rich sequences that cause plant transcription instability and Commonly used restriction enzyme sites (BamH I, EcoR I, Hind III, Nco I, Xho I, Xba I, etc.), and then correct and eliminate them by replacing codons; and add rice ACT1-intron sequence at the 5' end (No. 7-460 of ...
Embodiment 2
[0077] Embodiment 2, the construction of anti-glyphosate fusion gene plant expression vector
[0078] Artificially synthesize the GmEPSPS gene shown in Sequence 2 in the sequence listing, double-enzyme-cut the GmEPSPS gene with Xba I and Sac I respectively, and simultaneously double-enzyme-cut the plant expression vectors CPB and CUB with Xba I and Sac I, and recover with gel The purification kit recovers the digested GmEPSPS fragment and the backbone fragments of CPB vector and CUB vector respectively. The GmEPSPS fragment was ligated with the backbone fragments of the CPB vector and the CUB vector respectively to obtain the target plasmid. Transfer the target plasmid into Escherichia coli, screen for resistance, pick positive single clones, carry out liquid culture of positive single clones, extract positive clone plasmids for Xba I and Sac I double enzyme digestion identification, and initially determine the correctness after enzyme digestion identification The recombinant...
Embodiment 3
[0081] Embodiment 3, glyphosate herbicide tolerance test
[0082] Preparation of M9 medium:
[0083] (1) First prepare 1M MgSO 4 : Weigh MgSO 4 ·7H 2 Add 2.46g of O to 10mL of double distilled water to dissolve, autoclave for later use;
[0084] (2) Prepare 1M CaCl 2 : CaCl 2 ·6H 2 O 2.191g was dissolved in 10mL of double-distilled water, and autoclaved for later use;
[0085] (3) Prepare 5×M9 salt solution again:
[0086] Na 2 PO 4 ·7H 2 O 12.8g
[0087] K H 2 PO 4 3.0g
[0088] NaCl 0.5g
[0089] NH 4 Cl 1.0g
[0090] Add 200ml of double distilled water to dissolve, and sterilize at 121°C for 15 minutes.
[0091] Remarks: The above three samples are prepared separately, bottled separately, and can be sent to high pressure together.
[0092] (4) Prepare 20% glucose solution: 4g glucose is dissolved in 20ml of double distilled water, and sterilized by filtering through a 0.22 micron filter;
[0093] (5) Preparation of M9 medium by aseptic operation
[009...
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