gat3 Application of Gene and Its Mutants in Breeding Glyphosate Resistant Crops
A glyphosate-resistant and mutant technology, applied in the field of plant biology, can solve the problem of losing herbicide activity
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Embodiment 1
[0027] Example 1 Vector construction
[0028] gat Selection of resistance genes: Download the information of the PF00583 (Pfam: Family: Acetyltransf_1 (PF00583) (xfam.org)) protein family in the Pfam database (http: / / pfam.xfam.org / ), and use hmmsearch in the Uniparc database 3043 sequences were found. The above sequences are clustered according to 90% similarity, and they can be divided into 1196 categories. The sequence values of the class centers are sorted from large to small, and 18 sequences (clusternumber > 20) are selected from them and named as gat 1 - gat 18 .
[0029] 18 sequences and gat c11 (The 11th round of screening results) The sequence fragments were optimized and synthesized by Changzhou Jiyu Company according to E. coli codons. The synthetic vector and the pET28a+(R) vector were digested with BamHI / SacI double enzymes. The former was used to recover the synthetic fragments, and the latter was used to provide the vector backbone. Each synthetic f...
Embodiment 2
[0031] Example 2 Resistance screening of transformed strains
[0032] Bacterial activation: Take 50 μL of the preserved bacterial solution in 5 mL of LK liquid medium, and culture at 37°C, 200 rpm shaker for 8-10 h. The inoculation ring was burned and sterilized on the outer flame of an alcohol lamp. After cooling to room temperature, the bacterial solution was dipped and streaked on the LK solid plate. After sealing, it was placed in an incubator at 37 °C for overnight culture.
[0033] Add 5 mL of LK liquid medium to the test tube for use, clamp a 10 μL small pipette tip with sterilized tweezers, pick 3 single colonies of appropriate size on the plate and put them in 3 test tubes. Shaker culture. If the OD600 of the bacterial solution reaches 1.0, it needs to be cultured for 12-13 h. If the OD600 of the bacterial liquid reaches 0.6, it needs to be cultured for 6-8 h.
[0034] Plate qualitative screening: in plate screening, the bacterial solution needs to be activated to ...
Embodiment 3
[0046] Example 3 Screening of mutation sites
[0047] Mutation site primary selection:
[0048] According to the experimental data of 18 transformed strains, 18 gat The sequences are graded as shown in Table 1, and the gat3 Each amino acid site in the gene was scored, and 12 mutation sites as shown in Table 2 below were selected.
[0049] Table 1 gat Genetic grading
[0050]
[0051] Table 2 Selected gat3 intragenic mutation site
[0052]
[0053] Construction of single mutation vector:
[0054] Take pET28a- gat3 Plasmids were used as templates, and the sequences shown in Table 3 were used as primers for PCR amplification. The recovered PCR product was used as a large fragment primer, and pET28a- gat3 The plasmid is used as a template for PCR amplification of the complete plasmid to obtain a single mutation vector.
[0055] Table 3 Primer information for single mutation vector construction
[0056]
[0057] The constructed 12 single mutation vectors were ...
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