GGPPS (GGPPS) directed single-point mutant protein and application thereof
A technology of directed mutation and mutant protein is applied in the field of tobacco genetic engineering, which can solve the problems of short development time and unused plant genetic modification, and achieve the effect of improving the synthesis amount of beta-carotene.
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Embodiment 1
[0023] Since the acquisition of the existing geranyl geranyl diphosphate synthase (GGPPS) gene is the basis for related sequence analysis and directed evolution mutation, this example firstly analyzes the existing geranyl geranyl diphosphate synthase (GGPPS) gene. The cloning process of the diphosphate synthase (GGPPS) gene is briefly described as follows.
[0024] First, according to the gene sequence shown in GenBank accession number NM_001325177.1, the primer sequences for PCR amplification were designed as follows:
[0025] Forward primer: 5'-atgagatctatgaatcttgt-3',
[0026] Reverse primer: 5'-attttcacgataagcaatgt-3';
[0027] Subsequently, carry out PCR amplification;
[0028] After the PCR amplification product is detected by electrophoresis, it is recovered, purified, and the recovered and purified PCR product is connected to the pGEMT plasmid;
[0029] Subsequently, the ligation product was transformed into Escherichia coli DH5α competent cells, and after overnight...
Embodiment 2
[0038] In order to facilitate the detection and analysis of relevant mutation sites, the geranyl geranyl diphosphate synthase (GGPPS) recombinant engineering strain was used for experimental verification in this application. Therefore, the construction process of this engineering strain is discussed in this embodiment. The introduction is as follows.
[0039] First, the recombinant vector pET-32b(+)-GGPPS constructed in Example 1 was co-transformed with the PAC-94N plasmid to express the strain E.coli BL21(DE3), and at the same time, the empty vector pET-32b(+) and PAC- 94N plasmid co-transformation as a negative control strain;
[0040] Subsequently, after culturing overnight, positive clones were selected for identification, and positive clone strains with correct sequencing were preserved or further tested for β-carotene content.
[0041] The detection principle of β-carotene content is: Escherichia coli cannot produce GGPP by itself, while the PAC-94N plasmid contains all...
Embodiment 3
[0043] Since there is no report on the crystal structure of tobacco GGPPS, in order to analyze the protein, based on its amino acid sequence, the inventors performed homology modeling on the enzyme. During the modeling process, the optimal template is 3kro, with a score of 0.993 (TM-score is used to measure the matching degree of two protein structure models, the score ranges from 0 to 1, and 1 means a perfect match), root mean square error The deviation value (RMSD) was 0.36 Å, the sequence identity (IDEN) was 74.9%, and the protein structure coverage (Cov) was 99.7%.
[0044] The substrates C5-DMAPP, C10-GPP, and C15-FPP were docked to the GGPPS catalytic pocket using the Rosetta_docking program, respectively. By looking for the best binding position of receptor small molecule compound and enzyme action, so as to predict its binding mode. The final analysis results show that the 154th, 161st and 218th positions are located in the catalytic pocket of the enzyme, and the bind...
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