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Directed five-site mutant protein GGPPS in enzyme pocket and on enzyme molecule surface

A site mutation and pocket technology, applied in the field of tobacco genetic engineering, can solve the problems of short development time and genetic modification of unused plants

Active Publication Date: 2020-08-28
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In general, although directed evolution technology can speed up the evolution of crop genes and provide high-quality genes for the improvement of crop quality, due to the short development time and other technical difficulties, directed evolution technology has not yet been used for plant gene transformation

Method used

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  • Directed five-site mutant protein GGPPS in enzyme pocket and on enzyme molecule surface
  • Directed five-site mutant protein GGPPS in enzyme pocket and on enzyme molecule surface
  • Directed five-site mutant protein GGPPS in enzyme pocket and on enzyme molecule surface

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] 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.

[0048] First, according to the gene sequence shown in GenBank accession number NM_001325177.1, the primer sequences for PCR amplification were designed as follows:

[0049] Forward primer: 5'-atgagatctatgaatcttgt-3',

[0050] Reverse primer: 5'-attttcacgataagcaatgt-3';

[0051] Tobacco K326 leaf cDNA was used as a template for PCR amplification;

[0052] 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;

[0053] Subsequently, the ligation product was transformed into Escherichia coli DH5α competent ...

Embodiment 2

[0062] 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.

[0063] 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;

[0064] 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.

[0065] The detection principle of β-carotene content is: Escherichia coli cannot produce GGPP by itself, while the PAC-94N plasmid contains all...

Embodiment 3

[0067] 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%.

[0068] 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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Abstract

The invention belongs to the technical field of tobacco gene engineering, and particularly relates to patent application matters of a directional mutant protein GGPPS. 154, 161 and 218 sites of an existing GGPPS protein are positioned in a catalytic pocket of an enzyme, and 209 and 233 sites are positioned on an enzyme molecule surface; based on the five sites, the invention provides a GGPPS-series directional mutant protein, which comprises a series of single-site mutant proteins, a double-site mutant protein, a three-site mutant protein, a four-site mutant protein and a five-site mutant protein. The inventor constructs a 'small and fine' mutant library by using a CAST technology, and performs detailed analysis on the amino acid mutation type of a specific site through further screening on the basis of directed evolution requirements. Preliminary experiment results show that after amino acid mutation at the specific site, the synthesis amount of beta-carotene is obviously increased, and a certain technical foundation is laid for further cultivation of new crop varieties.

Description

technical field [0001] This application belongs to the technical field of tobacco genetic engineering, and specifically relates to the patent application for GGPPS directional mutant protein. Background technique [0002] Carotenoid is an important plastid pigment, which has important physiological functions, is closely related to plant growth and photosynthesis, and affects crop quality traits. Geranyl geranyl diphosphate (GGPP) is a common precursor of carotenoids, chlorophyll and vitamin E phytol side chains, gibberellins and diterpene phytoalexins. GGPP is catalyzed by geranylgeranyl diphosphate synthase (GGPP synthase, GGPPS), 3 molecules of isopentenyl pyrophosphate (IPP) and 1 molecule of allyl isomer dimethylallyl pyrophosphate Phosphoric acid (DMAPP) is condensed under the action of GGPPS to generate C20 GGPP. [0003] GGPP is the initial substrate of carotenoid synthesis, which can be catalyzed by the port enzyme phytoene synthase (phytoene synthase, PSY) of the ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/70C12N15/54C12N15/82C12P23/00A01H5/00A01H6/82A01H6/46A01H6/38A01H6/74A01H6/34A01H6/06A01H6/00A01H6/78A01H6/88A01H6/76A01H6/50A01H6/26
CPCC12N9/1085C12N15/825C12P23/00C12Y205/01029
Inventor 王燃董臣李锋魏攀金立锋
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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