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GGPPS directional single-point mutant protein GGPPS-218

A GGPPS-218, mutant protein technology, applied in the field of GGPPS-directed mutant protein patent applications, can solve the problems of short development time and no plant genetic modification

Active Publication Date: 2020-08-07
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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  • GGPPS directional single-point mutant protein GGPPS-218
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  • GGPPS directional single-point mutant protein GGPPS-218

Examples

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Effect test

Embodiment 1

[0047] Since the acquisition of the existing geranyl geranyl diphosphate synthase (GGPPS) gene is the basis of related sequence analysis and directed evolutionary mutation, this example firstly discusses the existing geranyl geranyl diphosphate synthase (GGPPS) gene. The process of cloning and obtaining the diphosphate synthase (GGPPS) gene is introduced as follows.

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

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

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

[0051] Use tobacco K326 leaf cDNA as template for PCR amplification;

[0052] After performing electrophoresis detection on the PCR amplification product, recover and purify it, and connect the recovered and purified PCR product to the pGEMT plasmid;

[0053] Subsequently, the ligation product was transformed into E. coli DH5α competent cells, and after ove...

Embodiment 2

[0062] In order to facilitate the detection and analysis of related mutation sites, the geranyl geranyl diphosphate synthase (GGPPS) recombinant engineering strain is used in this application for experimental verification. Therefore, this example describes the construction process of the engineering strain The introduction is as follows.

[0063] First, the recombinant vector pET-32b(+)-GGPPS constructed in Example 1 and the PAC-94N plasmid were co-transformed into the expression strain E.coli BL21(DE3), and the empty vector pET-32b(+) and PAC- Co-transformation with 94N plasmid serves as a negative control strain;

[0064] Subsequently, after overnight culture, positive clones were selected for identification, and the positive clones with correct sequencing were preserved or tested for further 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 genes of β-carotene synth...

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 of 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, and the score is from 0 to 1, and 1 means a perfect match), root mean square error The deviation value (RMSD) is 0.36 Å, the sequence identity (IDEN) is 74.9%, and the protein structure coverage (Cov) is 99.7%.

[0068] The Rosetta_docking program was used to dock the substrates C5-DMAPP, C10-GPP, and C15-FPP to the GGPPS catalytic pocket. By finding the best binding position of receptor small molecule compounds and enzymes, the binding mode can be predicted. The final analysis results show that the 154th, 161st and 218th positions are located in the catalytic pocket of the enzyme. The substitution of amino acids can furthe...

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Abstract

The invention belongs to the technical field of tobacco gene engineering, and particularly relates to patent application affairs of GGPPS (geranylgeranyl diphosphate synthase) directional mutant protein. 154, 161 and 218 sites of existing GGPPS protein are positioned in a catalytic pocket of an enzyme, and 209 and 233 sites are positioned on the surface of an enzyme molecule; based on the five sites, the invention provides GGPPS series directional mutant protein, which comprises: a series of single-site mutant protein, double-site mutant protein, three-site mutant protein, four-site mutant protein and 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 and on the basis of directed evolution requirements. Preliminary experiment results show that after amino acid mutation at a specific site, the beta-carotene synthesis amount 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 patent applications for GGPPS targeted mutant proteins. Background technique [0002] Carotenoids are important plastid pigments, which have important physiological functions, are closely related to plant growth and photosynthesis, and affect crop quality traits. Geranylgeranyldiphosphate (GGPP) is the common precursor of carotenoids, phytol side chains of chlorophyll and vitamin E, gibberellin and diterpene phytoales. GGPP is catalyzed by geranyl geranyl diphosphate synthase (GGPP synthase, GGPPS), 3 molecules of isopentenyl pyrophosphate (IPP) and 1 molecule of allyl isomer dimethylallyl pyrophosphate Phosphoric acid (DMAPP), under the action of GGPPS, condenses to generate C20 GGPP. [0003] GGPP is the starting substrate for carotenoid synthesis. It can be catalyzed by the port enzyme phytoene synthase (PSY) of the carotenoid synthesis pathway to ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12P23/00C12N15/70C12N15/82A01H5/00A01H6/82C12R1/19
CPCC12N9/1085C12Y205/01029C12P23/00C12N15/70C12N15/825
Inventor 王燃董臣李锋金立锋魏攀都菲王宇博宋卫娜
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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