CN111549009B - Directional three-point mutant protein GGPPS-154-161-218 - Google Patents

Abstract

The present application belongs to the technical field of tobacco genetic engineering, and specifically relates to a GGPPS-directed mutein. The three sites 154, 161 and 218 of the existing GGPPS protein are located in the catalytic pocket of the enzyme, and the two sites 209 and 233 are located on the surface of the enzyme molecule; based on these five sites, this application provides a series of GGPPS directed muteins. , including: a series of single-site muteins, double-site muteins, three-site muteins, four-site muteins, and five-site muteins. The inventor used CAST technology to construct a "small but refined" mutant library. Through further screening and based on the needs of directed evolution, the inventor made a detailed analysis of the types of amino acid mutations at specific sites. Preliminary experimental results showed that the synthesis of β-carotene was significantly improved after the amino acid mutation at a specific site, which laid a certain technical foundation for further cultivation of new crop varieties.

Description

Directional three-point mutant protein GGPPS-154-161-218

Technical Field

The application belongs to the technical field of tobacco genetic engineering, and particularly relates to GGPPS directional mutant protein patent application matters.

Background

The carotenoid is an important plastid pigment, has important physiological action, is closely related to the growth and development and photosynthesis of plants, and influences the quality and the character of crops. Geranylgeranyl diphosphate (GGPP) is a common precursor for carotenoids, the phytol side chains of chlorophyll and vitamin E, gibberellins, and diterpene phytochemicals. GGPP is produced by catalysis of geranylgeranyl diphosphate synthase (GGPP synthsase, GGPPS), and 3 molecules of isopentenyl pyrophosphate (IPP) and 1 molecule of allyl isomer dimethylallyl pyrophosphate (DMAPP) are condensed under the action of GGPPS to produce C20 GGPP.

GGPP is a starting substrate for carotenoid synthesis, and can be catalyzed by Phytoene Synthase (PSY), a terminal enzyme of the carotenoid synthesis pathway, to form phytoene, which is used for carotenoid synthesis. A great number of research examples on plant GGPPS gene family show that the family members not only encode important enzyme proteins at the upstream of a terpene synthesis pathway, but also directly participate in regulating and controlling various pathways of plant terpene synthesis, and play a central role in regulation and control.

In the existing research, although the synthesis routes of carotenoids have been studied more, from the aspect of plant improvement, if the oriented breeding and cultivation can be realized by using the genetic engineering technology, the method is an important technical premise for realizing the maximum utilization of plants.

As one of the key technologies in synthetic biology, directed biological evolution technology has acquired the 2018 nobel prize on chemistry. The technology is widely used for the activity design of enzyme, and can effectively overcome the defects of natural enzyme in the aspects of environmental tolerance, stereo/regioselectivity, substrate specificity, catalytic efficiency, product inhibition and the like through directed evolution, so that the evolution process of thousands of years in the nature is completed in a laboratory in a short time.

In the development process of the directed evolution technology of the enzyme, when the traditional directed evolution is carried out, such as error-prone PCR, DNA mixed group, sequence saturation mutation, random initiation of in vitro recombination and other technologies, the defects of low mutation efficiency, large screening workload and the like exist, and the application of the in vitro directed evolution of enzyme molecules is restricted. The combined active-site mutation strategy (CAST) is a relatively new directed evolution mutation strategy, and the technology is based on the structural information of protein, firstly, by means of the simulation basis of a computer, then, amino acid residues which have direct interaction with a substrate are selected around an enzyme catalytic active center, and the screening scale of a mutant library is reduced by constructing a small and fine mutant library.

Generally, although the directed evolution technology can accelerate the evolution process of crop genes and provide high-quality genes for improving the quality of crops, the directed evolution technology is not used for modifying plant genes at present due to short development time and other technical difficulties.

Tobacco is one of model crops for genetic engineering research, and meanwhile, the carotenoid content in tobacco directly influences the quality and the character of the tobacco. Therefore, if the directional evolution can be combined to carry out the directional transformation on the terpene synthetic genes of the tobacco, the method has very important application significance for accelerating the tobacco breeding, and simultaneously, a certain technical foundation can be laid for the genetic breeding of other crops.

Disclosure of Invention

Through the research on the structure of geranylgeranyl diphosphate synthase (GGPP synthsase, GGPPS) enzyme and the combination of the directed evolution technology, the application aims to provide a plurality of GGPPS and tobacco GGPPS genes with better enzyme activity, thereby providing a new technical thought for tobacco breeding and new tobacco variety cultivation and simultaneously laying a certain technical foundation for breeding and variety improvement of other crops.

The technical scheme adopted by the application is detailed as follows.

In the process of researching the existing GGPPS structure, the inventor thinks that the 154 th site, the 161 th site and the 218 th site of the existing GGPPS protein (the corresponding gene sequence is NM-001325177.1) are positioned in a catalytic pocket of enzyme, so after the directional mutation is carried out on the amino acids of the three sites, the combination of a substrate and the enzyme activity pocket is facilitated, and the enzyme activity performance of GGPPS is improved; the 209 th site and the 233 th site are positioned on the surface of enzyme molecules, and after directional mutation is carried out on the amino acids of the two sites, GGPPS homodimers are favorably formed, so that the stability of the protein is enhanced; based on the five sites, the inventors provide the GGPPS series of directional muteins, which include: the specific introduction of the series of single-site mutant proteins, the series of double-site mutant proteins, the series of three-site mutant proteins, the series of four-site mutant proteins and the series of five-site mutant proteins is described as follows.

Single site mutations:

GGPPS directional single-point mutant protein GGPPS-154, compared with the existing tobacco GGPPS protein, val (V) amino acid at the 154 th position is mutated into neutral amino acid Ala (A) or cysteine Cys (C);

GGPPS directional single-point mutant protein GGPPS-161, compared with the existing tobacco GGPPS protein, ile (I) amino acid at the 161 position is mutated into neutral aliphatic amino acid Leu (L) or into amino acid Met (M) containing sulfhydryl;

GGPPS is a directional single-point mutant protein GGPPS-209, and compared with the existing tobacco GGPPS protein, the 209 th Ile (I) amino acid of the GGPPS protein is mutated into a basic amino acid Lys (K) and a hydroxyl-containing amino acid Ser (S), or is mutated into any one of aspartic acid Asp (D), asparagine Asn (N), alanine Ala (A) or proline Pro (P);

GGPPS directional single point mutant protein GGPPS-218, compared with the existing tobacco GGPPS protein, the 218 th Phe (F) amino acid of the GGPPS directional single point mutant protein is mutated into aromatic amino acid tyrosine Tyr (Y) or leucine Leu (L);

GGPPS directional single-point mutant protein GGPPS-233, compared with the existing tobacco GGPPS protein, the 233 th amino acid Val of which is mutated into the acidic amino acid Glu (E) or tyrosine Tyr (Y);

the amino acid sequence of the existing tobacco GGPPS protein is shown in SEQ ID NO. 1.

In the two-site mutation:

on the basis of the unit mutation, the mutant protein formed by combining any two of the unit sites shows better enzyme activity effect, and specifically, the double-site mutant protein is, for example:

GGPPS directional double-site mutant protein 209-233, wherein the Ile amino acid at the 209 th site and the Val amino acid at the 233 th site are simultaneously mutated compared with the existing tobacco GGPPS protein; the 209 th amino acid Ile (I) is mutated into any one of basic amino acid Lys (K), amino acid Ser (S) containing hydroxyl, aspartic acid Asp (D), asparagine Asn (N), alanine Ala (A) or proline Pro (P), and the 233 th amino acid Val is mutated into acidic amino acid Glu (E) or tyrosine Tyr (Y);

that is, the GGPPS directional double-site mutant protein 209-233 is combined at the sites 209 and 233 into the following parts: 209K-233E, 209S-233E, 209P-233E, 209S-233Y, 209P-233Y, or the like.

In the three-site mutation:

on the basis of the unit mutation, mutant proteins formed by any three-site combination show better enzyme activity effect, and specifically, the two-site mutant proteins are, for example:

the directional three-point mutant protein GGPPS-154-161-218 has simultaneous mutation of Val amino acid in 154 th position, ile amino acid in 161 th position and Phe amino acid in 218 th position compared with the existing tobacco GGPPS protein; the Val (V) amino acid at the 154 th position is mutated into a neutral amino acid Ala (A) or is mutated into a cysteine Cys (C); the Ile (I) amino acid at the 161 st position is mutated into neutral aliphatic amino acid Leu (L) or is mutated into amino acid Met (M) containing sulfydryl; the 218 th Phe (F) amino acid is mutated into an aromatic amino acid tyrosine Tyr (Y) or into leucine Leu (L);

namely, the GGPPS directional three-point mutant protein GGPPS-154-161-218 is combined as follows: 154A-161M-218L, 154A-161M-218Y, 154A-161L-218L, 154A-161L-218Y, 154C-161M-218L, 154C-161M-218Y, 154C-161L-218L, 154C-161L-218Y, and the like.

In the five-site mutation:

on the basis of the unit mutation, the mutant protein formed by any mutation combination of five sites shows better enzyme activity effect, and specifically, the double-site mutant protein is, for example:

compared with the existing tobacco GGPPS protein, the directional five-site mutant protein GGPPS on the surfaces of the enzyme pocket and the enzyme molecule simultaneously mutates Val amino acid at position 154, ile amino acid at position 161, ile amino acid at position 209, phe amino acid at position 218 and Val amino acid at position 233;

the five-site mutant protein GGPPS combination is as follows: 154A-161M-209K-218Y-233E, 154A-161M-209S-218Y-233E, 154A-161L-209S-218Y-233E, or 154A-161L-209K-218Y-233E, and the like.

The GGPPS directional mutant protein is applied to pigment synthesis, and is used for catalyzing GGPP to synthesize beta-carotene.

The novel crop variety breeding method based on the GGPPS series directional mutant protein coding gene is characterized in that the content of pigment substances in crops can be adjusted (increased) by recombining the GGPPS series directional mutant protein coding gene into a crop genome by using a genetic engineering technical means;

the pigment substance is beta-carotene;

the crops are as follows: tobacco, pepper, tomato, rice, corn, wheat, potato, castor-oil plant, apple, cucumber, watermelon, carrot, banana, orange, grape, coffee, ginkgo, salvia, chrysanthemum or rubber tree, etc.

GGPP is used as a precursor substance of downstream carotenoid, and the inventors show that 154, 161, 209, 218 and 233 are key sites through structural analysis of key amino acid sites of an enzyme active pocket of GGPPS. Furthermore, the inventor utilizes CAST technology to construct a small and fine mutant library, thereby overcoming the defects of low mutation rate, large screening amount and the like in the traditional directed evolution screening process. Through further screening and based on the requirement of directed evolution, the inventors have conducted detailed analysis on the types of amino acid mutations at specific sites. Preliminary experiment results show that the synthesis amount of the beta-carotene is obviously improved after the amino acid at a specific site is mutated, and a certain technical basis is laid for further cultivating new varieties of crops.

Drawings

FIG. 1 is a bacterial color experiment of GGPPS-154 mutant, in which: a double plasmid bacteria color experiment, PAC-94N plasmid contains PSY, PDS and LCY-B three genes, IPP and DMAPP can be synthesized in Escherichia coli, but GGPP can not be produced, after GGPPS plasmid with enzyme activity and PAC-94N are jointly transformed into Escherichia coli, beta-carotene can be catalytically produced in Escherichia coli, and Escherichia coli is transformed from white to yellow; B. in the GGPPS-154 single-site mutation bacterial color experiment, the 154 th amino acid site is mutated, after the 154 th amino acid site and PAC-94N double plasmids are jointly transformed into escherichia coli BL21 (DE) 3, the color of the bacterial liquid is obviously changed after IPTG induction, the lower graph is the light absorption value at OD440, and empty pET32b is used as negative control, so that the light absorption value of the mutant at OD440 is obviously improved compared with wild type GGPPS;

FIG. 2 shows bacterial color experiments of GGPPS-161 mutant, wherein A is the same as that in FIG. 1A and is a synthetic schematic diagram, and B is bacterial color experiments of single-site mutation of GGPPS-161;

FIG. 3 shows bacterial color experiments of GGPPS-218 mutant, wherein A is the same as that in FIG. 1A and is a synthetic schematic diagram, and B is bacterial color experiments of single-site mutation of GGPPS-218;

FIG. 4 shows bacterial color experiments of GGPPS-209 mutant, wherein A is the same as that in FIG. 1A and is a synthetic principle diagram, and B is bacterial color experiments of single-site mutation of GGPPS-209;

FIG. 5 shows bacterial color experiments of GGPPS-233 mutant, wherein A is the same as FIG. 1A and is a synthetic schematic diagram, and B is bacterial color experiments of single-site mutation of GGPPS-233;

FIG. 6 shows bacterial color experiments of GGPPS-154/161/218 mutant, in which FIG. A is the same as FIG. 1A and is a synthetic schematic diagram, and FIG. B shows bacterial color experiments of GGPPS-154/161/218 three-site combined mutation;

FIG. 7 shows bacterial color experiments of GGPPS-209/233 mutant, wherein A is the same as that in FIG. 1A and is a synthetic schematic diagram, and B is bacterial color experiments of GGPPS-209/233 two-site combined mutation;

FIG. 8 shows bacterial color experiments of GGPPS-154/161/209/218/233 mutant, wherein the A diagram is the same as the A diagram in FIG. 1 and is a synthetic schematic diagram, and the B diagram shows bacterial color experiments of GGPPS-154/161/209/218/233 five-site combined mutation.

FIG. 9 is a comparison of GGPPS-154 and the main stream crop GGPPS loci, in which: hot pepper (cagggpps 1), potato (StGGPPS 1), wheat (tagggpps 1), corn (ZmGGPPS 1), salvia miltiorrhiza (SmGGPPS 1), coffee (CanGGPPS 1), carrot (DcGGPPS 1), grape (VvGGPPS), cucumber (CsaGGPPS), watermelon (ClGGPPS), apple (MdGGPPS 1), orange (csigpps), rubber tree (HbGGPPS), chrysanthemum (CmGGPPS), rice (OsGGPPS 1), tomato (SlGGPPS 1), castor bean (rcgggpps), banana (magggpps), and ginkgo biloba (gggbpps).

Detailed Description

The present application is further explained below with reference to the drawings and examples.

Example 1

Since the acquisition of the existing geranylgeranyl diphosphate synthase (GGPPS) gene is the basis of the analysis of the relevant sequences, as well as directed evolutionary mutation, this example is summarized below with respect to the cloning of the existing geranylgeranyl diphosphate synthase (GGPPS) gene.

First, based on the gene sequence shown in GenBank accession No. NM-001325177.1, primer sequences for PCR amplification were designed as follows:

a forward primer: 5 'atgagattattatgatcttgt-5',

reverse primer: 5 'attttcacgataagcaatgate-doped 3';

carrying out PCR amplification by taking cDNA of K326 leaves of tobacco as a template;

carrying out electrophoresis detection on the PCR amplification product, recovering and purifying, and connecting the recovered and purified PCR product with pGEMT plasmid;

subsequently, the ligation product was transformed into E.coli DH 5. Alpha. Competent cells, after overnight culture, positive clones were selected and identified to obtain the recombinant correct cloning plasmid pGEMT-GGPPS.

In order to facilitate the subsequent expression of GGPPS, pET-32b (+) plasmids are utilized, and the inventor further constructs a recombinant expression vector, wherein the specific process is as follows:

first, a pair of primer sequences containing Nde I and Xho I cleavage sites was designed as follows:

a forward primer: 5 'gctaatccatatgGAGCAATTCAATTTCAAAACT-3',

reverse primer: 5 'cagctccgaggaATTTTCACGATAAGCAATGTAAT-3';

then, using the clone plasmid pGEMT-GGPPS as a template to carry out PCR amplification so as to obtain a GGPPS gene, and recycling and purifying PCR amplification products;

subsequently, carrying out Nde I and Xho I double enzyme digestion on the GGPPS gene product obtained by PCR amplification and the pET-32b (+) plasmid respectively, and recovering enzyme digestion products for connection;

finally, the ligation product is transformed into DH5 alpha competent cells, after overnight culture, positive clones are selected for identification, and the correctly identified recombinant expression plasmid vector is renamed as: pET-32b (+) -GGPPS.

Example 2

In order to facilitate the detection and analysis of relevant mutation sites, the present application has been experimentally verified using a geranylgeranyl diphosphate synthase (GGPPS) recombinant engineered strain, and therefore, the construction process of this engineered strain is briefly described below in this example.

Firstly, the recombinant vector pET-32b (+) -GGPPS constructed in the example 1 and the PAC-94N plasmid are co-transformed into an expression strain E.coli BL21 (DE 3), and meanwhile, an empty vector pET-32b (+) and the PAC-94N plasmid are co-transformed into a negative control strain;

subsequently, after overnight culture, positive clones are selected for identification, and strains of the positive clones with correct sequencing are preserved or further subjected to beta-carotene content detection.

The principle of detecting the content of the beta-carotene comprises the following steps: coli cannot produce GGPP by itself, and PAC-94N plasmid contains all genes of a beta-carotene synthesis pathway but does not contain geranylgeranyl diphosphate synthase encoding genes, so that when the recombinant vector pET-32b (+) -GGPPS and the PAC-94N plasmid are co-transformed, the strain which can catalytically produce GGPP is changed from white to yellow, the strain with stronger enzyme activity has deeper yellow, and the absorbance value of OD440 is further measured by a spectrophotometer, so that the content of the final product beta-carotene can be measured.

Example 3

Since no crystal structure report is available for tobacco GGPPS, the inventors have performed homologous modeling on the enzyme based on its amino acid sequence in order to analyze the protein. During modeling, the optimal template was 3kro, the score was 0.993 (TM-score was used to measure the degree of matching between two protein structure models, a score from 0 to 1,1 means perfect match), the Root Mean Square Deviation (RMSD) was 0.36 a, the sequence Identity (IDEN) was 74.9%, and the protein structure coverage (Cov) was 99.7%.

The substrates C5-DMAPP, C10-GPP, C15-FPP were docked to the GGPPS catalytic pocket using the Rosetta _ docking program, respectively. The binding mode of the receptor small molecule compound is predicted by searching the optimal binding position acted by the receptor small molecule compound and the enzyme. The final analysis results show that: the 154 th site, the 161 th site and the 218 th site are positioned in a catalytic pocket of the enzyme, the binding capacity of a substrate and an enzyme activity pocket can be further improved after amino acid substitution, and the sites participate in enzyme activity regulation and control; and the 209 th and 233 th amino acid sites are positioned on the surface of the enzyme molecule, which is favorable for the protein to form a dimer.

It should be noted that, the existing GGPPS consists of 296 amino acids, and the specific sequence is shown in SEQ ID No.1, which is as follows:

EQFNFKTYVAEKAISVNKALDEAVIVKDPPVIHEAMRYSLLAGGKRVRPMLCLAACELVGGDQSNAMPAACAVEMIHTMSLIHDDLPCMDNDDLRRGKPTNHKVYGEDVAVLAGDSLLAFAFEYIATATAGVSPSRILAAIGELAKSIGTEGLVAGQVADIACTGNPNVGLDTLEFIHIHKTAALLEASVVLGAILGGGTDEEVEKLRIFARCIGLLFQVVDDILDVTKSSEVLGKTAGKDLAVDKTTYPKLLGLEKAKEFAAELNRDAKQQLVEFDPHKAAPLIALADYIAYREN

the gene for coding GGPPS consists of 888 nucleotides, and the specific sequence is shown as SEQ ID No.2, and specifically comprises the following steps:

GAGCAATTCAATTTCAAAACTTACGTAGCTGAAAAGGCTATTTCTGTAAATAAAGCTTTAGATGAGGCTGTTATAGTAAAAGACCCACCTGTGATCCACGAAGCAATGCGCTATTCACTTCTCGCCGGCGGCAAAAGAGTCCGACCGATGCTCTGCCTCGCCGCCTGCGAGCTCGTCGGCGGCGACCAATCCAACGCCATGCCGGCTGCTTGCGCCGTCGAGATGATCCACACTATGTCCCTCATTCACGACGATTTACCTTGTATGGATAACGACGATCTCCGCCGTGGAAAGCCGACGAACCACAAAGTCTACGGCGAGGACGTGGCGGTCCTCGCCGGAGACTCGCTCCTCGCTTTCGCCTTCGAGTACATCGCCACCGCTACCGCCGGAGTTTCACCGTCGAGGATCCTCGCCGCCATCGGCGAACTGGCGAAATCCATCGGAACTGAAGGGTTAGTAGCTGGACAAGTAGCGGATATAGCTTGTACTGGTAACCCTAATGTTGGACTCGACACACTCGAATTCATTCACATACACAAAACGGCGGCGCTTCTAGAAGCTTCCGTAGTTCTCGGAGCAATCCTCGGCGGCGGAACAGATGAAGAAGTGGAGAAATTGAGGATATTCGCGAGATGTATTGGATTATTGTTTCAAGTAGTAGATGATATACTCGATGTTACAAAGTCGTCGGAGGTGCTCGGAAAAACCGCCGGAAAAGATTTGGCAGTAGATAAAACGACGTATCCAAAACTGCTGGGATTGGAAAAGGCTAAGGAATTTGCGGCGGAGCTCAACCGAGATGCTAAACAACAGCTGGTGGAATTTGATCCACACAAAGCTGCTCCCTTGATTGCTTTGGCGGATTACATTGCTTATCGTGAAAAT

based on the above analysis, the inventors further constructed a GGPPS saturation mutant library aiming at the 5 sites, and the specific construction process is briefly described as follows.

First, degenerate primer sequences were designed according to the above 5 sites as follows:

V154-F：5’-ggaactgaagggttannkgctggacaagtagcg-3’，

I161-F: 5’-ggacaagtagcggatnnkgcttgtactggtaac-3’，

I209-F: 5’-gtggagaaattgaggnnkttcgcgagatgtatt-3’，

F218-F: 5’-tgtattggattattgnnkcaagtagtagatgat-3’，

V233-F: 5’-acaaagtcgtcggagnnkctcggaaaaaccgcc-3’，

general-R: 5’-cacgataagcaatgtaatccg-3’；

subsequently, whole plasmid PCR was performed using pET-32b (+) -GGPPS constructed in example 1 as a template, and then V154-F, I161-F, I209-F, F218-F and V233-F as forward primers and general-R as reverse primers, respectively;

and then, dpnI digestion is carried out on the PCR amplification product, the digested product and PAC-94N double plasmid are jointly transformed into E.coli BL21 (DE 3) competent cells, and after the transformation is finished, the bacterial liquid is coated on a flat plate containing 100 mu g/L ampicillin and 34 mu g/L chloramphenicol, and finally, a saturated mutation library is obtained.

For further screening, positive monoclonals were selected, cultured overnight in 20ml of LB liquid medium containing ampicillin (100. Mu.g/L) and chloramphenicol (34. Mu.g/L), induced at 18 ℃ for expression with IPTG at a final concentration of 0.1mM, centrifuged after completion of the culture, the supernatant was discarded, photographed, and resuspended in 3ml of acetone, and the OD440 value was measured.

And finally screening and determining the mutator with obviously enhanced absorbance by adopting the high-throughput screening method and further sequencing. The results show that when: the Val (V) amino acid at the 154 th position is mutated into a neutral amino acid Ala (A) or is mutated into a cysteine Cys (C); the Ile (I) amino acid at the 161 th position is mutated into neutral aliphatic amino acid Leu (L) or is mutated into amino acid Met (M) containing sulfydryl; mutation of Ile (I) amino acid at position 209 to basic amino acid Lys (K), amino acid Ser (S) containing hydroxyl, aspartic acid Asp (D), asparagine Asn (N), alanine Ala (A) or proline Pro (P); the 218 th Phe (F) amino acid is mutated into an aromatic amino acid tyrosine Tyr (Y) or into leucine Leu (L); when the 233 th amino acid Val is mutated into the acidic amino acid, the acidic amino acid is Glu (E) or tyrosine Tyr (Y); the geranylgeranyl diphosphate synthase GGPPS of tobacco has obvious change.

For position 154:

when the 154 site is mutated to A, the amino acid sequence is as follows:

EQFNFKTYVAEKAISVNKALDEAVIVKDPPVIHEAMRYSLLAGGKRVRPMLCLAACELVGGDQSNAMPAACAVEMIHTMSLIHDDLPCMDNDDLRRGKPTNHKVYGEDVAVLAGDSLLAFAFEYIATATAGVSPSRILAAIGELAKSIGTEGLAAGQVADIACTGNPNVGLDTLEFIHIHKTAALLEASVVLGAILGGGTDEEVEKLRIFARCIGLLFQVVDDILDVTKSSEVLGKTAGKDLAVDKTTYPKLLGLEKAKEFAAELNRDAKQQLVEFDPHKAAPLIALADYIAYREN；

the corresponding base coding sequence is as follows:

GAGCAATTCAATTTCAAAACTTACGTAGCTGAAAAGGCTATTTCTGTAAATAAAGCTTTAGATGAGGCTGTTATAGTAAAAGACCCACCTGTGATCCACGAAGCAATGCGCTATTCACTTCTCGCCGGCGGCAAAAGAGTCCGACCGATGCTCTGCCTCGCCGCCTGCGAGCTCGTCGGCGGCGACCAATCCAACGCCATGCCGGCTGCTTGCGCCGTCGAGATGATCCACACTATGTCCCTCATTCACGACGATTTACCTTGTATGGATAACGACGATCTCCGCCGTGGAAAGCCGACGAACCACAAAGTCTACGGCGAGGACGTGGCGGTCCTCGCCGGAGACTCGCTCCTCGCTTTCGCCTTCGAGTACATCGCCACCGCTACCGCCGGAGTTTCACCGTCGAGGATCCTCGCCGCCATCGGCGAACTGGCGAAATCCATCGGAACTGAAGGGTTAGCGGCTGGACAAGTAGCGGATATAGCTTGTACTGGTAACCCTAATGTTGGACTCGACACACTCGAATTCATTCACATACACAAAACGGCGGCGCTTCTAGAAGCTTCCGTAGTTCTCGGAGCAATCCTCGGCGGCGGAACAGATGAAGAAGTGGAGAAATTGAGGATATTCGCGAGATGTATTGGATTATTGTTTCAAGTAGTAGATGATATACTCGATGTTACAAAGTCGTCGGAGGTGCTCGGAAAAACCGCCGGAAAAGATTTGGCAGTAGATAAAACGACGTATCCAAAACTGCTGGGATTGGAAAAGGCTAAGGAATTTGCGGCGGAGCTCAACCGAGATGCTAAACAACAGCTGGTGGAATTTGATCCACACAAAGCTGCTCCCTTGATTGCTTTGGCGGATTACATTGCTTATCGTGAAAAT；

when the 154 th site is mutated to C, the coding base sequence is:

GAGCAATTCAATTTCAAAACTTACGTAGCTGAAAAGGCTATTTCTGTAAATAAAGCTTTAGATGAGGCTGTTATAGTAAAAGACCCACCTGTGATCCACGAAGCAATGCGCTATTCACTTCTCGCCGGCGGCAAAAGAGTCCGACCGATGCTCTGCCTCGCCGCCTGCGAGCTCGTCGGCGGCGACCAATCCAACGCCATGCCGGCTGCTTGCGCCGTCGAGATGATCCACACTATGTCCCTCATTCACGACGATTTACCTTGTATGGATAACGACGATCTCCGCCGTGGAAAGCCGACGAACCACAAAGTCTACGGCGAGGACGTGGCGGTCCTCGCCGGAGACTCGCTCCTCGCTTTCGCCTTCGAGTACATCGCCACCGCTACCGCCGGAGTTTCACCGTCGAGGATCCTCGCCGCCATCGGCGAACTGGCGAAATCCATCGGAACTGAAGGGTTATGCGCTGGACAAGTAGCGGATATAGCTTGTACTGGTAACCCTAATGTTGGACTCGACACACTCGAATTCATTCACATACACAAAACGGCGGCGCTTCTAGAAGCTTCCGTAGTTCTCGGAGCAATCCTCGGCGGCGGAACAGATGAAGAAGTGGAGAAATTGAGGATATTCGCGAGATGTATTGGATTATTGTTTCAAGTAGTAGATGATATACTCGATGTTACAAAGTCGTCGGAGGTGCTCGGAAAAACCGCCGGAAAAGATTTGGCAGTAGATAAAACGACGTATCCAAAACTGCTGGGATTGGAAAAGGCTAAGGAATTTGCGGCGGAGCTCAACCGAGATGCTAAACAACAGCTGGTGGAATTTGATCCACACAAAGCTGCTCCCTTGATTGCTTTGGCGGATTACATTGCTTATCGTGAAAAT。

for position 161:

when the 161 site is mutated to L, the coding base sequence is as follows:

GAGCAATTCAATTTCAAAACTTACGTAGCTGAAAAGGCTATTTCTGTAAATAAAGCTTTAGATGAGGCTGTTATAGTAAAAGACCCACCTGTGATCCACGAAGCAATGCGCTATTCACTTCTCGCCGGCGGCAAAAGAGTCCGACCGATGCTCTGCCTCGCCGCCTGCGAGCTCGTCGGCGGCGACCAATCCAACGCCATGCCGGCTGCTTGCGCCGTCGAGATGATCCACACTATGTCCCTCATTCACGACGATTTACCTTGTATGGATAACGACGATCTCCGCCGTGGAAAGCCGACGAACCACAAAGTCTACGGCGAGGACGTGGCGGTCCTCGCCGGAGACTCGCTCCTCGCTTTCGCCTTCGAGTACATCGCCACCGCTACCGCCGGAGTTTCACCGTCGAGGATCCTCGCCGCCATCGGCGAACTGGCGAAATCCATCGGAACTGAAGGGTTAGTAGCTGGACAAGTAGCGGATCTGGCTTGTACTGGTAACCCTAATGTTGGACTCGACACACTCGAATTCATTCACATACACAAAACGGCGGCGCTTCTAGAAGCTTCCGTAGTTCTCGGAGCAATCCTCGGCGGCGGAACAGATGAAGAAGTGGAGAAATTGAGGATATTCGCGAGATGTATTGGATTATTGTTTCAAGTAGTAGATGATATACTCGATGTTACAAAGTCGTCGGAGGTGCTCGGAAAAACCGCCGGAAAAGATTTGGCAGTAGATAAAACGACGTATCCAAAACTGCTGGGATTGGAAAAGGCTAAGGAATTTGCGGCGGAGCTCAACCGAGATGCTAAACAACAGCTGGTGGAATTTGATCCACACAAAGCTGCTCCCTTGATTGCTTTGGCGGATTACATTGCTTATCGTGAAAAT

when the 161 site is mutated to M, the coding base sequence is as follows:

GAGCAATTCAATTTCAAAACTTACGTAGCTGAAAAGGCTATTTCTGTAAATAAAGCTTTAGATGAGGCTGTTATAGTAAAAGACCCACCTGTGATCCACGAAGCAATGCGCTATTCACTTCTCGCCGGCGGCAAAAGAGTCCGACCGATGCTCTGCCTCGCCGCCTGCGAGCTCGTCGGCGGCGACCAATCCAACGCCATGCCGGCTGCTTGCGCCGTCGAGATGATCCACACTATGTCCCTCATTCACGACGATTTACCTTGTATGGATAACGACGATCTCCGCCGTGGAAAGCCGACGAACCACAAAGTCTACGGCGAGGACGTGGCGGTCCTCGCCGGAGACTCGCTCCTCGCTTTCGCCTTCGAGTACATCGCCACCGCTACCGCCGGAGTTTCACCGTCGAGGATCCTCGCCGCCATCGGCGAACTGGCGAAATCCATCGGAACTGAAGGGTTAGTAGCTGGACAAGTAGCGGATATGGCTTGTACTGGTAACCCTAATGTTGGACTCGACACACTCGAATTCATTCACATACACAAAACGGCGGCGCTTCTAGAAGCTTCCGTAGTTCTCGGAGCAATCCTCGGCGGCGGAACAGATGAAGAAGTGGAGAAATTGAGGATATTCGCGAGATGTATTGGATTATTGTTTCAAGTAGTAGATGATATACTCGATGTTACAAAGTCGTCGGAGGTGCTCGGAAAAACCGCCGGAAAAGATTTGGCAGTAGATAAAACGACGTATCCAAAACTGCTGGGATTGGAAAAGGCTAAGGAATTTGCGGCGGAGCTCAACCGAGATGCTAAACAACAGCTGGTGGAATTTGATCCACACAAAGCTGCTCCCTTGATTGCTTTGGCGGATTACATTGCTTATCGTGAAAAT。

for position 218:

when the 218 site is mutated to L, the coding base sequence is as follows:

GAGCAATTCAATTTCAAAACTTACGTAGCTGAAAAGGCTATTTCTGTAAATAAAGCTTTAGATGAGGCTGTTATAGTAAAAGACCCACCTGTGATCCACGAAGCAATGCGCTATTCACTTCTCGCCGGCGGCAAAAGAGTCCGACCGATGCTCTGCCTCGCCGCCTGCGAGCTCGTCGGCGGCGACCAATCCAACGCCATGCCGGCTGCTTGCGCCGTCGAGATGATCCACACTATGTCCCTCATTCACGACGATTTACCTTGTATGGATAACGACGATCTCCGCCGTGGAAAGCCGACGAACCACAAAGTCTACGGCGAGGACGTGGCGGTCCTCGCCGGAGACTCGCTCCTCGCTTTCGCCTTCGAGTACATCGCCACCGCTACCGCCGGAGTTTCACCGTCGAGGATCCTCGCCGCCATCGGCGAACTGGCGAAATCCATCGGAACTGAAGGGTTAGTAGCTGGACAAGTAGCGGATATAGCTTGTACTGGTAACCCTAATGTTGGACTCGACACACTCGAATTCATTCACATACACAAAACGGCGGCGCTTCTAGAAGCTTCCGTAGTTCTCGGAGCAATCCTCGGCGGCGGAACAGATGAAGAAGTGGAGAAATTGAGGATATTCGCGAGATGTATTGGATTATTGCTGCAAGTAGTAGATGATATACTCGATGTTACAAAGTCGTCGGAGGTGCTCGGAAAAACCGCCGGAAAAGATTTGGCAGTAGATAAAACGACGTATCCAAAACTGCTGGGATTGGAAAAGGCTAAGGAATTTGCGGCGGAGCTCAACCGAGATGCTAAACAACAGCTGGTGGAATTTGATCCACACAAAGCTGCTCCCTTGATTGCTTTGGCGGATTACATTGCTTATCGTGAAAAT

when the 218 site is mutated to Y, the coding base sequence is as follows:

GAGCAATTCAATTTCAAAACTTACGTAGCTGAAAAGGCTATTTCTGTAAATAAAGCTTTAGATGAGGCTGTTATAGTAAAAGACCCACCTGTGATCCACGAAGCAATGCGCTATTCACTTCTCGCCGGCGGCAAAAGAGTCCGACCGATGCTCTGCCTCGCCGCCTGCGAGCTCGTCGGCGGCGACCAATCCAACGCCATGCCGGCTGCTTGCGCCGTCGAGATGATCCACACTATGTCCCTCATTCACGACGATTTACCTTGTATGGATAACGACGATCTCCGCCGTGGAAAGCCGACGAACCACAAAGTCTACGGCGAGGACGTGGCGGTCCTCGCCGGAGACTCGCTCCTCGCTTTCGCCTTCGAGTACATCGCCACCGCTACCGCCGGAGTTTCACCGTCGAGGATCCTCGCCGCCATCGGCGAACTGGCGAAATCCATCGGAACTGAAGGGTTAGTAGCTGGACAAGTAGCGGATATAGCTTGTACTGGTAACCCTAATGTTGGACTCGACACACTCGAATTCATTCACATACACAAAACGGCGGCGCTTCTAGAAGCTTCCGTAGTTCTCGGAGCAATCCTCGGCGGCGGAACAGATGAAGAAGTGGAGAAATTGAGGATATTCGCGAGATGTATTGGATTATTGTATCAAGTAGTAGATGATATACTCGATGTTACAAAGTCGTCGGAGGTGCTCGGAAAAACCGCCGGAAAAGATTTGGCAGTAGATAAAACGACGTATCCAAAACTGCTGGGATTGGAAAAGGCTAAGGAATTTGCGGCGGAGCTCAACCGAGATGCTAAACAACAGCTGGTGGAATTTGATCCACACAAAGCTGCTCCCTTGATTGCTTTGGCGGATTACATTGCTTATCGTGAAAAT。

for site 209:

when the 209 th site is mutated to P, the coding base sequence is as follows:

GAGCAATTCAATTTCAAAACTTACGTAGCTGAAAAGGCTATTTCTGTAAATAAAGCTTTAGATGAGGCTGTTATAGTAAAAGACCCACCTGTGATCCACGAAGCAATGCGCTATTCACTTCTCGCCGGCGGCAAAAGAGTCCGACCGATGCTCTGCCTCGCCGCCTGCGAGCTCGTCGGCGGCGACCAATCCAACGCCATGCCGGCTGCTTGCGCCGTCGAGATGATCCACACTATGTCCCTCATTCACGACGATTTACCTTGTATGGATAACGACGATCTCCGCCGTGGAAAGCCGACGAACCACAAAGTCTACGGCGAGGACGTGGCGGTCCTCGCCGGAGACTCGCTCCTCGCTTTCGCCTTCGAGTACATCGCCACCGCTACCGCCGGAGTTTCACCGTCGAGGATCCTCGCCGCCATCGGCGAACTGGCGAAATCCATCGGAACTGAAGGGTTAGTAGCTGGACAAGTAGCGGATATAGCTTGTACTGGTAACCCTAATGTTGGACTCGACACACTCGAATTCATTCACATACACAAAACGGCGGCGCTTCTAGAAGCTTCCGTAGTTCTCGGAGCAATCCTCGGCGGCGGAACAGATGAAGAAGTGGAGAAATTGAGGCCGTTCGCGAGATGTATTGGATTATTGTTTCAAGTAGTAGATGATATACTCGATGTTACAAAGTCGTCGGAGGTGCTCGGAAAAACCGCCGGAAAAGATTTGGCAGTAGATAAAACGACGTATCCAAAACTGCTGGGATTGGAAAAGGCTAAGGAATTTGCGGCGGAGCTCAACCGAGATGCTAAACAACAGCTGGTGGAATTTGATCCACACAAAGCTGCTCCCTTGATTGCTTTGGCGGATTACATTGCTTATCGTGAAAAT

when the 209 th site is mutated to K, the coding base sequence is as follows:

GAGCAATTCAATTTCAAAACTTACGTAGCTGAAAAGGCTATTTCTGTAAATAAAGCTTTAGATGAGGCTGTTATAGTAAAAGACCCACCTGTGATCCACGAAGCAATGCGCTATTCACTTCTCGCCGGCGGCAAAAGAGTCCGACCGATGCTCTGCCTCGCCGCCTGCGAGCTCGTCGGCGGCGACCAATCCAACGCCATGCCGGCTGCTTGCGCCGTCGAGATGATCCACACTATGTCCCTCATTCACGACGATTTACCTTGTATGGATAACGACGATCTCCGCCGTGGAAAGCCGACGAACCACAAAGTCTACGGCGAGGACGTGGCGGTCCTCGCCGGAGACTCGCTCCTCGCTTTCGCCTTCGAGTACATCGCCACCGCTACCGCCGGAGTTTCACCGTCGAGGATCCTCGCCGCCATCGGCGAACTGGCGAAATCCATCGGAACTGAAGGGTTAGTAGCTGGACAAGTAGCGGATATAGCTTGTACTGGTAACCCTAATGTTGGACTCGACACACTCGAATTCATTCACATACACAAAACGGCGGCGCTTCTAGAAGCTTCCGTAGTTCTCGGAGCAATCCTCGGCGGCGGAACAGATGAAGAAGTGGAGAAATTGAGGAAATTCGCGAGATGTATTGGATTATTGTTTCAAGTAGTAGATGATATACTCGATGTTACAAAGTCGTCGGAGGTGCTCGGAAAAACCGCCGGAAAAGATTTGGCAGTAGATAAAACGACGTATCCAAAACTGCTGGGATTGGAAAAGGCTAAGGAATTTGCGGCGGAGCTCAACCGAGATGCTAAACAACAGCTGGTGGAATTTGATCCACACAAAGCTGCTCCCTTGATTGCTTTGGCGGATTACATTGCTTATCGTGAAAAT

when the 209 site is mutated to D, the coding base sequence is as follows:

GAGCAATTCAATTTCAAAACTTACGTAGCTGAAAAGGCTATTTCTGTAAATAAAGCTTTAGATGAGGCTGTTATAGTAAAAGACCCACCTGTGATCCACGAAGCAATGCGCTATTCACTTCTCGCCGGCGGCAAAAGAGTCCGACCGATGCTCTGCCTCGCCGCCTGCGAGCTCGTCGGCGGCGACCAATCCAACGCCATGCCGGCTGCTTGCGCCGTCGAGATGATCCACACTATGTCCCTCATTCACGACGATTTACCTTGTATGGATAACGACGATCTCCGCCGTGGAAAGCCGACGAACCACAAAGTCTACGGCGAGGACGTGGCGGTCCTCGCCGGAGACTCGCTCCTCGCTTTCGCCTTCGAGTACATCGCCACCGCTACCGCCGGAGTTTCACCGTCGAGGATCCTCGCCGCCATCGGCGAACTGGCGAAATCCATCGGAACTGAAGGGTTAGTAGCTGGACAAGTAGCGGATATAGCTTGTACTGGTAACCCTAATGTTGGACTCGACACACTCGAATTCATTCACATACACAAAACGGCGGCGCTTCTAGAAGCTTCCGTAGTTCTCGGAGCAATCCTCGGCGGCGGAACAGATGAAGAAGTGGAGAAATTGAGGGATTTCGCGAGATGTATTGGATTATTGTTTCAAGTAGTAGATGATATACTCGATGTTACAAAGTCGTCGGAGGTGCTCGGAAAAACCGCCGGAAAAGATTTGGCAGTAGATAAAACGACGTATCCAAAACTGCTGGGATTGGAAAAGGCTAAGGAATTTGCGGCGGAGCTCAACCGAGATGCTAAACAACAGCTGGTGGAATTTGATCCACACAAAGCTGCTCCCTTGATTGCTTTGGCGGATTACATTGCTTATCGTGAAAAT

when the 209 th site is mutated to N, the coding base sequence is as follows:

GAGCAATTCAATTTCAAAACTTACGTAGCTGAAAAGGCTATTTCTGTAAATAAAGCTTTAGATGAGGCTGTTATAGTAAAAGACCCACCTGTGATCCACGAAGCAATGCGCTATTCACTTCTCGCCGGCGGCAAAAGAGTCCGACCGATGCTCTGCCTCGCCGCCTGCGAGCTCGTCGGCGGCGACCAATCCAACGCCATGCCGGCTGCTTGCGCCGTCGAGATGATCCACACTATGTCCCTCATTCACGACGATTTACCTTGTATGGATAACGACGATCTCCGCCGTGGAAAGCCGACGAACCACAAAGTCTACGGCGAGGACGTGGCGGTCCTCGCCGGAGACTCGCTCCTCGCTTTCGCCTTCGAGTACATCGCCACCGCTACCGCCGGAGTTTCACCGTCGAGGATCCTCGCCGCCATCGGCGAACTGGCGAAATCCATCGGAACTGAAGGGTTAGTAGCTGGACAAGTAGCGGATATAGCTTGTACTGGTAACCCTAATGTTGGACTCGACACACTCGAATTCATTCACATACACAAAACGGCGGCGCTTCTAGAAGCTTCCGTAGTTCTCGGAGCAATCCTCGGCGGCGGAACAGATGAAGAAGTGGAGAAATTGAGGAATTTCGCGAGATGTATTGGATTATTGTTTCAAGTAGTAGATGATATACTCGATGTTACAAAGTCGTCGGAGGTGCTCGGAAAAACCGCCGGAAAAGATTTGGCAGTAGATAAAACGACGTATCCAAAACTGCTGGGATTGGAAAAGGCTAAGGAATTTGCGGCGGAGCTCAACCGAGATGCTAAACAACAGCTGGTGGAATTTGATCCACACAAAGCTGCTCCCTTGATTGCTTTGGCGGATTACATTGCTTATCGTGAAAAT

when the 209 site is mutated to S, the coding base sequence is as follows:

GAGCAATTCAATTTCAAAACTTACGTAGCTGAAAAGGCTATTTCTGTAAATAAAGCTTTAGATGAGGCTGTTATAGTAAAAGACCCACCTGTGATCCACGAAGCAATGCGCTATTCACTTCTCGCCGGCGGCAAAAGAGTCCGACCGATGCTCTGCCTCGCCGCCTGCGAGCTCGTCGGCGGCGACCAATCCAACGCCATGCCGGCTGCTTGCGCCGTCGAGATGATCCACACTATGTCCCTCATTCACGACGATTTACCTTGTATGGATAACGACGATCTCCGCCGTGGAAAGCCGACGAACCACAAAGTCTACGGCGAGGACGTGGCGGTCCTCGCCGGAGACTCGCTCCTCGCTTTCGCCTTCGAGTACATCGCCACCGCTACCGCCGGAGTTTCACCGTCGAGGATCCTCGCCGCCATCGGCGAACTGGCGAAATCCATCGGAACTGAAGGGTTAGTAGCTGGACAAGTAGCGGATATAGCTTGTACTGGTAACCCTAATGTTGGACTCGACACACTCGAATTCATTCACATACACAAAACGGCGGCGCTTCTAGAAGCTTCCGTAGTTCTCGGAGCAATCCTCGGCGGCGGAACAGATGAAGAAGTGGAGAAATTGAGGAGCTTCGCGAGATGTATTGGATTATTGTTTCAAGTAGTAGATGATATACTCGATGTTACAAAGTCGTCGGAGGTGCTCGGAAAAACCGCCGGAAAAGATTTGGCAGTAGATAAAACGACGTATCCAAAACTGCTGGGATTGGAAAAGGCTAAGGAATTTGCGGCGGAGCTCAACCGAGATGCTAAACAACAGCTGGTGGAATTTGATCCACACAAAGCTGCTCCCTTGATTGCTTTGGCGGATTACATTGCTTATCGTGAAAAT

when the 209 th site is mutated to A, the coding base sequence is as follows:

GAGCAATTCAATTTCAAAACTTACGTAGCTGAAAAGGCTATTTCTGTAAATAAAGCTTTAGATGAGGCTGTTATAGTAAAAGACCCACCTGTGATCCACGAAGCAATGCGCTATTCACTTCTCGCCGGCGGCAAAAGAGTCCGACCGATGCTCTGCCTCGCCGCCTGCGAGCTCGTCGGCGGCGACCAATCCAACGCCATGCCGGCTGCTTGCGCCGTCGAGATGATCCACACTATGTCCCTCATTCACGACGATTTACCTTGTATGGATAACGACGATCTCCGCCGTGGAAAGCCGACGAACCACAAAGTCTACGGCGAGGACGTGGCGGTCCTCGCCGGAGACTCGCTCCTCGCTTTCGCCTTCGAGTACATCGCCACCGCTACCGCCGGAGTTTCACCGTCGAGGATCCTCGCCGCCATCGGCGAACTGGCGAAATCCATCGGAACTGAAGGGTTAGTAGCTGGACAAGTAGCGGATATAGCTTGTACTGGTAACCCTAATGTTGGACTCGACACACTCGAATTCATTCACATACACAAAACGGCGGCGCTTCTAGAAGCTTCCGTAGTTCTCGGAGCAATCCTCGGCGGCGGAACAGATGAAGAAGTGGAGAAATTGAGGGCGTTCGCGAGATGTATTGGATTATTGTTTCAAGTAGTAGATGATATACTCGATGTTACAAAGTCGTCGGAGGTGCTCGGAAAAACCGCCGGAAAAGATTTGGCAGTAGATAAAACGACGTATCCAAAACTGCTGGGATTGGAAAAGGCTAAGGAATTTGCGGCGGAGCTCAACCGAGATGCTAAACAACAGCTGGTGGAATTTGATCCACACAAAGCTGCTCCCTTGATTGCTTTGGCGGATTACATTGCTTATCGTGAAAAT。

for position 233:

when the 233 site is mutated to E, it encodes the base sequence:

GAGCAATTCAATTTCAAAACTTACGTAGCTGAAAAGGCTATTTCTGTAAATAAAGCTTTAGATGAGGCTGTTATAGTAAAAGACCCACCTGTGATCCACGAAGCAATGCGCTATTCACTTCTCGCCGGCGGCAAAAGAGTCCGACCGATGCTCTGCCTCGCCGCCTGCGAGCTCGTCGGCGGCGACCAATCCAACGCCATGCCGGCTGCTTGCGCCGTCGAGATGATCCACACTATGTCCCTCATTCACGACGATTTACCTTGTATGGATAACGACGATCTCCGCCGTGGAAAGCCGACGAACCACAAAGTCTACGGCGAGGACGTGGCGGTCCTCGCCGGAGACTCGCTCCTCGCTTTCGCCTTCGAGTACATCGCCACCGCTACCGCCGGAGTTTCACCGTCGAGGATCCTCGCCGCCATCGGCGAACTGGCGAAATCCATCGGAACTGAAGGGTTAGTAGCTGGACAAGTAGCGGATATAGCTTGTACTGGTAACCCTAATGTTGGACTCGACACACTCGAATTCATTCACATACACAAAACGGCGGCGCTTCTAGAAGCTTCCGTAGTTCTCGGAGCAATCCTCGGCGGCGGAACAGATGAAGAAGTGGAGAAATTGAGGATATTCGCGAGATGTATTGGATTATTGTTTCAAGTAGTAGATGATATACTCGATGTTACAAAGTCGTCGGAGGAACTCGGAAAAACCGCCGGAAAAGATTTGGCAGTAGATAAAACGACGTATCCAAAACTGCTGGGATTGGAAAAGGCTAAGGAATTTGCGGCGGAGCTCAACCGAGATGCTAAACAACAGCTGGTGGAATTTGATCCACACAAAGCTGCTCCCTTGATTGCTTTGGCGGATTACATTGCTTATCGTGAAAAT

when the 233 site is mutated to Y, the coding base sequence is:

GAGCAATTCAATTTCAAAACTTACGTAGCTGAAAAGGCTATTTCTGTAAATAAAGCTTTAGATGAGGCTGTTATAGTAAAAGACCCACCTGTGATCCACGAAGCAATGCGCTATTCACTTCTCGCCGGCGGCAAAAGAGTCCGACCGATGCTCTGCCTCGCCGCCTGCGAGCTCGTCGGCGGCGACCAATCCAACGCCATGCCGGCTGCTTGCGCCGTCGAGATGATCCACACTATGTCCCTCATTCACGACGATTTACCTTGTATGGATAACGACGATCTCCGCCGTGGAAAGCCGACGAACCACAAAGTCTACGGCGAGGACGTGGCGGTCCTCGCCGGAGACTCGCTCCTCGCTTTCGCCTTCGAGTACATCGCCACCGCTACCGCCGGAGTTTCACCGTCGAGGATCCTCGCCGCCATCGGCGAACTGGCGAAATCCATCGGAACTGAAGGGTTAGTAGCTGGACAAGTAGCGGATATAGCTTGTACTGGTAACCCTAATGTTGGACTCGACACACTCGAATTCATTCACATACACAAAACGGCGGCGCTTCTAGAAGCTTCCGTAGTTCTCGGAGCAATCCTCGGCGGCGGAACAGATGAAGAAGTGGAGAAATTGAGGATATTCGCGAGATGTATTGGATTATTGTTTCAAGTAGTAGATGATATACTCGATGTTACAAAGTCGTCGGAGTATCTCGGAAAAACCGCCGGAAAAGATTTGGCAGTAGATAAAACGACGTATCCAAAACTGCTGGGATTGGAAAAGGCTAAGGAATTTGCGGCGGAGCTCAACCGAGATGCTAAACAACAGCTGGTGGAATTTGATCCACACAAAGCTGCTCCCTTGATTGCTTTGGCGGATTACATTGCTTATCGTGAAAAT。

other two, three or five site specific base sequences can be referred to above, for example, for the five site mutant protein GGPPS-154A/161L/209S/218Y/233E, the amino acid sequence is:

EQFNFKTYVAEKAISVNKALDEAVIVKDPPVIHEAMRYSLLAGGKRVRPMLCLAACELVGGDQSNAMPAACAVEMIHTMSLIHDDLPCMDNDDLRRGKPTNHKVYGEDVAVLAGDSLLAFAFEYIATATAGVSPSRILAAIGELAKSIGTEGLAAGQVADLACTGNPNVGLDTLEFIHIHKTAALLEASVVLGAILGGGTDEEVEKLRSFARCIGLLYQVVDDILDVTKSSEELGKTAGKDLAVDKTTYPKLLGLEKAKEFAAELNRDAKQQLVEFDPHKAAPLIALADYIAYREN

in this case, the corresponding coding base sequence is:

GAGCAATTCAATTTCAAAACTTACGTAGCTGAAAAGGCTATTTCTGTAAATAAAGCTTTAGATGAGGCTGTTATAGTAAAAGACCCACCTGTGATCCACGAAGCAATGCGCTATTCACTTCTCGCCGGCGGCAAAAGAGTCCGACCGATGCTCTGCCTCGCCGCCTGCGAGCTCGTCGGCGGCGACCAATCCAACGCCATGCCGGCTGCTTGCGCCGTCGAGATGATCCACACTATGTCCCTCATTCACGACGATTTACCTTGTATGGATAACGACGATCTCCGCCGTGGAAAGCCGACGAACCACAAAGTCTACGGCGAGGACGTGGCGGTCCTCGCCGGAGACTCGCTCCTCGCTTTCGCCTTCGAGTACATCGCCACCGCTACCGCCGGAGTTTCACCGTCGAGGATCCTCGCCGCCATCGGCGAACTGGCGAAATCCATCGGAACTGAAGGGTTAGCGGCTGGACAAGTAGCGGATCTGGCTTGTACTGGTAACCCTAATGTTGGACTCGACACACTCGAATTCATTCACATACACAAAACGGCGGCGCTTCTAGAAGCTTCCGTAGTTCTCGGAGCAATCCTCGGCGGCGGAACAGATGAAGAAGTGGAGAAATTGAGGAGCTTCGCGAGATGTATTGGATTATTGTATCAAGTAGTAGATGATATACTCGATGTTACAAAGTCGTCGGAGGAACTCGGAAAAACCGCCGGAAAAGATTTGGCAGTAGATAAAACGACGTATCCAAAACTGCTGGGATTGGAAAAGGCTAAGGAATTTGCGGCGGAGCTCAACCGAGATGCTAAACAACAGCTGGTGGAATTTGATCCACACAAAGCTGCTCCCTTGATTGCTTTGGCGGATTACATTGCTTATCGTGAAAAT。

furthermore, the inventors have performed different combination type mutations at amino acid positions 154, 161, 209, 218 and 233, synthesized expression vectors with different combination mutations by using the existing genetic engineering technology, further constructed different strains by using the construction method of the geranylgeranyl diphosphate synthase (GGPPS) recombinant engineering strain in example 2, and examined the synthetic amount of beta-carotene. The specific test results are summarized below.

The results of the single-site experiments shown in FIGS. 1 to 5 show that:

when Val at 154 th position is mutated into Ala or Cys, ile at 161 th position is mutated into Leu or Met, ile at 209 th position is mutated into Lys, ser, aspartic acid Asp, asparagine Asn, alanine Ala and proline Pro, phe at 218 th position is mutated into Leu or Tyr, and Val at 233 th position is mutated into Glu or Tyr, the color of the thallus or the synthetic amount of beta-carotene is obviously improved;

as shown in FIGS. 6 to 8, the results of the two-site, three-site and five-site experiments show that the color of the cells or the amount of synthesized beta-carotene is significantly increased under the condition of different site combinations in a specific mutation direction.

The results show that the GGPPS enzyme activity can be better provided by aiming at the directional mutation evolution mode of a specific site, so that a good foundation is laid for further providing the beta-carotene content in crops.

Example 4

Based on the analysis and verification of example 3, the inventors further analyzed the GGPPS modification sites of the existing mainstream crops. The results of comparative analysis of amino acid sequences (as shown in FIG. 9, FIG. 9 shows only the 154-position comparison results) indicate that: similar to GGPPS of tobacco in the present application, pepper (cagggpps 1), potato (StGGPPS 1), wheat (tagggpps 1), corn (ZmGGPPS 1), salvia miltiorrhiza (SmGGPPS 1), coffee (CanGGPPS 1), carrot (DcGGPPS 1), grape (VvGGPPS), cucumber (csagpps), watermelon (ClGGPPS), apple (MdGGPPS 1), orange (csigpps), rubber tree (HbGGPPS), chrysanthemum (CmGGPPS), rice (OsGGPPS 1), tomato (SlGGPPS 1), castor (rcgggpps), banana (magggpps), and ginkgo (GbGGPPS) are all conserved in these 5 sites of GGPPS enzyme, and thus, mutation based on the same site is also feasible, or directed mutant protein obtained by the present application is also feasible directly, which provides a new idea for breeding new varieties of these mainstream crops.

SEQUENCE LISTING

<110> Zhengzhou tobacco institute of China tobacco general Co

<120> three-point directed mutein GGPPS-154-161-218

<130> none

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 296

<212> PRT

<213> Nicotiana tabacum

<400> 1

Glu Gln Phe Asn Phe Lys Thr Tyr Val Ala Glu Lys Ala Ile Ser Val

1 5 10 15

Asn Lys Ala Leu Asp Glu Ala Val Ile Val Lys Asp Pro Pro Val Ile

20 25 30

His Glu Ala Met Arg Tyr Ser Leu Leu Ala Gly Gly Lys Arg Val Arg

35 40 45

Pro Met Leu Cys Leu Ala Ala Cys Glu Leu Val Gly Gly Asp Gln Ser

50 55 60

Asn Ala Met Pro Ala Ala Cys Ala Val Glu Met Ile His Thr Met Ser

65 70 75 80

Leu Ile His Asp Asp Leu Pro Cys Met Asp Asn Asp Asp Leu Arg Arg

85 90 95

Gly Lys Pro Thr Asn His Lys Val Tyr Gly Glu Asp Val Ala Val Leu

100 105 110

Ala Gly Asp Ser Leu Leu Ala Phe Ala Phe Glu Tyr Ile Ala Thr Ala

115 120 125

Thr Ala Gly Val Ser Pro Ser Arg Ile Leu Ala Ala Ile Gly Glu Leu

130 135 140

Ala Lys Ser Ile Gly Thr Glu Gly Leu Val Ala Gly Gln Val Ala Asp

145 150 155 160

Ile Ala Cys Thr Gly Asn Pro Asn Val Gly Leu Asp Thr Leu Glu Phe

165 170 175

Ile His Ile His Lys Thr Ala Ala Leu Leu Glu Ala Ser Val Val Leu

180 185 190

Gly Ala Ile Leu Gly Gly Gly Thr Asp Glu Glu Val Glu Lys Leu Arg

195 200 205

Ile Phe Ala Arg Cys Ile Gly Leu Leu Phe Gln Val Val Asp Asp Ile

210 215 220

Leu Asp Val Thr Lys Ser Ser Glu Val Leu Gly Lys Thr Ala Gly Lys

225 230 235 240

Asp Leu Ala Val Asp Lys Thr Thr Tyr Pro Lys Leu Leu Gly Leu Glu

245 250 255

Lys Ala Lys Glu Phe Ala Ala Glu Leu Asn Arg Asp Ala Lys Gln Gln

260 265 270

Leu Val Glu Phe Asp Pro His Lys Ala Ala Pro Leu Ile Ala Leu Ala

275 280 285

Asp Tyr Ile Ala Tyr Arg Glu Asn

290 295

<210> 2

<211> 888

<212> DNA

<213> Nicotiana tabacum

<400> 2

gagcaattca atttcaaaac ttacgtagct gaaaaggcta tttctgtaaa taaagcttta 60

gatgaggctg ttatagtaaa agacccacct gtgatccacg aagcaatgcg ctattcactt 120

ctcgccggcg gcaaaagagt ccgaccgatg ctctgcctcg ccgcctgcga gctcgtcggc 180

ggcgaccaat ccaacgccat gccggctgct tgcgccgtcg agatgatcca cactatgtcc 240

ctcattcacg acgatttacc ttgtatggat aacgacgatc tccgccgtgg aaagccgacg 300

aaccacaaag tctacggcga ggacgtggcg gtcctcgccg gagactcgct cctcgctttc 360

gccttcgagt acatcgccac cgctaccgcc ggagtttcac cgtcgaggat cctcgccgcc 420

atcggcgaac tggcgaaatc catcggaact gaagggttag tagctggaca agtagcggat 480

atagcttgta ctggtaaccc taatgttgga ctcgacacac tcgaattcat tcacatacac 540

aaaacggcgg cgcttctaga agcttccgta gttctcggag caatcctcgg cggcggaaca 600

gatgaagaag tggagaaatt gaggatattc gcgagatgta ttggattatt gtttcaagta 660

gtagatgata tactcgatgt tacaaagtcg tcggaggtgc tcggaaaaac cgccggaaaa 720

gatttggcag tagataaaac gacgtatcca aaactgctgg gattggaaaa ggctaaggaa 780

tttgcggcgg agctcaaccg agatgctaaa caacagctgg tggaatttga tccacacaaa 840

gctgctccct tgattgcttt ggcggattac attgcttatc gtgaaaat 888

Claims (4)

1. A directional three-point mutant protein GGPPS-154-161-218, which is characterized in that, compared with the existing tobacco GGPPS protein, val amino acid at position 154, ile amino acid at position 161 and Phe amino acid at position 218 are simultaneously mutated; the Val amino acid at position 154 is mutated into a neutral amino acid Ala or is mutated into a cysteine Cys; the Ile amino acid at the 161 th position is mutated into neutral aliphatic amino acid Leu or mutated into amino acid Met containing sulfydryl; phe amino acid 218 is mutated into aromatic amino acid tyrosine Tyr or leucine Leu;

the amino acid sequence of the existing tobacco GGPPS protein is shown as SEQ ID NO. 1;

the GGPPS directional three-point mutant protein GGPPS-154-161-218 comprises the following components in percentage by weight: 154A-161M-218L, 154A-161M-218Y, 154A-161L-218L, 154A-161L-218Y, 154C-161M-218L, 154C-161M-218Y, 154C-161L-218L, or 154C-161L-218Y.

2. Use of the directional three-point mutant protein GGPPS-154-161-218 of claim 1 in pigment synthesis for catalyzing the synthesis of beta-carotene from GGPP.

3. The recombinant expression vector pET-32b (+) -GGPPS for expressing the directional Triplex mutant protein GGPPS-154-161-218 of claim 1, wherein the coding gene of the directional Triplex mutant protein GGPPS-154-161-218 is recombined in a pET-32b (+) plasmid.

4. The application of the directional triple point mutant protein GGPPS-154-161-218 in the cultivation of tobacco varieties as claimed in claim 1, which is characterized in that three sites directionally designed by the GGPPS-154-161-218 are used for modifying the tobacco GGPPS gene by using a genetic engineering technical means, or the directional triple point mutant protein GGPPS-154-161-218 is recombined into the tobacco genome to regulate the content of pigment substances in tobacco;

the pigment substance is beta-carotene.

Images