CN105820990B - A method for in vivo evolution of target proteins using iterative homologous recombination - Google Patents

Abstract

The present invention relates to a method for in vivo evolution of a target protein using iterative homologous recombination. It is characterized in that: firstly, the single-stranded nucleotide with mutation is prepared by two-step PCR in vitro and digested with DNase I, and the 80-100nt fragment is recovered to form the single-stranded nucleotide mutation library of the target protein; thus, the single-stranded mutation library Transform Escherichia coli, and establish an E. coli colony containing the target protein mutant through λ-Red homologous recombination technology; transform the above single-chain mutant library into the E. coli colony containing the target protein mutant again to form a larger library capacity of E. coli Bacillus flora; retransforming E. coli for 2-6 cycles to construct an E. coli mutation library containing target gene mutations; finally, obtain target protein mutants through high-efficiency screening or high-throughput screening. This technology can be applied to the in situ evolution of microbial genes, to improve the catalytic activity of enzymes, and to increase the yield of microbial metabolites.

Description

A method for in vivo evolution of target proteins using iterative homologous recombination

field of invention

The invention relates to a new method for evolving a target protein in vivo by iterative homologous recombination, which belongs to the field of biotechnology.

Background technique

Directed evolution technology is a simulation of Darwinian evolution in the laboratory to evolve proteins that do not exist in nature or have better properties, and can change the metabolic flow by changing the catalytic efficiency of enzymes, expand or build new metabolic pathways , weaken or eliminate unnecessary or harmful metabolic pathways, so as to achieve the purpose of increasing the yield of certain metabolites or degrading harmful substances. Directed evolution technology mainly has two steps: firstly, modern molecular biological methods are used to construct a gene mutation library, and then coupled screening method is used to screen the library. The construction of mutant library can provide enough diversity for screening, which is a key step in directed evolution. According to the place where the DNA mutation occurs, the techniques for constructing a mutation library can be divided into in vitro mutation and in vivo mutation.

In vitro mutagenesis approaches introduce diversity at the gene of interest through standard experimental techniques such as error-prone PCR, saturation point mutagenesis, or DNA rearrangement. The in vitro mutation method can effectively control the mutation rate and mutation spectrum, while the in vivo mutation method can couple mutation and screening, and can bypass the transformation efficiency bottleneck, avoiding time-consuming and laborious library construction, cloning, and operation steps.

The most commonly used means of in vivo mutation include chemical mutagens, base analogs, ultraviolet light and hypermutated strains. Chemical mutagens produce a narrow spectrum of mutations and may be carcinogenic in humans. UV radiation produces a broad spectrum of mutations with little sequence preference. However, the killing effect of ultraviolet light on cells limits its effect. The most widely used method of in vivo mutagenesis is the use of hypermutated strains such as XL1-Red. This strain improves the probability of mutation by deleting and modifying genes related to DNA replication and repair. However, its mutation frequency cannot be controlled, the gene is unstable, and its growth is slow, making it difficult to isolate mutants.

The George Church laboratory invented MAGE (Multiple Automated Genome Engineering). It simultaneously designs a series of single-stranded oligonucleotides for different regions of the genome, and uses the λ-Red homologous recombination system to integrate these oligonucleotides into the genome to realize the transformation of multiple sites in the genome of a single cell or a cell population Diversity in genome modification. Use this technology to directional transform the RBS regions of 20 genes in the lycopene synthesis process in E. coli, design different degenerate primers, and make them directional evolve to the classic SD sequence (TAAGGAGGT) that can increase the expression level, and finally screen High-yielding strains were obtained. MAGE technology uses 90nt single-stranded oligonucleotides synthesized in vitro to achieve the purpose of efficient homologous recombination, and the transformation targets are all regulatory genes, and it does not realize the directed evolution of protein structural genes.

Contents of the invention

The technical problem to be solved by the present invention is to provide a new method for evolving target protein in vivo by iterative homologous recombination. For this reason, the present invention adopts following technical scheme:

Construct an 80-100nt single-stranded nucleotide target gene mutation library, and use the mutation library to perform in situ iterative recombination of protein genes on the E. The Escherichia coli mutation library containing the target gene mutation achieves the purpose of evolving the molecular structure of the enzyme and improving the catalytic efficiency of the enzyme with a higher mutation rate.

Further, the method is to construct a TA cloning vector of the target protein gene in vitro, use the vector as a template, and use error-prone PCR to prepare double-stranded nucleotides with base mutations; The single-stranded nucleotides with mutations are prepared by primer PCR; the above single-stranded enzymes are digested with DNase I and the 80-100nt fragments are recovered to form the single-stranded nucleotide target gene mutation library of the target protein.

Further, the iterative steps are:

Transform E. coli with the obtained single-stranded nucleotide target gene mutation library, establish an E. coli colony containing target protein mutants through λ-Red homologous recombination technology, and obtain the initial E. coli mutation library containing target gene mutations;

The above-mentioned single-stranded nucleotide target gene mutation library is transformed into the initial E. coli mutation library containing the target gene mutation to obtain a new E. coli mutation library containing the target gene mutation, forming a larger library capacity of E. coli flora, and constructing E. coli mutant library containing the mutation of the gene of interest; or:

Then use the above-mentioned single-stranded nucleotide target gene mutation library to transform the newly formed E. coli mutation library containing the target gene mutation to obtain an updated E. coli mutation library containing the target gene mutation, thereby continuously increasing the E. coli mutation library containing the target gene mutation. The library capacity of the bacillus mutant library is used to construct the E. coli mutant library containing the target gene mutation.

Methods for obtaining single-stranded nucleotides include but are not limited to T7 reverse transcription method, exonuclease method, denaturing high performance liquid chromatography, magnetic bead capture method, asymmetric PCR, two-step PCR method, etc.

80-100nt single-stranded nucleotides are obtained by degrading long single-stranded nucleotides, and its acquisition methods include but are not limited to DNase I digestion, sonication, etc.

Single-stranded nucleotides can enter cells through transformation or transfection. Transformation or transfection methods include but not limited to calcium phosphate method, calcium chloride method, lipofection, electroporation, photoporation, etc. Transformation media include, but are not limited to, water, calcium chloride, liposomes, and the like.

Before transformation or transfection, E. coli cells were grown to a certain concentration at 30°C and induced at 42°C. The λ-Red homologous recombination system on the cell genome is under the control of the pL promoter and is regulated by the cI857 repressor protein. At 30°C, the pL promoter is repressed and does not express, and at 42°C, the expression of the three proteins required for homologous recombination of λ-Red is induced. After 15 minutes of induction, the cells were stored at 4°C to prevent the degradation of induced proteins.

The medium needs to be replaced with transformation medium to remove ions. The methods that can be used in this process include but are not limited to centrifugation, membrane filtration and so on.

In order to achieve a higher mutation rate on the Escherichia coli genome and mutate one or more protein genes, the present invention achieves the purpose of protein evolution through iterative λ-Red homologous recombination strategy. This method can evolve a single gene on the genome, or modify multiple genes at the same time, and couple screening to find out the best effect achieved by the synergistic effect of multiple mutant proteins.

The new method of evolving target protein by iterative homologous recombination provided by the present invention is a method for effectively obtaining desired protein mutation, which is simpler and more efficient than previous methods.

The present invention provides a new method for in vivo evolution of target proteins by iterative homologous recombination, through mutation of the 1-deoxy-D-xylulose-5-phosphate synthase (DXS) gene on the E. Lycopene levels. 5 mutants of 1-deoxy-D-xylulose-5-phosphate synthetase were obtained by screening, the gene sequences of which were selected from the sequence table SEQ ID NO.5, NO.7, NO.9, NO.11, NO . One of the 13 sequences respectively increases the level of lycopene synthesized by corresponding Escherichia coli by 50%-300%.

The present invention provides a new method for in vivo evolution of a target protein by iterative homologous recombination, through mutation of the σ ^s factor (Rpos) gene on the Escherichia coli genome, the level of lycopene produced by the Escherichia coli is increased. Two σ ^s factor mutants were obtained through screening, and their sequences were selected from one of the sequences of SEQ ID NO.19 and NO.21 in the sequence table, which respectively increased the levels of lycopene synthesized by corresponding Escherichia coli by 80% and 205%.

Description of drawings

Fig. 1: schematic flow chart of the present invention. Among them, Gene: gene; Error-Prone PCR: error-prone PCR; SinglePrimer PCR: single-stranded PCR; DNase I Digestion: DNase I digestion; cycles of Recombination: multiple recombination; Genome: genome; E.coli: Escherichia coli.

Figure 2: Comparison chart of lycopene production capacity of wild-type and E. coli strains containing DXS mutants; among them, Lycopene Production: lycopene production.

Figure 3: Comparison chart of lycopene production capacity of wild-type and E. coli strains containing Rpos mutants; wherein, Lycopene Production: lycopene production.

Detailed ways

The present invention mutates the structural genes on the Escherichia coli genome through the strategy of iterative homologous recombination to achieve the purpose of in situ evolution of functional proteins. The following applies this strategy to two examples of directly evolving proteins on the Escherichia coli genome , confirming that the present invention is a method for efficiently obtaining desired protein mutants. The operation process of the present invention is as shown in Figure 1, and two examples are respectively 1-deoxy-D-xylulose-5-phosphate synthase (DXS) gene on the mutant Escherichia coli genome and the σ ^s factor on the mutant Escherichia coli genome ( Rpos) gene increases the level of lycopene produced in Escherichia coli.

Example 1, Escherichia coli genome mutation 1-deoxy-D-xylulose-5-phosphate synthase (DXS) gene

1-deoxy-D-xylulose-5-phosphate synthase (DXS) is a key enzyme in the isoprene synthesis pathway of Escherichia coli, which uses 3-phosphate-glyceraldehyde and pyruvate as substrates to synthesize 1-deoxy- D-xylulose-5-phosphate, improving its catalytic efficiency can enhance isoprene metabolic flux and ultimately increase lycopene production.

In Escherichia coli EcNR2 (Harris H. Wang et al. (2009) Nature. 460:894-890), exogenous genes crtE, crtB, crtI were introduced, so that the Escherichia coli could produce lycopene. Since lycopene has a visible red color, strains with increased lycopene production can be screened in high throughput according to the shade of red color of the colonies.

In vitro preparation of 80-100nt dxs gene single-stranded mutation library:

1. The dxs gene was obtained by PCR from the Escherichia coli genome and connected with a T vector to construct a TA cloning vector of the dxs gene.

2. Using the upper primer dxs-for (SEQ ID NO.1) and the lower primer dxs-rev (SEQ ID NO.2) phosphorylated at the 5' end, use the TA cloning vector of the dxs gene as a template, and add Mn ² Under the condition of ⁺ , 94°C for 45s, 54°C for 50s, and 72°C for 2min were cycled 30 times to do error-prone PCR, and the double-stranded nucleotides obtained were purified and recovered by tapping rubber;

3. Using the above-mentioned double-stranded nucleotide containing mutation as a template, add only primer dxs-for (SEQID NO.1) to the PCR system, cycle 15 times at 94°C for 30s, 53°C for 30s, and 72°C for 4min to prepare dxs single-stranded nucleotide;

4. Treat the PCR product obtained above with Lambda EXO exonuclease, and cut the dxs double-strand enzyme into single-stranded nucleotides;

5. Ethanol precipitation of the enzyme-cut products obtained above to obtain dxs single-stranded nucleotides;

6. Digest the single-stranded nucleotides obtained above with DNase I, and cut the gel to recover 80-100nt single-stranded nucleotide fragments to form a single-stranded mutation library.

Iterative Homologous Recombination Evolution Target Protein:

1. Insert a single colony of Escherichia coli picked from the plate into a shake flask of 50mL LB medium, add 25μL of kanamycin, and culture overnight on a shaker at 30°C;

2. Transfer 1% of the bacterial solution to a test tube of 2mL LB medium, and place it in a shaking table at 30°C for 2.5-3h until the OD reaches about 0.7;

3. Place the test tube in a 42°C water bath and shake for 15 minutes to induce the full expression of λ-Red recombinant protein; place the test tube on ice for 5 minutes;

4. Transfer 1mL of bacterial liquid into a 1.5mL pre-cooled centrifuge tube, centrifuge at 13000r/min for 30s at 4°C, discard the supernatant, add 1ml of pre-cooled sterile water to resuspend and wash, discard the supernatant, and use Pre-cooled sterile water 1ml to resuspend and wash twice;

5. Remove the supernatant, resuspend the bacteria with 100 μL pre-cooled sterile water, and add the 80-100nt dxs single-stranded mutation library;

6. Transfer the above mixture to a 2mm electric cup, and perform electric shock under the conditions of a capacitance of 25μF, a voltage of 2.5kV, and a resistance of 200Ω;

7. Quickly add the prepared 1ml SOC medium into the electric shock cup, and gently blow to suspend the cells. Transfer the mixture in the electric shock cup to a test tube containing 1ml of culture medium, and place it in a shaker at 30°C to revive the cells;

8. When the cells recover to an OD of about 0.7, repeat the above steps for the second round of transformation. The conversion process was carried out for 4 rounds in total;

9. After the last round of electroporation, recover for 12 hours and paint the plate. Colonies with darker red color were screened out on the plate. Wild E. coli strain (wild-type DXS gene sequence SEQ ID NO.3; amino acid sequence SEQ ID NO.4) and E. coli strains containing DXS mutants (DXS-M1, DXS-M2, DXS-M3, DXS-M4, DXS-M5, the summary of DXS mutation sites are shown in Table 1) The comparison of lycopene production ability is shown in Figure 2.

Table 1 Summary of DXS mutation sites

It can be seen from the figure that the lycopene accumulation of each E. coli strain containing the DXS mutant has been increased relative to the wild-type strain, and the range is 50%-300%.

Embodiment 2, mutation σ ^s factor (Rpos) gene on Escherichia coli genome

The σ ^s factor (Rpos) plays a crucial role in Escherichia coli cells and can respond to different environmental stresses. Because large accumulations of lycopene are toxic to E. coli, mutation of the σ ^s factor can make E. coli better adapt to stress.

In vitro preparation of 80-100nt rpos gene single-stranded mutation library:

1. Obtain the Rpos gene from the Escherichia coli genome by PCR and connect it with the T vector to construct the TA cloning vector of the Rpos gene.

2. Using the upper primer rpos-for (SEQ ID NO.15) and the lower primer rpos-rev (SEQ ID NO.16) phosphorylated at the 5' end, use the TA cloning vector of the rpos gene as a template, and add Mn ² Under the condition of ⁺ , 94°C for 45s, 54°C for 50s, and 72°C for 2min were cycled 30 times to do error-prone PCR, and the double-stranded nucleotides obtained were purified and recovered by tapping rubber;

3. Using the above-mentioned double-stranded nucleotide containing mutation as a template, add only primer rpos-for (SEQ ID NO.15) to the PCR system, cycle 15 times at 94°C for 30s, 53°C for 30s, and 72°C for 4min to prepare Get rpos single-stranded nucleotides;

4. Treat the PCR product obtained above with Lambda EXO exonuclease, and cut the rpos double-strand enzyme into single-stranded nucleotides;

5. Precipitate the enzyme-cut product obtained above with ethanol to obtain rpos single-stranded nucleotides;

6. Digest the single-stranded nucleotides obtained above with DNase I, and cut the gel to recover 80-100nt single-stranded nucleotide fragments to form a single-stranded mutation library.

Iterative Homologous Recombination Evolution Target Protein:

1. Insert a single colony of Escherichia coli picked from the plate into a shake flask of 50mL LB medium, add 25μL of kanamycin, and culture overnight on a shaker at 30°C;

2. Transfer 1% of the bacterial solution to a test tube of 2mL LB medium, and place it in a shaking table at 30°C for 2.5-3h until the OD reaches about 0.7;

3. Place the test tube in a 42°C water bath and shake for 15 minutes to induce the full expression of λ-Red recombinant protein; place the test tube on ice for 5 minutes;

4. Transfer 1mL of bacterial liquid into a 1.5mL pre-cooled centrifuge tube, centrifuge at 13000r/min for 30s at 4°C, discard the supernatant, add 1ml of pre-cooled sterile water to resuspend and wash, discard the supernatant, and use Pre-cooled sterile water 1ml to resuspend and wash twice;

5. Remove the supernatant, resuspend the bacteria with 100 μL pre-cooled sterile water, and add the 80-100nt dxs single-stranded mutation library;

6. Transfer the above mixture to a 2mm electric cup, and perform electric shock under the conditions of a capacitance of 25μF, a voltage of 2.5kV, and a resistance of 200Ω;

7. Quickly add the prepared 1ml SOC medium into the electric shock cup, and gently blow to suspend the cells. Transfer the mixture in the electric shock cup to a test tube containing 1ml of culture medium, and place it in a shaker at 30°C to revive the cells;

8. When the cells recover to an OD of about 0.7, repeat the above steps for the second round of transformation. The transformation process was carried out 6 rounds in total;

9. After the last round of electroporation, recover for 12 hours and paint the plate. Colonies with darker red color were screened out on the plate. Wild Escherichia coli strain (gene sequence SEQ ID NO.17 of wild-type Rpos; amino acid sequence SEQ ID NO.18) and Escherichia coli strain containing Rpos mutant (Rpos-M1, Rpos-M2, Rpos mutation sites are summarized in the table 2) The comparison of lycopene producing capacity is shown in Figure 2.

Table 2 Summary of Rpos mutation sites

It can be seen from the figure that the lycopene accumulation of each Escherichia coli strain containing the Rpos mutant has been increased relative to the wild-type strain, and the ranges are 80%-205%, respectively.

sequence:

Primer dxs-for on SEQ ID NO.1

5'-GAGTTTTGATATTGCCAAATACCCGACCCTGGC-3'

The lower primer dxs-rev phosphorylated at the 5' end of SEQ ID NO.2

5'-TTATGCCAGCCAGGCCTTGATTTTG-3' (phosphorylated at the 5' end)

SEQ ID NO.3 DXS gene sequence

atgagttttgatattgccaaatacccgaccctggcactggtcgactccacccaggagttacgactgttgccgaaagagagtttaccgaaactctgcgacgaactgcgccgctatttactcgacagcgtgagccgttccagcgggcacttcgcctccgggctgggcacggtcgaactgaccgtggcgctgcactatgtctacaacaccccgtttgaccaattgatttgggatgtggggcatcaggcttatccgcataaaattttgaccggacgccgcgacaaaatcggcaccatccgtcagaaaggcggtctgcacccgttcccgtggcgcggcgaaagcgaatatgacgtattaagcgtcgggcattcatcaacctccatcagtgccggaattggtattgcggttgctgccgaaaaagaaggcaaaaatcgccgcaccgtctgtgtcattggcgatggcgcgattaccgcaggcatggcgtttgaagcgatgaatcacgcgggcgatatccgtcctgatatgctggtgattctcaacgacaatgaaatgtcgatttccgaaaatgtcggcgcgctcaacaaccatctggcacagctgctttccggtaagctttactcttcactgcgcgaaggcgggaaaaaagttttctctggcgtgccgccaattaaagagctgctcaaacgcaccgaagaacatattaaaggcatggtagtgcctggcacgttgtttgaagagctgggctttaactacatcggcccggtggacggtcacgatgtgctggggcttatcaccacgctaaagaacatgcgcgacctgaaaggcccgcagttcctgcatatcatgaccaaaaaaggtcgtggttatgaaccggcagaaaaagacccgatcactttccacgccgtgcctaaatttgatccctccagcggttgtttgccgaaaagtagcggcggtttgccgagctattcaaaaatctttggcgactggttgtgcgaaacggcag cgaaagacaacaagctgatggcgattactccggcgatgcgtgaaggttccggcatggtcgagttttcacgtaaattcccggatcgctacttcgacgtggcaattgccgagcaacacgcggtgacctttgctgcgggtctggcgattggtgggtacaaacccattgtcgcgatttactccactttcctgcaacgcgcctatgatcaggtgctgcatgacgtggcgattcaaaagcttccggtcctgttcgccatcgaccgcgcgggcattgttggtgctgacggtcaaacccatcagggtgcttttgatctctcttacctgcgctgcataccggaaatggtcattatgaccccgagcgatgaaaacgaatgtcgccagatgctctataccggctatcactataacgatggcccgtcagcggtgcgctacccgcgtggcaacgcggtcggcgtggaactgacgccgctggaaaaactaccaattggcaaaggcattgtgaagcgtcgtggcgagaaactggcgatccttaactttggtacgctgatgccagaagcggcgaaagtcgccgaatcgctgaacgccacgctggtcgatatgcgttttgtgaaaccgcttgatgaagcgttaattctggaaatggccgccagccatgaagcgctggtcaccgtagaagaaaacgccattatgggcggcgcaggcagcggcgtgaacgaagtgctgatggcccatcgtaaaccagtacccgtgctgaacattggcctgccggacttctttattccgcaaggaactcaggaagaaatgcgcgccgaactcggcctcgatgccgctggtatggaagccaaaatcaaggcctggctggcataa

SEQ ID NO.4 DXS amino acid sequence

msfdiakyptlalvdstqelrllpkeslpklcdelrrylldsvsrssghfasglgtveltvalhyvyntpfdqliwdvghqayphkiltgrrdkigtirqkgglhpfpwrgeseydvlsvghsstsisagigiavaaekegknrrtvcvigdgaitagmafeamnhagdirpdmlvilndnemsisenvgalnnhlaqllsgklysslreggkkvfsgvppikellkrteehikgmvvpgtlfeelgfnyigpvdghdvlglittlknmrdlkgpqflhimtkkgrgyepaekdpitfhavpkfdpssgclpkssgglpsyskifgdwlcetaakdnklmaitpamregsgmvefsrkfpdryfdvaiaeqhavtfaaglaiggykpivaiystflqraydqvlhdvaiqklpvlfaidragivgadgqthqgafdlsylrcipemvimtpsdenecrqmlytgyhyndgpsavryprgnavgveltpleklpigkgivkrrgeklailnfgtlmpeaakvaeslnatlvdmrfvkpldealilemaashealvtveenaimggagsgvnevlmahrkpvpvlniglpdffipqgtqeemraelgldaagmeakikawla

SEQ ID NO.5 DXS-M1 gene sequence

atgagttttgatattgccaaatacccgaccctggcactggtcgactccacccaggagttacgactgttgccgaaagagagtttaccgaaactctgcgacgaactgcgccgctatttactcgacagcgtgagccgttccagcgggcacttcgcctccgggctgggcacggtcgaactgaccgtggcgctgcactatgtctacaacaccccgtttgaccaattgatttgggatgtggggcatcaggcttatccgcataaaattttgaccggacgccgcgacaaaatcggcaccatccgtcagaaaggcggtctgcacccgttcccgtggcgcggcgaaagcgaatatgacgtattaagcgtcgggcattcatcaacctccatcagtgccggaattggtattgcggttgctgccgaaaaagaaggcaaaaatcgccgcaccgtctgtgtcattggcgatggcgcgattaccgcaggcatggcgtttgaagcgatgaatcacgcgggcgatatccgtcctgatatgctggtgattctcaacgacaatgaaatgtcgatttccgaaaatgtcggcgcgctcaacaaccatctggcacagctgctttccggtaagctttactcttcactgcgcgaaggcgggaaaaaagttttctctggcgtgccgccaattaaagagctgcccaaacgcaccgaagaacatattaaaggcatggtagtgcctggcacgttgtttgaagagctgggctttaactacatcggcccggtggacggtcacgatgtgctggggcttatcaccacgctaaagaacatgcgcgacctgaaaggcccgcagttcctgcatatcatgaccaaaaaaggtcgtggttatgaaccggcagaaaaagacccgatcactttccacgccgtgcctaaatttgatccctccagcggttgtttgccgaaaagtagcggcggtttgccgagctattcaaaaatctttggcgactggttgtgcgaaacggcag cgaaagacaacaagctgatggcgattactccggcgatgcgtgaaggttccggcatggtcgagttttcacgtaaattcccggatcgctacttcgacgtggcaattgccgagcaacacgcggtgacctttgctgcgggtctggcgattggtgggtacaaacccattgtcgcgatttactccactttcctgcaacgcgcctatgatcaggtgctgcatgacgtggcgattcaaaagcttccggtcctgttcgccatcgaccgcgcgggcattgttggtgctgacggtcaaacccatcagggtgcttttgatctctcttacctgcgctgcataccggaaatggtcattatgaccccgagcgatgaaaacgaatgtcgccagatgctctataccggctatcactataacgatggcccgtcagcggtgcgctacccgcgtggcaacgcggtcggcgtggaactgacgccgctggaaaaactaccaattggcaaaggcattgtgaagcgtcgtggcgagaaactggcgatccttaactttggtacgctgatgccagaagcggcgaaagtcgccgaatcgctgaacgccacgctggtcgatatgcgttttgtgaaaccgcttgatgaagcgttaattctggaaatggccgccagccatgaagcgctggtcaccgtagaagaaaacgccattatgggcggcgcaggcagcggcgtgaacgaagtgctgatggcccatcgtaaaccagtacccgtgctgaacattggcctgccggacttctttattccgcaaggaactcaggaagaaatgcgcgccgaactcggcctcgatgccgctggtatggaagccaaaatcaaggcctggctggcataa

SEQ ID NO.6 DXS-M1 amino acid sequence

msfdiakyptlalvdstqelrllpkeslpklcdelrrylldsvsrssghfasglgtveltvalhyvyntpfdqliwdvghqayphkiltgrrdkigtirqkgglhpfpwrgeseydvlsvghsstsisagigiavaaekegknrrtvcvigdgaitagmafeamnhagdirpdmlvilndnemsisenvgalnnhlaqllsgklysslreggkkvfsgvppikelpkrteehikgmvvpgtlfeelgfnyigpvdghdvlglittlknmrdlkgpqflhimtkkgrgyepaekdpitfhavpkfdpssgclpkssgglpsyskifgdwlcetaakdnklmaitpamregsgmvefsrkfpdryfdvaiaeqhavtfaaglaiggykpivaiystflqraydqvlhdvaiqklpvlfaidragivgadgqthqgafdlsylrcipemvimtpsdenecrqmlytgyhyndgpsavryprgnavgveltpleklpigkgivkrrgeklailnfgtlmpeaakvaeslnatlvdmrfvkpldealilemaashealvtveenaimggagsgvnevlmahrkpvpvlniglpdffipqgtqeemraelgldaagmeakikawla

SEQ ID NO.7 DXS-M2 gene sequence

atgagttttgatattgccaaatacccgaccctggcactggtcgactccacccaggagttacgactgttgccgaaagagagtttaccgaaactctgcgacgaactgcgccgctatttactcgacagcgtgagccgttccagcgggcacttcgcctccgggctgggcacggtcgaactgaccgtggcgctgcactatgtctacaacaccccgtttgaccaattgatttgggatgtggggcatcaggcttatccgcataaaattttgaccggacgccgcgacaaaatcggcaccatccgtcagaaaggcggtctgcacccgttcccgtggcgcggcgaaagcgaatatgacgtattaagcgtcgggcattcatcaacctccatcagtgccggaattggtattgcggttgctgccgaaaaagaaggcaaaaatcgccgcaccgtctgtgtcattggcgatggcgcgattaccgcaggcatggcgtttgaagcgatgaatcacgcgggcgatatccgtcctgatatgctggtgattctcaacgacaatgaaatgtcgatttccgaaaatgtcggcgcgctcaacaaccatctggcacagctgctttccggtaagccttactcttcactgcgcgaaggcgggaaaaaagttttctctggcgtgccgccaattaaagagctgctcaaacgcaccgacgaacatattaaaggcatggtagtgcctggcacgttgtttgaagagctgggctttaactacatcggcccggtggacggtcacgatgtgctggggcttatcaccacgctaaagaacatgcgcgacctgaaaggcccgcagttcctgcatatcatgaccaaaaaaggtcgtggttatgaaccggcagaaaaagacccgatcactttccacgccgtgcctaaatttgatccctccagcggttgtttgccgaaaagtagcggcggtttgccgagctattcaaaaatctttggcgactggttgtgcgaaacggcag cgaaagacaacaagctgatggcgattactccggcgatgcgtgaaggttccggcatggtcgagttttcacgtaaattcccggatcgctacttcgacgtggcaattgccgagcaacacgcggtgacctttgctgcgggtctggcgattggtgggtacaaacccattgtcgcgatttactccactttcctgcaacgcgcctatgatcaggtgctgcatgacgtggcgattcaaaagcttccggtcctgttcgccatcgaccgcgcgggcattgttggtgctgacggtcaaacccatcagggtgcttttgatctctcttacctgcgctgcataccggaaatggtcattatgaccccgagcgatgaaaacgaatgtcgccagatgctctataccggctatcactataacgatggcccgtcagcggtgcgctacccgcgtggcaacgcggtcggcgtggaactgacgccgctggaaaaactaccaattggcaaaggcattgtgaagcgtcgtggcgagaaactggcgatccttaactttggtacgctgatgccagaagcggcgaaagtcgccgaatcgctgaacgccacgctggtcgatatgcgttttgtgaaaccgcttgatgaagcgttaattctggaaatggccgccagccatgaagcgctggtcaccgtagaagaaaacgccattatgggcggcgcaggcagcggcgtgaacgaagtgctgatggcccatcgtaaaccagtacccgtgctgaacattggcctgccggacttctttattccgcaaggaactcaggaagaaatgcgcgccgaactcggcctcgatgccgctggtatggaagccaaaatcaaggcctggctggcataa

SEQ ID NO.8 DXS-M2 amino acid sequence

msfdiakyptlalvdstqelrllpkeslpklcdelrrylldsvsrssghfasglgtveltvalhyvyntpfdqliwdvghqayphkiltgrrdkigtirqkgglhpfpwrgeseydvlsvghsstsisagigiavaaekegknrrtvcvigdgaitagmafeamnhagdirpdmlvilndnemsisenvgalnnhlaqllsgkpysslreggkkvfsgvppikellkrtdehikgmvvpgtlfeelgfnyigpvdghdvlglittlknmrdlkgpqflhimtkkgrgyepaekdpitfhavpkfdpssgclpkssgglpsyskifgdwlcetaakdnklmaitpamregsgmvefsrkfpdryfdvaiaeqhavtfaaglaiggykpivaiystflqraydqvlhdvaiqklpvlfaidragivgadgqthqgafdlsylrcipemvimtpsdenecrqmlytgyhyndgpsavryprgnavgveltpleklpigkgivkrrgeklailnfgtlmpeaakvaeslnatlvdmrfvkpldealilemaashealvtveenaimggagsgvnevlmahrkpvpvlniglpdffipqgtqeemraelgldaagmeakikawla

SEQ ID NO.9 DXS-M3 gene sequence

atgagttttgatattgccaaatacccgaccctggcactggtcgactccacccaggagttacgactgtcgccgaaagagagtttaccgaaactctgcgacgaactgcgccgctatttactcgacagcgtgagccgttccagcgggcacttcgcctccgggctgggcacggtcgaactgaccgtggcgctgcactatgtctacaacaccccgtttgaccaattgatttgggatgtggggcatcaggcttatccgcataaaattttgaccggacgccgcgacaaaatcggcaccatccgtcagaaaggcggtctgcacccgttcccgtggcgcggcgaaagcgaatatgacgtattaagcgtcgggcattcatcaacctccatcagtgccggaactggtattgcggttgctgccgaaaaagaaggcaaaaatcgccgcaccgtctgtgtcattggcgatggcgcgattaccgcaggcatggcgtttgaagcgatgaatcacgcgggcgatatccgtcctgatatgctggtgattctcaacgacaatgaaatgtcgatttccgaaaatgtcggcgcgctcaacaaccatctggcacagctgctttccggtaagctttactcttcactgcgcgaaggcgggaaaaaagttttctctggcgcgccgccaattaaagagctgctcaaacgcaccgaagaacatattaaaggcatggtagtgcctggcacgttgtttgaagagctgggctttaactacatcggcccggtggacggtcacgatgtgctggggcttatcaccacgctaaagaacatgcgcgacctgaaaggcccgcagttcctgcatatcatgaccaaaaaaggtcgtggttatgaaccggcagaaaaagacccgatcactttccacgccgtgcctaaatttgatccctccagcggttgtttgccgaaaagtagcggcggtttgccgagctattcaaaaatctttggcgactggttgtgcgaaacggcag cgaaagacaacaagctgatggcgattactccggcgatgcgtgaaggttccggcatggtcgagttttcacgtaaattcccggatcgctacttcgacgtggcaattgccgagcaacacgcggtgacctttgctgcgggtctggcgattggtgggtacaaacccattgtcgcgatttactccactttcctgcaacgcgcctatgatcaggtgctgcatgacgtggcgattcaaaagcttccggtcctgttcgccatcgaccgcgcgggcattgttggtgctgacggtcaaacccatcagggtgcttttgatctctcttacctgcgctgcataccggaaatggtcattatgaccccgagcgatgaaaacgaatgtcgccagatgctctataccggctatcactataacgatggcccgtcagcggtgcgctacccgcgtggcaacgcggtcggcgtggaactgacgccgctggaaaaactaccaattggcaaaggcattgtgaagcgtcgtggcgagaaactggcgatccttaactttggtacgctgatgccagaagcggcgaaagtcgccgaatcgctgaacgccacgctggtcgatatgcgttttgtgaaaccgcttgatgaagcgttaattctggaaatggccgccagccatgaagcgctggtcaccgtagaagaaaacgccattatgggcggcgcaggcagcggcgtgaacgaagtgctgatggcccatcgtaaaccagtacccgtgctgaacattggcctgccggacttctttattccgcaaggaactcaggaagaaatgcgcgccgaactcggcctcgatgccgctggtatggaagccaaaatcaaggcctggctggcataa

SEQ ID NO.10 DXS-M3 amino acid sequence

msfdiakyptlalvdstqelrlspkeslpklcdelrrylldsvsrssghfasglgtveltvalhyvyntpfdqliwdvghqayphkiltgrrdkigtirqkgglhpfpwrgeseydvlsvghsstsisagtgiavaaekegknrrtvcvigdgaitagmafeamnhagdirpdmlvilndnemsisenvgalnnhlaqllsgklysslreggkkvfsgappikellkrteehikgmvvpgtlfeelgfnyigpvdghdvlglittlknmrdlkgpqflhimtkkgrgyepaekdpitfhavpkfdpssgclpkssgglpsyskifgdwlcetaakdnklmaitpamregsgmvefsrkfpdryfdvaiaeqhavtfaaglaiggykpivaiystflqraydqvlhdvaiqklpvlfaidragivgadgqthqgafdlsylrcipemvimtpsdenecrqmlytgyhyndgpsavryprgnavgveltpleklpigkgivkrrgeklailnfgtlmpeaakvaeslnatlvdmrfvkpldealilemaashealvtveenaimggagsgvnevlmahrkpvpvlniglpdffipqgtqeemraelgldaagmeakikawla

SEQ ID NO.11 DXS-M4 gene sequence

atgagttttgatattgccaaatacccgaccctggcactggtcgactccacccaggagttacgactgttgccgaaagagagtttaccgaaactctgcgacgaactgcgccgctatttactcgacagcgtgagccgttccagcgggcacttcgcctccgggctgggcacggtcgaactgaccgtggcgctgcactatgtctacaacaccccgtttgaccaattgatttgggatgtggggcatcaggcttatccgcataaaattttgaccggacgccgcgacaaaatcggcaccatccgtcagaaaggcggtctgcacccgttcccgtggcgcggcgaaagcgaatatgacgtattaagcgtcgggcattcatcaacctccatcagtgccggaattggtattgcggttgctgccgaaaaagaaggcaaaaatcgccgcaccgtctgtgtcattggcgatggcgcgattaccgcaggcatggcgtttgaagcgacgaatcacgcgggcgatatccgtcctgatatgctggtgattctcaacgacaatgagatgtcgatttccgaaaatgtcggcgcgctcaacaaccatctggcacagctgctttccggtaagctttactcttcactgcgcgaaggcgggaaaaaagttttctctggcgtgccgccaaataaagagctgctcaaacgcaccgaagaacatattaaaggcatggtagtgcctggcacgttgtttgaagagctgggctttaactacatcggcccggtggacggtcacgatgtgctggggcttatcaccacgctaaagaacatgcgcgacctgaaaggcccgcagttcctgcatatcatgaccaaaaaaggtcgtggttatgaaccggcagaaaaagacccgatcactttccacgccgtgcctaaatttgatccctccagcggttgtttgccgaaaagtagcggcggtttgccgagctattcaaaaatctttggcgactggttgtgcgaaacggcag cgaaagacaacaagctgatggcgattactccggcgatgcgtgaaggttccggcatggtcgagttttcacgtaaattcccggatcgctacttcgacgtggcaattgccgagcaacacgcggtgacctttgctgcgggtctggcgattggtgggtacaaacccattgtcgcgatttactccactttcctgcaacgcgcctatgatcaggtgctgcatgacgtggcgattcaaaagcttccggtcctgttcgccatcgaccgcgcgggcattgttggtgctgacggtcaaacccatcagggtgcttttgatctctcttacctgcgctgcataccggaaatggtcattatgaccccgagcgatgaaaacgaatgtcgccagatgctctataccggctatcactataacgatggcccgtcagcggtgcgctacccgcgtggcaacgcggtcggcgtggaactgacgccgctggaaaaactaccaattggcaaaggcattgtgaagcgtcgtggcgagaaactggcgatccttaactttggtacgctgatgccagaagcggcgaaagtcgccgaatcgctgaacgccacgctggtcgatatgcgttttgtgaaaccgcttgatgaagcgttaattctggaaatggccgccagccatgaagcgctggtcaccgtagaagaaaacgccattatgggcggcgcaggcagcggcgtgaacgaagtgctgatggcccatcgtaaaccagtacccgtgctgaacattggcctgccggacttctttattccgcaaggaactcaggaagaaatgcgcgccgaactcggcctcgatgccgctggtatggaagccaaaatcaaggcctggctggcataa

SEQ ID NO.12 DXS-M4 amino acid sequence

msfdiakyptlalvdstqelrllpkeslpklcdelrrylldsvsrssghfasglgtveltvalhyvyntpfdqliwdvghqayphkiltgrrdkigtirqkgglhpfpwrgeseydvlsvghsstsisagigiavaaekegknrrtvcvigdgaitagmafeatnhagdirpdmlvilndnemsisenvgalnnhlaqllsgklysslreggkkvfsgvppnkellkrteehikgmvvpgtlfeelgfnyigpvdghdvlglittlknmrdlkgpqflhimtkkgrgyepaekdpitfhavpkfdpssgclpkssgglpsyskifgdwlcetaakdnklmaitpamregsgmvefsrkfpdryfdvaiaeqhavtfaaglaiggykpivaiystflqraydqvlhdvaiqklpvlfaidragivgadgqthqgafdlsylrcipemvimtpsdenecrqmlytgyhyndgpsavryprgnavgveltpleklpigkgivkrrgeklailnfgtlmpeaakvaeslnatlvdmrfvkpldealilemaashealvtveenaimggagsgvnevlmahrkpvpvlniglpdffipqgtqeemraelgldaagmeakikawla

SEQ ID NO.13 DXS-M5 gene sequence

atgagttttgatattgccaaatacccgaccctggcactggtcgactccacccaggagttacgactgttgccgaaagagagtttaccgaaactctgcgacgaactgcgccgctatttactcgacagcgtgagccgttccagcgggcacttcgcctccgggctgggcacggtcgaactgaccgtggcgctgcactatgtctacaacaccccgtttgaccaattgatttgggatgtggggcatcaggcttatccgcataaaattttgaccggacgccgcgacaaaatcagcaccatccgtcagaaaggcggtctgcacccgttcccgtggcgcggcgaaagcgaatatgacgtattaagcgtcgggcattcatcaacctccatcagtgccggaattggtattgcggttgctgccgaaaaagaaggcaaaaatcgccgcaccgtctgtgtcattggcgatggcgcgattaccgcaggcatggcgtttgaagcgatgaatcacgcgggcgatatccgtcctgatatgctggtgattctcaacgacaatgaaatgtcgatttccgaaaatgtcggcgcgctcaacaaccatctggcacagctgctttccggtaagctttactcttcactgcgcgagggcgggaaaaaagttttctctggcgtgccgccaattaaagagctgctcaaacgcaccgaagaacatattaaaggcacggtagtgcctggcacgttgtttgaagagctgggctttaactacatcggcccggtggacggtcacgatgtgctggggcttatcaccacgctaaagaacatgcgcgacctgaaaggcccgcagttcctgcatatcatgaccaaaaaaggtcgtggttatgaaccggcagaaaaagacccgatcactttccacgccgtgcctaaatttgatccctccagcggttgtttgccgaaaagtagcggcggtttgccgagctattcaaaaatctttggcgactggttgtgcgaaacggcag cgaaagacaacaagctgatggcgattactccggcgatgcgtgaaggttccggcatggtcgagttttcacgtaaattcccggatcgctacttcgacgtggcaattgccgagcaacacgcggtgacctttgctgcgggtctggcgattggtgggtacaaacccattgtcgcgatttactccactttcctgcaacgcgcctatgatcaggtgctgcatgacgtggcgattcaaaagcttccggtcctgttcgccatcgaccgcgcgggcattgttggtgctgacggtcaaacccatcagggtgcttttgatctctcttacctgcgctgcataccggaaatggtcattatgaccccgagcgatgaaaacgaatgtcgccagatgctctataccggctatcactataacgatggcccgtcagcggtgcgctacccgcgtggcaacgcggtcggcgtggaactgacgccgctggaaaaactaccaattggcaaaggcattgtgaagcgtcgtggcgagaaactggcgatccttaactttggtacgctgatgccagaagcggcgaaagtcgccgaatcgctgaacgccacgctggtcgatatgcgttttgtgaaaccgcttgatgaagcgttaattctggaaatggccgccagccatgaagcgctggtcaccgtagaagaaaacgccattatgggcggcgcaggcagcggcgtgaacgaagtgctgatggcccatcgtaaaccagtacccgtgctgaacattggcctgccggacttctttattccgcaaggaactcaggaagaaatgcgcgccgaactcggcctcgatgccgctggtatggaagccaaaatcaaggcctggctggcataa

SEQ ID NO.14 DXS-M5 amino acid sequence

msfdiakyptlalvdstqelrllpkeslpklcdelrrylldsvsrssghfasglgtveltvalhyvyntpfdqliwdvghqayphkiltgrrdkistirqkgglhpfpwrgeseydvlsvghsstsisagigiavaaekegknrrtvcvigdgaitagmafeamnhagdirpdmlvilndnemsisenvgalnnhlaqllsgklysslreggkkvfsgvppikellkrteehikgtvvpgtlfeelgfnyigpvdghdvlglittlknmrdlkgpqflhimtkkgrgyepaekdpitfhavpkfdpssgclpkssgglpsyskifgdwlcetaakdnklmaitpamregsgmvefsrkfpdryfdvaiaeqhavtfaaglaiggykpivaiystflqraydqvlhdvaiqklpvlfaidragivgadgqthqgafdlsylrcipemvimtpsdenecrqmlytgyhyndgpsavryprgnavgveltpleklpigkgivkrrgeklailnfgtlmpeaakvaeslnatlvdmrfvkpldealilemaashealvtveenaimggagsgvnevlmahrkpvpvlniglpdffipqgtqeemraelgldaagmeakikawla

Primer rpos-for on SEQ ID NO.15

5'-gagtcagaatacgctgaaagttcatgatttaaatg-3'

The lower primer rpos-rev phosphorylated at the 5' end of SEQ ID NO.16

5'-tactcgcggaacagcgcttcgatattcagc-3'

SEQ ID NO.17 Rpos gene sequence

Atgagtcagaatacgctgaaagttcatgatttaaatgaagatgcggaatttgatgagaacggagttgaggtttttgacgaaaaggccttagtagaacaggaacccagtgataacgatttggccgaagaggaactgttatcgcagggagccacacagcgtgtgttggacgcgactcagctttaccttggtgagattggttattcaccactgttaacggccgaagaagaagtttattttgcgcgtcgcgcactgcgtggagatgtcgcctctcgccgccggatgatcgagagtaacttgcgtctggtggtaaaaattgcccgccgttatggcaatcgtggtctggcgttgctggaccttatcgaagagggcaacctggggctgatccgcgcggtagagaagtttgacccggaacgtggtttccgcttctcaacatacgcaacctggtggattcgccagacgattgaacgggcgattatgaaccaaacccgtactattcgtttgccgattcacatcgtaaaggagctgaacgtttacctgcgaaccgcacgtgagttgtcccataagctggaccatgaaccaagtgcggaagagatcgcagagcaactggataagccagttgatgacgtcagccgtatgcttcgtcttaacgagcgcattacctcggtagacaccccgctgggtggtgattccgaaaaagcgttgctggacatcctggccgatgaaaaagagaacggtccggaagataccacgcaagatgacgatatgaagcagagcatcgtcaaatggctgttcgagctgaacgccaaacagcgtgaagtgctggcacgtcgattcggtttgctggggtacgaagcggcaacactggaagatgtaggtcgtgaaattggcctcacccgtgaacgtgttcgccagattcaggttgaaggcctgcgccgtttgcgcgaaatcctgcaaacgcaggggctgaatatcgaagcgctgttccgcgagtaa

SEQ ID NO.18 Rpos amino acid sequence

msqntlkvhdlnedaefdengvevfdekalveqepsdndlaeeellsqgatqrvldatqlylgeigysplltaeeevyfarralrgdvasrrrmiesnlrlvvkiarrygnrglalldlieegnlgliravekfdpergfrfstyatwwirqtieraimnqtrtirlpihivkelnvylrtarelshkldhepsaeeiaeqldkpvddvsrmlrlneritsvdtplggdsekalldiladekengpedttqdddmkqsivkwlfelnakqrevlarrfgllgyeaatledvgreigltrervrqiqveglrrlreilqtqglniealfre

SEQ ID NO.19 Rpos-M1 gene sequence

Atgagtcagaatacgctgaaagttcatgatttaaatgaagatgcggaatttgatgagaacggagttgaggtttttgacgaaaaggccttagtagaacaggaacccagtgataacgatttggccgaagaggaactgttatcgcagggagccacacagcgtgtgttggacgcgactcagctttaccttggtgagattggttattcaccactgttaacggccgaagaagaagtttattttgcgcgtcgcgcactgcgtggagatgtcgcctctcgccgccggatgatcgagagtaacttgcgtctggtggtaaaaattgcccgccgttatggcaatcgtggtctggcgttgctggaccttatcgaagagggcaacctggggctgatccgcgcggtagagaagtttgacccggaacgtggtttccgcttctcaacatacgcaacctggtggattcgccagacgattgaacgggcgattatggaccaaacccgtactattcgtttgccgattcacatcgtaaaggagctgaacgtttacctgcgaaccgcacgtgggttgtcccataagctggaccatgaaccaagtgcggaagagatcgcagagcaactggataagccagttgatgacgtcagccgtatgcttcgtcttaacgagcgcattacctcggtagacaccccgctgggtggtgattccgaaaaagcgttgctggacatcctggccgatgaaaaagagaacggtccggaagataccacgcaagaggacgatatgaagcagagcatcgtcaaatggctgttcgagctgaacgccaaacagcgtgaagtgctggcacgtcgattcggtttgctggggtacgaagcggcaacactggaagatgtaggtcgtgaaattggcctcacccgtgaacgtgttcgccagattcaggttgaaggcctgcgccgtttgcgcgaaatcctgcaaacgcaggggctgaatatcgaagcgctgttccgcgagtaa

SEQ ID NO.20 Rpos-M1 amino acid sequence

msqntlkvhdlnedaefdengvevfdekalveqepsdndlaeeellsqgatqrvldatqlylgeigysplltaeeevyfarralrgdvasrrrmiesnlrlvvkiarrygnrglalldlieegnlgliravekfdpergfrfstyatwwirqtieraimdqtrtirlpihivkelnvylrtarglshkldhepsaeeiaeqldkpvddvsrmlrlneritsvdtplggdsekalldiladekengpedttqeddmkqsivkwlfelnakqrevlarrfgllgyeaatledvgreigltrervrqiqveglrrlreilqtqglniealfre

SEQ ID NO.21 Rpos-M2 gene sequence

atgagtcagaatacgctgaaagttcatgatttaaatgaagatgcggaatttgatgagaacggagttgaggtttttgacgaaaaggccttagtagaacaggaacccagtgataacgatttggccgaagaggaactgttatcgcagggagccacactgcgtgtgttggacgcgactcagctttaccttggtgagattggttattcaccactgttaacggccgaagaagaagtttattctgcgcgtcgcgcactgcgtggagatgtcgcctctcgccgccggatgatcgagagtaacttgcgtctggtggtaaaaattgcccgccgttatggcaatcgtggtctggcgttgctagaccttatcgaagagggcaacctggggctgatccgcgcggtagggaagtttgacccggaacgtggtttccgcttctcaacatacgcaacctggtggattcgccagacgattgaatgggcgattatgaaccaaacccgtactattcgtttgccgattcacatcgtaaaggagctgaacgtttacctgcgaaccgcacgtgagttgtcccataagctggaccatgaaccaagtgcggaagagatcgcagagcaactggataagccagttgatgacgtcagccgcatgcttcgtcttaacgagcgcattacctcggtagacaccccgctgggtggtgattccgaaaaagcgttgctggacatcctggccgatgaaaaagagaacggtccggaagataccacgcaagatgacgatatgaagcggagcatcgtcaaatggctgttcgagctgaacgccaaacagcgtgaagtgctggcacgtcgattcggtttgctggggtacgaagcggcaacactggaagatgtaggtcgtgaaattggcctcacccgtgaacgtgttcgccagattcaggttgaaggcctgcgccgtttgcgcgaaatcctgcaaacgcaggggctgaatatcgaagcgctgttccgcgagtaa

SEQ ID NO.22 Rpos-M2 amino acid sequence

msqntlkvhdlnedaefdengvevfdekalveqepsdndlaeeellsqgatlrvldatqlylgeigysplltaeeevysarralrgdvasrrrmiesnlrlvvkiarrygnrglalldlieegnlgliravgkfdpergfrfstyatwwirqtiewaimnqtrtirlpihivkelnvylrtarelshkldhepsaeeiaeqldkpvddvsrmlrlneritsvdtplggdsekalldiladekengpedttqdddmkrsivkwlfelnakqrevlarrfgllgyeaatledvgreigltrervrqiqveglrrlreilqtqglniealfre

Claims (1)

1. a kind of DXS gene, sequence is in sequence table SEQ ID NO.5, NO.7, NO.9, NO.11, NO.13 sequence It is a kind of.