CN106916799B - A Ppmar1 transposase D332S mutant with high catalytic activity and its application - Google Patents

Abstract

The invention discloses a Ppmar1 transposase D332S mutant with high catalytic activity, and the amino acid sequence of the Ppmar1 transposase D332S mutant is shown in SEQ ID NO.1. The nucleotide sequence of the gene encoding the Ppmar1 transposase D332S mutant is shown in SEQ ID NO.2. The aspartic acid at position 332 of the wild-type Ppmar1 transposase was mutated to serine. The activity of the Ppmar1 transposase D332S mutant to catalyze transposon transposition is 2.56 times that of the wild-type transposase, laying a foundation for the development of gene tags using MLE transposons for large-scale isolation and tagging in the post-genomic era Genes, provide new tools to study the function of genes.

Description

A Ppmar1 transposase D332S mutant with high catalytic activity and its application

technical field

The invention belongs to the field of biotechnology, in particular to a Ppmar1 transposase D332S mutant with high catalytic activity and an application thereof.

Background technique

A transposon is a DNA sequence that can be transferred from one site to another on the genome. Since the American geneticist McClintock first discovered transposons (Ac/Ds) in maize in the 1940s, scientists have discovered many types of transposons, which are widely found in bacteria, yeast, and higher plants and animals. With the deepening of the understanding of the structure and function of transposons at the molecular level, some transposons have been transformed into gene tags for gene analysis, and have gradually become one of the important methods for large-scale gene isolation.

Mariner-Like Elements (MLE) is an important family of transposons, which was first discovered in the study of an unstable mutation in the white-eye gene of Drosophila mauristiana. Since then, a large number of MLE transposons have also been found in other animal and plant genomes. Compared with other transposons, MLE transposons have the characteristics of simple structure, high heterologous transposition rate, and close to random insertion sites in the genome. other transposons.

The MLE transposon is composed of Terminal Inverted Repeats (TIRs) and a gene encoding a transposase. The transposase is responsible for catalyzing the transposition of the transposon, so the activity of the transposase affects the transposon. Major factor in transposition frequency. However, the MLE transposase isolated in nature has accumulated more or less mutations due to the "vertical inactivation" effect in the evolution process, partially or completely lost the ability to catalyze transposition, and become a low-activity or inactive transposase. It affects the application of MLE transposons, so it is very important to artificially construct highly active transposases.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a Ppmar1 transposase D332S mutant with high catalytic activity and its application, which solve the problem of low catalytic activity or no catalytic activity of the existing MLE transposase isolated from nature.

The present invention provides a Ppmar1 transposase D332S mutant with high catalytic activity, and the amino acid sequence of the Ppmar1 transposase D332S mutant is shown in SEQ ID NO.1.

The present invention also provides a gene encoding the Ppmar1 transposase D332S mutant, and the nucleotide sequence of the gene encoding the Ppmar1 transposase D332S mutant is shown in SEQ ID NO. 2.

The present invention also provides a recombinant plasmid carrying a gene encoding the Ppmar1 transposase D332S mutant.

The present invention also provides an engineering strain carrying the above-mentioned recombinant plasmid.

The invention also provides the application of a Ppmar1 transposase D332S mutant with high catalytic activity in constructing a yeast mutant.

Compared with the prior art, the Ppmar1 transposase D332S mutant with high catalytic activity provided by the present invention has the following beneficial effects:

The active transposase cloned from Phyllostachys edulis in the present invention is artificially transformed to obtain a highly active MLE transposase mutant (Ppmar1 transposase D332S mutant), and the Ppmar1 transposase D332S mutant catalyzes transposition The activity of the subtransposase is 2.56 times that of the wild-type transposase, which lays the foundation for the development of gene tags using MLE transposons, and provides a new tool for large-scale isolation and tagging of genes in the post-genome era and studying the function of genes.

Detailed ways

The present invention will be described in detail below with reference to the specific embodiments, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. The test methods without specific conditions in the following examples are usually operated under conventional conditions, such as the conditions described in the "Molecular Cloning Experiment Guide" edited by Sambrook et al., or according to the steps stated in the kit, because the invention is not involved. , so the steps are not described in detail.

When numerical ranges are given in the examples, it is to be understood that, unless otherwise indicated herein, both endpoints of each numerical range and any number between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment and materials used in the embodiments, according to the mastery of the prior art by those skilled in the art and the description of the present invention, the methods, equipment and materials described in the embodiments of the present invention can also be used Any methods, devices and materials similar or equivalent to those of the prior art can be used to implement the present invention.

1. Acquisition of wild-type MLE transposase and transposase-removed non-autonomous transposons

Step 1.1, collect fresh bamboo leaves (Phyllostachyspubescens, collected in the Botanical Garden of Zhejiang Agriculture and Forestry University, N30°15′14.67″N latitude E119°43′33.47″E longitude E119°43′33.47″), use the CTAB method to extract the bamboo genomic DNA, according to the MLE transposon TIR conserved sequence The primer Ppmar1-5-3 was designed (see Table 1 for the sequence information of Ppmar1-5-3), and PCR amplification was performed to obtain the MLE transposon amplification product.

The PCR amplification system was 20 μl, including 0.2 μl rTaq Polymerase (5U/μl), 1 μl Ppmar1-5-3 (10 μmol/L), 2 μl 10×rTaq Buffer (Mg ²⁺ plus), 1.6 μl dNTP mix (2.5 mmol/L) /L), 100ng of Phyllostachys pubescens genomic DNA, add sterile water to make up 20μl.

The reaction conditions of PCR amplification were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30s, 60°C for 30s, extension at 72°C for 40s, 35 cycles; 72°C for 2min, 4°C for 10min.

In step 1.2, after the sequence was amplified, the amplified product of the MLE transposon in step 1.1 was ligated to the pMD18-T vector using the method of TaKaRa company pMD ^TM 18-T Vector Cloning Kit. The transposon, the full-length sequence of the Ppmar1 transposon is shown in SEQ ID NO.3.

Step 1.3, using the RNeasy Mini Kit of QIAGEN company to extract the leaf RNA of Phyllostachys pubescens, reverse-transcribe the RNA into cDNA by the SuperScript ^TM VILO ^TM cDNA Synthesis Kit of Invitrogen company, and design a pair of primers PpTpase1- 5 and PpTpase1-3 (see Table 1 for the sequence information of PpTpase1-5 and PpTpase1-3), carry out PCR amplification, and recover the Ppmar1 transposase amplification product, which is the Ppmar1 transposase nucleotide sequence.

The PCR amplification system was 20 μl, including 0.2 μl rTaq Polymerase (5U/μl), 0.5 μl PpTpase1-5 (10 μmol/L), 0.5 μl PpTpase1-3 (10 μmol/L), 2 μl 10×rTaq Buffer (Mg ²⁺ plus), 1.6 μl of dNTPmix (2.5 mmol/L), 10 ng of Phyllostachys edulis leaf cDNA, and 20 μl of sterile water was added.

The reaction conditions of PCR amplification were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30s, 55°C for 30s, extension at 72°C for 40s, 35 cycles; 72°C for 2min, 4°C for 10min.

In step 1.4, the nucleotide sequence of the Ppmar1 transposase in step 1.3 was ligated into the pMD18-T vector clone using the method of the pMD ^TM 18-T Vector Cloning Kit of TaKaRa Company, and the nucleotide sequence of the Ppmar1 transposase was confirmed by sequencing. The corresponding amino acid sequences are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively.

The pMD18-T vector containing the full-length sequence of the Ppmar1 transposon was excised with BseR I for most of the sequence of the Ppmar1 intermediate transposase.

The digestion system was 50 μl, including 5 μl of 10×buffer, 1 μl of BseR I (1 U/μl), 1 μg of plasmid (pMD18-T vector containing the full-length sequence of Ppmar1), supplemented with 50 μl of sterile water, and incubated at 37°C for 6 hours. The large plasmid fragment was recovered and self-ligated with T ₄ DNA Ligase to obtain a non-autonomous transposon of Ppmar1 pMD18-T-Ppmar1-Tn (Tn represents a non-autonomous transposon).

The self-ligation system was 10 μl, including 1 μl of 10×T4 DNA Ligase buffer, ₁ μl of T4 DNA Ligase (10 U/μl), 50 ng of large plasmid fragment, supplemented with 10 μl of sterile water, and incubated at 16°C for 8 hours.

The sequence of the non-autonomous transposon of Ppmar1 is shown in SEQ ID NO.6.

2. Construction of yeast transposition expression vector

Step 2.1, Construction of Ppmar1 Transposase Expression Vector

The nucleotide sequence of the Ppmar1 transposase in step 1.3 was double digested by Not I and EcoR V, and the large fragment of the Ppmar1 transposase digested product was recovered; the pAG413-gal-ccdB vector was digested by Not I and EcoR V. , the large fragment of the pAG413-gal-ccdB vector digestion product was recovered; and the double digestion system and double digestion conditions of the Ppmar1 transposase nucleotide sequence were the same as the double digestion system and double digestion conditions of the pAG413-gal-ccdB vector. conditions are the same;

The double-enzyme digestion system is 50μl, including 5μl 10×buffer, 1μl Not I (1U/μl), 1μl EcoR (1U/μl), 1μg plasmid (Ppmar1 transposase nucleotide sequence or pAG413-gal-ccdB vector) , add sterile water to make up 50 μl, and the double-enzyme digestion conditions are: 37 ℃ warm bath for 6 hours.

Connect the large fragment of the digested product of Ppmar1 transposase to the large fragment of the digested product of pAG413-gal-ccdB vector;

The ligation system is 10μl, including 1μl 10×T4 DNA Ligase buffer, 1μl T4 DNA Ligase (10U/μl), 50ng large fragment of pAG413-gal-ccdB vector digested product, 20ng large fragment of Ppmar1 transposase digested product, Add sterile water to make up 10 μl, and incubate at 16°C for 8 hours.

At this point, the ccdB nucleotide sequence in the pAG413-gal-ccdB plasmid was replaced with the Ppmar1 transposase nucleotide sequence to obtain the recombinant plasmid pAG413-gal-Tpase (Tpase represents transposase);

The recombinant plasmid pAG413-gal-Tpase is the Ppmar1 transposase expression vector, which carries the gene encoding the Ppmar1 transposase. The expression vector has a His (histidine) selectable marker, which enables the host into which the pAG413-gal-Tpase vector is introduced to grow on a His-deficient medium.

Step 2.2, Construction of Ppmar1 Non-autonomous Transposon Donor Vector

Using the non-autonomous transposon of Ppmar1 pMD18-T-Ppmar1-Tn in step 1.4 as a template, the non-autonomous transposon of Ppmar1 was amplified by primers Ppmar1-5-3, and PCR amplification was performed to obtain the non-autonomous transposon of Ppmar1. Sexual transposon amplification products.

The PCR amplification system was 20 μl, including 0.2 μl rTaq Polymerase (5U/μl), 1 μl Ppmar1-5-3 (10 μmol/L), 2 μl 10×rTaq Buffer (Mg ²⁺ plus), 1.6 μl dNTP mix (2.5 mmol/L) /L), 10ng pMD18-T-Ppmar1-Tn, add sterile water to make up 20μl.

The reaction conditions of PCR amplification were: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, 60 °C for 30 s, extension at 72 °C for 40 s, 35 cycles; 72 °C for 2 min, 4 °C for 10 min.

At the same time, the vector pWL89a was digested with XhoI (the enzyme cleavage site is located in the ADE2 gene), and the vector pWL89a backbone was recovered. The digestion system was 50 μl, including 5 μl of 10×buffer, 1 μl of XhoI (1 U/μl), 1 μg of vector pWL89a, supplemented with 50 μl of sterile water, and incubated at 37°C for 6 hours.

Then, the non-autonomous transposon amplification product of Ppmar1 was inserted into the ADE2 gene of the vector pWL89a backbone using the In-Fusion Advantage PCR Cloning Kit (TaKaRa Company, Japan), resulting in the inactivation of the reporter gene ADE2 insertion to obtain pWL89a-Tn recombination The plasmid is the Ppmar1 non-autonomous transposon donor vector. If the transposon is transposed away from the ADE2 gene, the ADE2 gene reading frame is restored. This vector has a URA3 selectable marker that enables the host into which pWL89a-Tn has been introduced to grow on deletion medium lacking Ura (urea).

Third, the acquisition of Ppmar1 transposase D332S mutant

The nucleotide sequence of Ppmar1 transposase was compared with the nucleotide sequences of other plant MLE transposases, and the aspartic acid at position 332 of the nucleotide sequence of Ppmar1 transposase was selected for mutation. It was mutated to serine (D332S).

Step 3.1, design site-directed mutagenesis primers D332S-F and D332S-R (see Table 1 for the sequence information of D332S-F and D332S-R) according to the kit instructions of QuikChange ^TM Site-Directed Mutagenesis Kit (Stratagene, USA), according to QuikChange ^TM Site-Directed Mutagenesis Kit method, using the recombinant plasmid pAG413-gal-Tpase in step 2.1 as a template, using PfuTurbo ^TM DNA polymerase to re-synthesize plasmid DNA containing the Ppmar1 transposase D332S mutant;

In step 3.2, 2 μL of Dpn I restriction endonuclease was added to the synthesized plasmid DNA, and the reaction was carried out at 37°C for 5 min to completely degrade the original template sequence. The newly synthesized plasmid DNA was sequenced to confirm the Ppmar1 transposase D332S mutant;

The amino acid sequence of the Ppmar1 transposase D332S mutant is shown in SEQ ID NO. 1, and the nucleotide sequence of the gene encoding the Ppmar1 transposase D332S mutant is shown in SEQ ID NO. 2.

4. Detection of transposase activity

In the experimental group, the plasmid DNA containing the Ppmar1 transposase D332S mutant in step 3.1 and the pWL89a-Tn recombinant plasmid in step 2.2 were co-transformed into yeast by the PEG/LiAc method, and carried out on a His/Ura double-deficient solid medium. Choose to cultivate. Induction of transposase expression with galactose promotes transposition of non-autonomous transposons.

Taking the wild-type Ppmar1 transposase as the control group, the recombinant plasmid pAG413-gal-Tpase with the wild-type Ppmar1 transposase in step 2.1 and the pWL89a-Tn recombinant plasmid in step 2.2 were co-transformed into yeast by PEG/LiAc method , using His/Ura double-deficient solid medium for selective culture. Induction of transposase expression with galactose promotes transposition of non-autonomous transposons.

The induced cultured yeasts of the experimental group and the control group were selected and cultured on the solid medium lacking His/Ura/Ade, and the yeast plaques grown on the medium were counted. If transposition occurs, the ADE2 gene on the pWL89a-Tn recombinant plasmid can be expressed, so positive yeast strains can grow on adenine-deficient medium.

Using the wild-type Ppmar1 transposase as a control, the number of yeast colonies transformed with the Ppmar1 transposase D332S mutant was compared, and the transposase mutant with higher activity was screened. The results are shown in Table 2.

It can be seen from Table 2 that the number of positive yeast colonies of the wild-type Ppmar1 transposase is significantly smaller than that of the Ppmar1 transposase D332S mutant, and the catalytic transposition ability of the Ppmar1 transposase D332S mutant is increased to 256% of the original. This highly active engineered Ppmar1 transposase D332S mutant will lay an important foundation for the development of gene tags using Ppmar1 transposons.

Table 1 Primer sequences used in the present invention

Table 2 The number and catalytic activity of positive yeast colonies induced by different transposases

Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be construed to include the preferred embodiment and all changes and modifications that fall within the scope of the present invention.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention. Thus, provided that these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

sequence listing

<110> Zhejiang Agriculture and Forestry University

<120> A Ppmar1 transposase D332S mutant with high catalytic activity and its application

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atggctgacc caatagattc tggcttcgat ctgaacgttc ggttagaaga agatgatgac 60

ggcaatcttc cctttgatct caacgagcca atattggaag atcacaacaa tggaattgat 120

ttgaacttgc cattagatga gtttggtgcc gtcgacttcg actatgtaca aaacctcgct 180

gaacaagatg ttgaggctcc cgttcaagta caccctccga agcatgacta tcctgaacat 240

gttagaaaac tagtgtacca agcattgttg atgagaagca agaatgggaa actaggcaat 300

catgatacaa caattgtttc cagtcaattt ggagtaaaga ttcgatcagt tcagcgcata 360

tggaagcaag gtaaaaacca acttgctcaa aacattccgg tcgtggttgc taatctaaag 420

aaaggtagaa gtggccgtaa agcaacccct cttgatttgg aacaattgcg caacattcct 480

ctcaagcaaa gaatgaccat agaagatgtg tctagtagac ttggtattag caaatctagg 540

atacaaaggt atttgaaaaa gggtttgctt aggcgccact ctagtagcat aaaaccttac 600

ctcaccgatg ctaacaagaa gactaggttg aagtggtgca ttgacatgat tgagcaaggt 660

ttggttgatg atccaaagtt cagggatttg tttgactttg tgtttattga tgagaagtgg 720

ttctacctct ctcaaaaatc cgagagatac tacttgctac ccgacgaaga tgaaccacat 780

cgcacttgca agaacaagaa ttacatccct aggatcatgt ttttgtgtgt ttgtgctcgg 840

ccaagattta gaaatggaga atgtgtgttt gatggcaaaa taggttgttt tccactagtc 900

acttttgaac aagctattag aggaagccaa aaccgtcttc gtggagaaca agtaatcaag 960

ccaattcaat caattaatag ggaagtgata agaagtttca tgataaatag agtgttgcct 1020

gcaattagag caaagtggcc aagagaagat gtacacaagc caattttcat acaacaagat 1080

aatgttccat ctcatttaaa ggtggatgat cctcagtttc gtgaggttgc taagcaagat 1140

gggtttgaca ttaggctcat atgtcaacca cccaattctc cagattttaa cattctagat 1200

ttgggttttt ttcgagctat tcaagcaatt caatacaaga aagatgctaa gacattgaaa 1260

gatctaattc cagcagtcca acaggcattt ttggagtact ctccatggaa agcaaatagg 1320

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atcccttgtg aagcttcctt gctagccgaa gcacttgcaa gccttcctgc agctaattag 1500

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tactccctcc atacccgaaa ttcctgacgt ttaggacatg attgtggtaa ccaaggagtg 60

attaattagg ggttagtttt ccatctttgc ccctaataaa tatggttacg ggtgctcttt 120

gtacgagaaa gtaaaccagc tcgactggct agcgcgcgga ggcctcagtc ctgtggtgcg 180

cgttcgatac ctcgcggacg caggtttttt tcttgttgct gtttattcat ttttgcatgg 240

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tggattttca aaattttgtc ctcgcgctgt ggaggctcgt ttgaggccgc gtttttttttc 420

atctggcgcg ctggaaggcc gacgtttgga gtgctcgttg cttgttctat ttaaacgcct 480

ggaaccttcc ttgttgtctt cctatgccgg actcctgtac tatggctgac ccaatagatt 540

ctggcttcga tctgaacgtt cggttagaag aagatgatga cggcaatctt ccctttgatc 600

tcaacgagcc aatattggaa gatcacaaca atggtaagca aaaacgtcaa attagtttct 660

cagtttctcg tttcctttttt tctttactga gcttgtcgtt tcctttttcg ataggaattg 720

atttgaactt gccattagat gagtttggtg ctgtcgactt cgactatgta caaaacctcg 780

ctggtaagca tggctagtat tatgaattcg cttgttttttt tatttccttt tgctggaaca 840

tgccgtgaat aatagtatta tgaactcgct tgtttttttat ttccttttac tagaacatgt 900

gcttgtttta ttcctatagc tagatcatga cgtcaatact ttttacgatg aatatgctcg 960

ttacagtata gctagaacat gccgtgacta catagtagta tgaatatgct tgttttattt 1020

ctataactat aacatgccgt gagtatattt agatcatgcc gtgagtacta agtactatta 1080

aaatgcttgt ttttatttc cttttgctag aacaagatgt tgaggctccc gttcaagtac 1140

accctccgaa gcatgactat cctgaacatg ttagaaaact agtgtaccaa gcattgttga 1200

tgagaagcaa gaatgggaaa ctaggcaatc atgatacaac aattgtttcc agtcaatttg 1260

gagtaaagat tcgatcagtt cagcgcatat ggaagcaagg taaaaaccaa cttgctcaaa 1320

acattccggt cgtggttgct aatctaaaga aaggtagaag tggccgtaaa gcaacccctc 1380

ttgatttgga acaattgcgc aacattcctc tcaagcaaag aatgaccata gaagatgtgt 1440

ctagtagact tggtattagc aaatctagga tacaaaggta tttgaaaaag ggtttgctta 1500

ggcgccactc tagtagcata aaaccttacc tcaccgatgc taacaagaag actaggttga 1560

agtggtgcat tgacatgatt gagcaaggtt tggttgatga tccaaagttc agggatttgt 1620

ttgactttgt gtttattgat gagaagtggt tctacctctc tcaaaaatcc gagagatact 1680

acttgctacc cgacgaagat gaaccacatc gcacttgcaa gaacaagaat tacatcccta 1740

ggatcatgtt tttgtgtgtt tgtgctcggc caagatttag aaatggagaa tgtgtgtttg 1800

atggcaaaat aggttgtttt ccactagtca cttttgaaca agctattaga ggaagccaaa 1860

accgtcttcg tggagaacaa gtaatcaagc caattcaatc aatcaatagg gaagtgataa 1920

gagatttcat gataaataga gtgttgcctg caattagagc aaagtggcca agagaagatg 1980

tacacaagcc aattttcata caacaagata atgctccatc tcatttaaag gtggatgatc 2040

ctcagttttg tgaggttgct aagcaagatg ggtttgacat taggctcata tgtcaaccac 2100

ccaattctcc agattttaac attctagatt tgggttttttt tcgagctatt caagcaattc 2160

aatacaagaa agatgctaag acattgaaag atctaattcc agcagtccaa caggtaaatg 2220

atcatccatt acagtgttta aattgatctt gaacaaataa tataatcact gatcttgaac 2280

atgttttgta ggcatttttg gagtactctc catggaaagc aaataggata tttgtgacac 2340

tacaaactgt tttgaaggaa gcaatgaaga taaaaggttg caacaaaatc aaaattcctc 2400

acatccagaa acaaagactt gagagagaag ataggctgcc attgcaaatc ccttgtgaag 2460

cttccttgct agccgaagca cttgcaagcc ttcctgcggc taattagaag atgcaagcat 2520

gttactcttt tgcagcagca agcatgtaag aagacgcgag catgttagta gcaaactatg 2580

aacaaactag tttatgcatg tagtagtatg ttagcttgtg caccttagtc atctcgtccc 2640

aaccgcttga taacatgctc aggaagaagt attgtgtcac catccatttc aagtttctcc 2700

acatcaggaa tgtagacctc acaatcaaac ttttccatgt catcgagcca cttcgctgtc 2760

atgtcgtagt cttcatgtaa aaggccacaa cgggcacaca tgcgagcttc gcggcgagct 2820

tggtagcagg cttctccgaa gacgccgccg gcgtggaacg taacacagcg aggacacaga 2880

gactcgacgg agtcgggatc gacggtgtcg ggcaccatct cgagggagtc tgcaaccatg 2940

tcgacggagt ccggcagctc ctcgacggag tccggcacca tgtcgacggt gtccggcagc 3000

tcctcgacgg agtctggcac ctcctgcggc gccatgtcca cggtgtccag cgacgctatg 3060

gagcccgacg agatgtcctg cacggcgacg tccagcgccg caacggactc cgtcgtttcc 3120

atctgatccg acgaggcatc gacgtcctgc gacgagcgtg gcggcgagag cacggcgagc 3180

gggcaggcga gcgggcaggc gagcgagcca ttcgcgcgag cgatgaatgc gagctgctgt 3240

accaggcgca cacacgcgca atcaatgcgg gcgagtaacg atgcgagcat gcgcggcgga 3300

agcgcaacag acgggcagca gcgcatggcc aggggcaaac gcgtgaaaag aagaccacgc 3360

gaggccacaa cgtcagcttt tgcgcaaacg ggcacttcgc ctagaacgtc aggaatttcg 3420

ggtatggagg gagta 3435

<210> 4

<211> 1500

<212> DNA

<213> Artificial sequences

<400> 4

atggctgacc caatagattc tggcttcgat ctgaacgttc ggttagaaga agatgatgac 60

ggcaatcttc cctttgatct caacgagcca atattggaag atcacaacaa tggaattgat 120

ttgaacttgc cattagatga gtttggtgcc gtcgacttcg actatgtaca aaacctcgct 180

gaacaagatg ttgaggctcc cgttcaagta caccctccga agcatgacta tcctgaacat 240

gttagaaaac tagtgtacca agcattgttg atgagaagca agaatgggaa actaggcaat 300

catgatacaa caattgtttc cagtcaattt ggagtaaaga ttcgatcagt tcagcgcata 360

tggaagcaag gtaaaaacca acttgctcaa aacattccgg tcgtggttgc taatctaaag 420

aaaggtagaa gtggccgtaa agcaacccct cttgatttgg aacaattgcg caacattcct 480

ctcaagcaaa gaatgaccat agaagatgtg tctagtagac ttggtattag caaatctagg 540

atacaaaggt atttgaaaaa gggtttgctt aggcgccact ctagtagcat aaaaccttac 600

ctcaccgatg ctaacaagaa gactaggttg aagtggtgca ttgacatgat tgagcaaggt 660

ttggttgatg atccaaagtt cagggatttg tttgactttg tgtttattga tgagaagtgg 720

ttctacctct ctcaaaaatc cgagagatac tacttgctac ccgacgaaga tgaaccacat 780

cgcacttgca agaacaagaa ttacatccct aggatcatgt ttttgtgtgt ttgtgctcgg 840

ccaagattta gaaatggaga atgtgtgttt gatggcaaaa taggttgttt tccactagtc 900

acttttgaac aagctattag aggaagccaa aaccgtcttc gtggagaaca agtaatcaag 960

ccaattcaat caattaatag ggaagtgata agagatttca tgataaatag agtgttgcct 1020

gcaattagag caaagtggcc aagagaagat gtacacaagc caattttcat acaacaagat 1080

aatgttccat ctcatttaaa ggtggatgat cctcagtttc gtgaggttgc taagcaagat 1140

gggtttgaca ttaggctcat atgtcaacca cccaattctc cagattttaa cattctagat 1200

ttgggttttt ttcgagctat tcaagcaatt caatacaaga aagatgctaa gacattgaaa 1260

gatctaattc cagcagtcca acaggcattt ttggagtact ctccatggaa agcaaatagg 1320

atatttgtga cactacaaac tgttttgaag gaagcaatga agataaaagg ttgcaacaaa 1380

atcaaaattc ctcacatcca gaaacaaaga cttgagagag aagataggct gccattgcaa 1440

atcccttgtg aagcttcctt gctagccgaa gcacttgcaa gccttcctgc agctaattag 1500

<210> 5

<211> 499

<212> PRT

<213> Artificial sequences

<400> 5

Met Ala Asp Pro Ile Asp Ser Gly Phe Asp Leu Asn Val Arg Leu Glu

1 5 10 15

Glu Asp Asp Asp Gly Asn Leu Pro Phe Asp Leu Asn Glu Pro Ile Leu

20 25 30

Glu Asp His Asn Asn Gly Ile Asp Leu Asn Leu Pro Leu Asp Glu Phe

35 40 45

Gly Ala Val Asp Phe Asp Tyr Val Gln Asn Leu Ala Glu Gln Asp Val

50 55 60

Glu Ala Pro Val Gln Val His Pro Pro Lys His Asp Tyr Pro Glu His

65 70 75 80

Val Arg Lys Leu Val Tyr Gln Ala Leu Leu Met Arg Ser Lys Asn Gly

85 90 95

Lys Leu Gly Asn His Asp Thr Thr Ile Val Ser Ser Gln Phe Gly Val

100 105 110

Lys Ile Arg Ser Val Gln Arg Ile Trp Lys Gln Gly Lys Asn Gln Leu

115 120 125

Ala Gln Asn Ile Pro Val Val Val Val Ala Asn Leu Lys Lys Gly Arg Ser

130 135 140

Gly Arg Lys Ala Thr Pro Leu Asp Leu Glu Gln Leu Arg Asn Ile Pro

145 150 155 160

Leu Lys Gln Arg Met Thr Ile Glu Asp Val Ser Ser Arg Leu Gly Ile

165 170 175

Ser Lys Ser Arg Ile Gln Arg Tyr Leu Lys Lys Gly Leu Leu Arg Arg

180 185 190

His Ser Ser Ser Ile Lys Pro Tyr Leu Thr Asp Ala Asn Lys Lys Thr

195 200 205

Arg Leu Lys Trp Cys Ile Asp Met Ile Glu Gln Gly Leu Val Asp Asp

210 215 220

Pro Lys Phe Arg Asp Leu Phe Asp Phe Val Phe Ile Asp Glu Lys Trp

225 230 235 240

Phe Tyr Leu Ser Gln Lys Ser Glu Arg Tyr Tyr Leu Leu Pro Asp Glu

245 250 255

Asp Glu Pro His Arg Thr Cys Lys Asn Lys Asn Tyr Ile Pro Arg Ile

260 265 270

Met Phe Leu Cys Val Cys Ala Arg Pro Arg Phe Arg Asn Gly Glu Cys

275 280 285

Val Phe Asp Gly Lys Ile Gly Cys Phe Pro Leu Val Thr Phe Glu Gln

290 295 300

Ala Ile Arg Gly Ser Gln Asn Arg Leu Arg Gly Glu Gln Val Ile Lys

305 310 315 320

Pro Ile Gln Ser Ile Asn Arg Glu Val Ile Arg Asp Phe Met Ile Asn

325 330 335

Arg Val Leu Pro Ala Ile Arg Ala Lys Trp Pro Arg Glu Asp Val His

340 345 350

Lys Pro Ile Phe Ile Gln Gln Asp Asn Val Pro Ser His Leu Lys Val

355 360 365

Asp Asp Pro Gln Phe Arg Glu Val Ala Lys Gln Asp Gly Phe Asp Ile

370 375 380

Arg Leu Ile Cys Gln Pro Pro Asn Ser Pro Asp Phe Asn Ile Leu Asp

385 390 395 400

Leu Gly Phe Phe Arg Ala Ile Gln Ala Ile Gln Tyr Lys Lys Asp Ala

405 410 415

Lys Thr Leu Lys Asp Leu Ile Pro Ala Val Gln Gln Ala Phe Leu Glu

420 425 430

Tyr Ser Pro Trp Lys Ala Asn Arg Ile Phe Val Thr Leu Gln Thr Val

435 440 445

Leu Lys Glu Ala Met Lys Ile Lys Gly Cys Asn Lys Ile Lys Ile Pro

450 455 460

His Ile Gln Lys Gln Arg Leu Glu Arg Glu Asp Arg Leu Pro Leu Gln

465 470 475 480

Ile Pro Cys Glu Ala Ser Leu Leu Ala Glu Ala Leu Ala Ser Leu Pro

485 490 495

Ala Ala Asn

<210> 6

<211> 779

<212> DNA

<213> Artificial sequences

<400> 6

tactccctcc atacccgaaa ttcctgacgt ttaggacatg attgtggtaa ccaaggagtg 60

attaattagg ggttagtttt ccatctttgc ccctaataaa tatggttacg ggtgctcttt 120

gtacgagaaa gtaaaccagc tcgactggct agcgcgcgga ggcctcagtc ctgtggtgcg 180

cgttcgatac ctcgcggacg caggtttttt tcttgttgct gtttattcat ttttgcatgg 240

cactgtttag gcaacgcacg tcgcgcgcgc ttagccgctg cgggcgttag ttttcgagtg 300

gatttgggcc tggcgcacgg aggaggttgc atggctccgg cagctcctcg acggagtctg 360

gcacctcctg cggcgccatg tccacggtgt ccagcgacgc tatggagccc gacgagatgt 420

cctgcacggc gacgtccagc gccgcaacgg actccgtcgt ttccatctga tccgacgagg 480

catcgacgtc ctgcgacgag cgtggcggcg agagcacggc gagcgggcag gcgagcgggc 540

aggcgagcga gccattcgcg cgagcgatga atgcgagctg ctgtaccagg cgcacacacg 600

cgcaatcaat gcgggcgagt aacgatgcga gcatgcgcgg cggaagcgca acagacgggc 660

agcagcgcat ggccaggggc aaacgcgtga aaagaagacc acgcgaggcc acaacgtcag 720

cttttgcgca aacgggcact tcgcctagaa cgtcaggaat ttcgggtatg gagggagta 779

Claims (5)

1. A Ppmar1 transposase D332S mutant with high catalytic activity, wherein the amino acid sequence of the transposase D332S mutant is shown in SEQ ID NO.1. 2 . A gene encoding the Ppmar1 transposase D332S mutant of claim 1 , wherein the nucleotide sequence of the gene encoding the transposase D332S mutant is shown in SEQ ID NO. 2. 3 . 3 . A recombinant plasmid, characterized in that, the recombinant plasmid carries the gene encoding the Ppmar1 transposase D332S mutant according to claim 2 . 4. An engineering strain, wherein the engineering strain carries the recombinant plasmid of claim 3. 5. The application of the Ppmar1 transposase D332S mutant with high catalytic activity according to claim 1 in the construction of yeast mutants.