CN114958798A - High catalytic activityPpmar1Transposase L477A-P478E-L479I mutant and application thereof - Google Patents

High catalytic activityPpmar1Transposase L477A-P478E-L479I mutant and application thereof Download PDF

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CN114958798A
CN114958798A CN202111383042.4A CN202111383042A CN114958798A CN 114958798 A CN114958798 A CN 114958798A CN 202111383042 A CN202111383042 A CN 202111383042A CN 114958798 A CN114958798 A CN 114958798A
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周明兵
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Zhejiang A&F University ZAFU
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Abstract

The invention discloses a catalyst with high catalytic activityPpmar1Transposase L477A-P478E-L479I mutant, the describedPpmar1The amino acid sequence of the transposase L477A-P478E-L479I mutant is shown in SEQ ID NO. 1. Encoding the samePpmar1The nucleotide sequence of the gene of the transposase L477A-P478E-L479I mutant is shown in SEQ ID NO. 2. Is prepared from wild typePpmar1Leucine, proline and leucine at positions 477, 478 and 479 of the transposase were mutated to alanine, glutamic acid and isoleucine, respectively. ThePpmar1Transposase L477A-P478E-L479I mutantThe activity of the catalytic transposon transposition is 3.35 times of that of the wild transposase, which lays a foundation for developing gene labels by utilizing MLE transposons and provides a new tool for large-scale gene separation and marking and gene function research in the later genome era.

Description

High catalytic activityPpmar1Transposase L477A-P478E-L479I mutant and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a catalyst with high catalytic activityPpmar1Transposase L477A-P478E-L479I mutant and application thereof.
Background
Transposon (transposon) refers to a DNA sequence that can be transferred from one site to another on the genome. Since the discovery of transposons, some transposons have been transformed into gene tags for gene analysis and become one of the important means for large-scale gene isolation, with the development of understanding of transposon structure and function at the molecular level.
Mariner-Like transposons (MLE) are an important family of transposons, and first studied Drosophila melanogaster (Michelia furiosa, Inc.)Drosophila mauristiana) An unstable mutation in the white-eye gene. Thereafter, the presence of a large number of MLE transposons was also found in the genomes of other animals as well as plants. Compared with other transposons, the MLE transposon has the characteristics of simple structure, high heterologous transposition rate, near random genomic insertion sites and the like, and is far superior to other transposons in developing gene tags, separating genes and researching gene functions.
MLE transposons are composed of Inverted Terminal Repeats (TIRs) and a gene encoding a transposase responsible for catalyzing transposon transposition, so that the activity of the transposase is a major factor affecting the transposition frequency of the transposon. However, in the course of evolution, MLE transposases isolated from nature accumulate more or less mutations due to the effect of "vertical inactivation", lose some or all of their catalytic transposability, become low-activity or inactive transposases, and seriously affect the application of MLE transposons, so that it is very important to artificially construct high-activity transposases.
Disclosure of Invention
The object of the present invention is to provide a catalyst having a high catalytic activityPpmar1The transposase L477A-P478E-L479I mutant and the application thereof solve the problem that the MLE transposase separated from the natural world has low catalytic activity or does not have catalytic activity.
The invention provides a catalyst with high catalytic activityPpmar1Transposase L477A-P478E-L479I mutant, and its usePpmar1The amino acid sequence of the transposase L477A-P478E-L479I mutant is shown in SEQ ID NO. 1.
The invention also provides a method for encoding the codePpmar1Gene of transposase L477A-P478E-L479I mutant, encoding the samePpmar1The nucleotide sequence of the gene of the transposase L477A-P478E-L479I mutant is shown in SEQ ID NO. 2.
The invention also provides a recombinant plasmid carrying the codePpmar1The gene of transposase L477A-P478E-L479I mutant.
The invention also provides an engineering strain, and the engineering strain carries the recombinant plasmid.
The invention also provides a catalyst with high catalytic activityPpmar1Application of transposase L477A-P478E-L479I mutant in constructing yeast mutant.
Compared with the prior art, the catalyst provided by the invention has high catalytic activityPpmar1The transposase L477A-P478E-L479I mutant has the following beneficial effects:
the invention relates to a bamboo made from moso bamboo (A)Phyllostachys pubescens) The cloned active transposase is artificially modified to obtain MLE transposase mutants with higher activity (Ppmar1Transposase L477A-P478E-L479I mutant),Ppmar1the activity of transposase catalyzed by the transposase L477A-P478E-L479I mutant is 3.35 times of that of wild transposase, which lays a foundation for developing gene labels by using MLE transposons and provides a new tool for large-scale gene separation and labeling in the later genome era and gene function research.
Detailed Description
The present invention will be described in detail with reference to specific embodiments, but it should be understood that the scope of the present invention is not limited by the specific embodiments. The test methods in the following examples, in which specific conditions are not specified, are generally conducted under conventional conditions such as those described in molecular cloning instructions, which are mainly compiled by Sambrook et al, or according to the procedures set forth in the kit, and the procedures are not described in detail since they do not relate to the invention.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any number between the two endpoints are optional unless otherwise specified in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
First, obtaining a wild-type MLE transposase and a transposase-deleted nonautonomous transposon
Step 1.1, collecting fresh moso bamboo leaves: (Phyllostachys pubescens) The method comprises the steps of collecting DNA of a phyllostachys pubescens genome in a plantary of agriculture and forestry university in Zhejiang, N30 degrees 15 '14.67' east longitude E119 degrees 43 '33.47'), extracting Ppmar1-5-3 (the sequence information of Ppmar1-5-3 is shown in table 1) according to a MLE transposon TIR conserved sequence, and carrying out PCR amplification to obtain an MLE transposon amplification product.
The PCR amplification system is 20 μ L, including 0.2 μ L rTaq Polymerase (5U/μ L), 1 μ L Ppmar1-5-3 (10 μmol/L), 2 μ L10 XrTaq Buffer (Mg) 2+ plus), 1.6 μ L dNTPmix (2.5 mmol/L), 100 ng moso bamboo genome DNA, and sterile water is added to fill up 20 μ L.
The reaction conditions for PCR amplification are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s, denaturation at 60 ℃ for 30 s, elongation at 72 ℃ for 40 s, and 35 cycles; 72 ℃ for 2 min and 4 ℃ for 10 min.
Step 1.2, after the sequence is amplified, the MLE transposon amplification product of step 1.1 is connected to a pMD18-T Vector by adopting the method of TaKaRa company pMD18-T Vector Cloning Kit, and after the sequencing confirmation, the MLE transposon amplification product is named as pMD18-T VectorPpmar1A transposon.
Step 1.3, using RNeasy Mini Kit of QIAGEN company to extract the RNA of Mao bamboo leaves, reverse transcribing the RNA into cDNA using SuperScript-VILO-cDNA Synthesis Kit of Invitrogen company, according to the methodPpmar1A pair of primers PpTase 1-5 and PpTase 1-3 (the sequence information of PpTase 1-5 and PpTase 1-3 is shown in Table 1) are designed for transposase sequences to carry out PCR amplification and are recovered to obtainPpmar1The transposase amplification product isPpmar1A transposase nucleotide sequence.
The PCR amplification system is 20 mu L, and comprises 0.2 mu L rTaq Polymerase (5U/mu L), 0.5 mu L PpTpase1-5 (10 mu mol/L), 0.5 mu L PpTpase1-3 (10 mu mol/L), and 2 mu L10 xrTaq Buffer (Mg) 2+ plus), 1.6 mul dNTPmix (2.5 mmol/L), 10 ng Phyllostachys pubescens leaf cDNA, and adding sterile water to make up 20 mul.
The reaction conditions for PCR amplification are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s, denaturation at 55 ℃ for 30 s, elongation at 72 ℃ for 40 s, and 35 cycles; 72 ℃ for 2 min and 4 ℃ for 10 min.
Step 1.4, the method of using TaKaRa corporation pMD18-T Vector Cloning Kit to mix the plasmid of step 1.3Ppmar1The transposase nucleotide sequence is connected to pMD18-T vector clone, and sequencing is confirmed,Ppmar1the transposase nucleotide sequence and the corresponding amino acid sequence are shown as SEQ ID number 3 and SEQ ID number 4, respectively.
Will containPpmar1pMD18-T vector for transposon full-length sequenceBseR IExcision ofPpmar1Most of the sequence of the intermediate transposase.
The enzyme digestion system is 50 mu l, and comprises 5 mu l 10 Xbuffer and 1 mu lBseR I (1U/ul), 1 ug plasmid (containingPpmar1pMD18-T vector of full-length sequence), adding sterile water to make up 50 mul, and carrying out warm bath at 37 ℃ for 6 hours. Recovering the large plasmid fragment with T 4 The DNA Ligase self-connects the large plasmid fragment to obtain the plasmid connected to pMD18-T vectorPpmar1Nonautonomous transposon-Ppmar1NA。
Wherein the self-connection system is 10 mu l，Including 1 μ l 10 × T 4 DNA Ligase buffer, 1 mu l T4 DNA Ligase (10U/mu l), 50ng plasmid large fragment, adding sterile water to supplement 10 mu l, and carrying out warm bath at 16 ℃ for 8 hours.
Ppmar1NAThe sequence of (3) is shown as SEQ ID number 5.
Second, construction of yeast transposition expression vector
In the step 2.1, the method comprises the following steps of,Ppmar1construction of transposase expression vector
To step 1.3Ppmar1Transposase nucleotide sequenceNot
Figure 212012DEST_PATH_IMAGE001
AndEcoR
Figure 939797DEST_PATH_IMAGE002
double digestion and recoveryPpmar1Large fragments of transposase enzyme digestion products; the pAG413-gal-ccdB vector is subjected toNot
Figure 697773DEST_PATH_IMAGE001
AndEcoR
Figure 903626DEST_PATH_IMAGE002
double enzyme digestion, recovering the large fragment of the enzyme digestion product of the pAG413-gal-ccdB vector; and isPpmar1The double enzyme digestion system and the double enzyme digestion condition of the transposase nucleotide sequence are the same as those of the pAG413-gal-ccdB carrier;
wherein the double enzyme digestion system is 50 mu l, and comprises 5 mu l 10 Xbuffer and 1 mu lNot
Figure 895853DEST_PATH_IMAGE001
(1U/µl), 1µl EcoR V (1U/. mu.l), 1. mu.g plasmid (c) (1U/. mu.l)Ppmar1Transposase nucleotide sequence or pAG413-gal-ccdB vector), sterile water is added to supplement 50 mu l, and double-enzyme cutting conditions are as follows: the mixture is incubated at 37 ℃ for 6 hours.
Will be provided withPpmar1The large fragment of the transposase enzyme digestion product is connected with the large fragment of the pAG413-gal-ccdB vector enzyme digestion product;
the connector system is 10 mu l, and comprises 1 mu l 10 xT 4 DNA Ligase buffer, 1 mu l T4 DNA Ligase (10U/mu l), 50ng pAG413-gal-ccdB vector enzyme digestion product large fragment and 20ngPpmar1Adding sterile water to fill 10 mu l of large fragments of the transposase enzyme digestion product, and carrying out warm bath at 16 ℃ for 8 hours.
At this point finish usingPpmar1Replacing the ccdB nucleotide sequence in the pAG413-gal-ccdB plasmid by the transposase nucleotide sequence to obtain a recombinant plasmid pAG 413-gal-Tpass (Tpass represents transposase);
the recombinant plasmid pAG 413-gal-Tpass isPpmar1Transposase expression vector carrying the codePpmar1A gene of transposase. The expression vector has His (histidine) screening marker, so that the host introduced into pAG413-gal-Tpase vector can grow on the deletion culture medium lacking His.
In the step 2.2, the step of the method,Ppmar1construction of non-autonomous transposon Donor vectors
In step 1.4Ppmar1The nonautonomous transposon pMD18-T-Ppmar1NAAmplification Using Ppmar1-5-3 primer as templatePpmar1NAPerforming PCR amplification to obtainPpmar1NAAnd (4) amplifying the product.
The PCR amplification system is 20 μ L, including 0.2 μ L rTaq Polymerase (5U/μ L), 1 μ L Ppmar1-5-3 (10 μmol/L), 2 μ L10 XrTaq Buffer (Mg) 2+ plus），1.6µl dNTP mix (2.5 mmol/L)，10 ng pMD18-T-Ppmar1NAAnd adding sterile water to replenish 20 mu l.
The PCR amplification reaction conditions are that the pre-denaturation is carried out for 5 min at 94 ℃; denaturation at 94 ℃ for 30 s, denaturation at 60 ℃ for 30 s, elongation at 72 ℃ for 40 s, and 35 cycles; 72 ℃ for 2 min and 4 ℃ for 10 min.
At the same time, vector pWL89a was usedXhoⅠCleavage (cleavage site located atADE2Intragenic), vector pWL89a backbone was recovered. The enzyme digestion system is 50 mu l, and comprises 5 mu l 10 Xbuffer and 1 mu lXhoⅠ(1U/mul), 1 mug carrier pWL89a, and sterile water is added to supplement 50 mul, and the temperature bath is carried out for 6 hours at 37 ℃.
Then, the resulting mixture was subjected to In-Fusion Advantage PCR Cloning Kit (TaKaRa Co., Japan)Ppmar1NAInsertion of amplification product into vector pWL89a backboneADE2Among the genes, resulting in a reporter geneADE2Insertional inactivation to obtain pWL89a-Ppmar1NARecombinant plasmid, namelyPpmar1A non-autonomous transposon donor vector. If it isPpmar1NATo make a transposition fromADE2Leaves on the gene, thenADE2The gene reading frame is restored. The vector has URA3 selection marker, so that pWL89a-Ppmar1NACan be grown on deletion medium lacking Ura (uracil).
III,Ppmar1Obtaining of transposase L477A-P478E-L479I mutant
Will be provided withPpmar1Carrying out homology comparison on the transposase nucleotide sequence and nucleotide sequences of MLE transposases of other plants, and selectingPpmar1The transposase nucleotide sequences 477, 478, and the leucine at positions 479 were mutated to plan for mutations to alanine, glutamic acid, and isoleucine (L477A-P478E-L479I).
Step 3.1, design Site-Directed Mutagenesis primers L477A-P478E-L479I-F and L477A-P478E-L479I-R (L477A-P478E-L479I-F and L477A-P based on QuikChange Site-Directed Mutagenesis Kit (Stratagene, USA) instructions478E-L479I-R, see Table 1), according to the QuikChange Site-Directed Mutagenesis Kit method, using the recombinant plasmid pAG 413-gal-Tpass of step 2.1 as a templatePfuTurboResynthesis of the mutant DNA polymerasePpmar1Plasmid DNA of transposase L477A-P478E-L479I mutant;
step 3.2, then 2. mu.L ofDpn
Figure 794539DEST_PATH_IMAGE001
And (3) reacting the restriction enzyme for 5 min at 37 ℃ to completely degrade the original template sequence. Sequencing and confirming newly synthesized plasmid DNAPpmar1Transposase L477A-P478E-L479I mutant;
Ppmar1the amino acid sequence of the transposase L477A-P478E-L479I mutant is shown in SEQ ID NO.1, and the transposase L477A-P478E-L479I mutant is codedPpmar1The nucleotide sequence of the gene of the transposase L477A-P478E-L479I mutant is shown in SEQ ID NO. 2.
Detection of transposase Activity
The experimental group is the compound of step 3.2Ppmar1Plasmid DNA of transposase L477A-P478E-L479I mutant and pWL89 a-of step 2.2Ppmar1NARecombinant plasmids were co-transformed into yeast by PEG/LiAc method, and selectively cultured on His/Ura double-deficient solid medium. Galactose is used for inducing the expression of transposase, so that the transposition of the non-autonomous transposon is promoted.
In the wild typePpmar1Transposase as control, step 2.1 with wild typePpmar1Recombinant plasmid pAG 413-gal-Tpass for transposase and pWL89a-Ppmar1NARecombinant plasmids were co-transformed into yeast by PEG/LiAc method, and selectively cultured on His/Ura double-deficient solid medium. Galactose is used for inducing the expression of transposase, so that the transposition of the non-autonomous transposon is promoted.
The induced yeast of the experimental group and the control group are selectively cultured by using a His/Ura/Ade deletion solid medium, and the yeast bacterial plaque growing on the medium is calculated. If transposition occurs pWL89a-Ppmar1NAThe ADE2 gene on the recombinant plasmid can be expressed, so that the positive yeast strain can be deletedGrowth on adenine-deficient medium.
In the wild typePpmar1Transposase as control, comparative transformationPpmar1The transposase mutants with higher activity were selected by the number of yeast colonies of the transposase L477A-P478E-L479I mutant, and the results are shown in Table 2.
As can be seen from Table 2, the wild typePpmar1The number of positive yeast colonies of transposase is significantly less thanPpmar1A transposase L477A-P478E-L479I mutant, andPpmar1the transposase L477A-P478E-L479I mutant catalyzes the transposition ability to 335% of the original ability. This high activity is artificially modifiedPpmar1The transposase L477A-P478E-L479I mutant will be utilizedPpmar1The transposon lays an important foundation for developing gene labels.
TABLE 1 primer sequences for use in the invention
Figure 770585DEST_PATH_IMAGE003
TABLE 2 number of Positive Yeast colonies induced by different transposases and catalytic Activity
Figure 514550DEST_PATH_IMAGE004
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such 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 such modifications and variations.
Sequence listing
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<210> 3
<211> 1500
<212> DNA
<213> Phyllostachys pubescens (Phyllostachys pubescens)
<400> 3
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> 4
<211> 499
<212> PRT
<213> Phyllostachys pubescens (Phyllostachys pubescens)
<400> 4
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 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> 5
<211> 779
<212> DNA
<213> Phyllostachys pubescens (Phyllostachys pubescens)
<400> 5
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 catalyst with high catalytic activityPpmar1The transposase L477A-P478E-L479I mutant is characterized in that the amino acid sequence of the transposase L477A-P478E-L479I mutant is shown as SEQ ID NO. 1.
2. A method for encoding thePpmar1The gene of the transposase L477A-P478E-L479I mutant is characterized in that the nucleotide sequence of the gene coding the transposase L477A-P478E-L479I mutant is shown as SEQ ID NO. 2.
3. A recombinant plasmid carrying the code of claim 2Ppmar1The gene of transposase L477A-P478E-L479I mutant.
4. An engineered strain carrying the recombinant plasmid of claim 3.
5. The catalyst of claim 1 having high catalytic activityPpmar1Application of transposase L477A-P478E-L479I mutant in constructing yeast mutant.
CN202111383042.4A 2021-11-22 2021-11-22 High catalytic activityPpmar1Transposase L477A-P478E-L479I mutant and application thereof Pending CN114958798A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103627684A (en) * 2013-11-20 2014-03-12 浙江农林大学 Manually optimized high-activity Mariner-Like transposase
CN106754815A (en) * 2017-01-20 2017-05-31 浙江农林大学 A kind of Ppmar1 transposase C296I mutant and its application with high catalytic activity

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103627684A (en) * 2013-11-20 2014-03-12 浙江农林大学 Manually optimized high-activity Mariner-Like transposase
CN106754815A (en) * 2017-01-20 2017-05-31 浙江农林大学 A kind of Ppmar1 transposase C296I mutant and its application with high catalytic activity

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MUTHUSAMY RAMAKRISHNAN等: "Nuclear export signal (NES) of transposases affects the transposition activity of marinerlike elements Ppmar1 and Ppmar2 of moso bamboo", MOBILE DNA *
TANG, D.-Q.等: "transposase [Phyllostachys edulis]", GENBANK *
刘政捷: "毛竹Mariner-like转座子的转座特性分析", 中国优秀硕士学位论文全文数据库农业科技辑 *

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