CN114854710A - A catalyst having high activityPpmar2NATransposon G25A-C27A mutant and application thereof - Google Patents
A catalyst having high activityPpmar2NATransposon G25A-C27A mutant and application thereof Download PDFInfo
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Abstract
The invention discloses a high-activityPpmar2NATransposon G25A-C27A mutant, saidPpmar2NAThe base sequence of the transposon G25A-C27A mutant is shown in SEQ ID NO. 1. Is prepared from wild typePpmar2NABoth G (guanine) and C (cytosine) at positions 25 and 27 of the transposons are mutated to A (adenine). ThePpmar2NAThe activity of the transposon G25A-C27A mutant is 1.88 times of that of wild transposase, lays a foundation for developing a gene label by utilizing an MLE transposon, and provides a large-scale gene separation and labeling and research medium for the post-genome eraThe function of the tool provides a new tool.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a high-activity antibacterial compositionPpmar2NATransposon G25A-C27A 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 gradually become one of the important means for large-scale gene isolation, as people have increasingly developed knowledge 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 transposons haveSimple structure, high heterologous transposition rate, near random insertion sites in genome and the like, and is far superior to other transposons in developing gene labels, 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, and thus the activity of the transposase is a major factor affecting the transposition frequency of the transposon. However, in the course of evolution, MLE transposase isolated from nature accumulates more or less mutations due to the effect of "vertical inactivation", loses some or all of its ability to catalyze transposition, becomes a transposase with low activity or no activity, and seriously affects the application of MLE transposase, so that it is very important to artificially construct a transposase with high activity.
Disclosure of Invention
It is an object of the present invention to provide a composition having high activityPpmar2NAThe transposon G25A-C27A mutant and the application thereof solve the problem that the prior MLE separated from the nature has low or no activity.
The invention provides a composition with high activityPpmar2NATransposon G25A-C27A mutant, saidPpmar2NAThe base sequence of the transposon G25A-C27A mutant is shown in SEQ ID NO. 1.
The invention also provides a recombinant plasmid carrying the codePpmar2NASequences of the transposon G25A-C27A mutant.
The invention also provides an engineering strain, and the engineering strain carries the recombinant plasmid.
The invention also provides a composition with high activityPpmar2NAThe use of the transposon G25A-C27A mutant for constructing yeast mutants.
Compared with the prior art, the invention provides the high-activityPpmar2NATransposon G25A-C27A 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 transposition with higher activityEnzyme mutants (A)Ppmar2NATransposon G25A-C27A mutant),Ppmar2NAthe activity of the transposon G25A-C27A mutant is 1.88 times of that of a wild transposon, lays a foundation for developing a gene label by utilizing the MLE transposon, and provides a new tool for large-scale gene separation and marking and gene function research in the post-genome era.
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 protocols, which are mainly compiled by Sambrook et al, or according to procedures set forth in kits, and the procedures are not described in detail since they do not refer 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 value therebetween can be selected unless the invention otherwise indicated. 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 Ppmar2-5-3 (the sequence information of Ppmar2-5-3 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.
Amplified by PCRThe system is 20 mu L and comprises 0.2 mu L rTaq Polymerase (5U/mu L), 1 mu L Ppmar2-5-3 (10 mu mol/L) and 2 mu 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 50 ℃ 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 VectorPpmar2A transposon.
Step 1.3, using RNeasy Mini Kit of QIAGEN company to extract the RNA of the bamboo leaves, using SuperScript VILO cDNA Synthesis Kit of Invitrogen company to reverse transcribe the RNA into cDNA, and performing reverse transcription on the cDNA by using the SuperScript VILO cDNA Synthesis Kit of Invitrogen companyPpmar2Transposase sequence design A pair of primers PpTpase2-5 and PpTpase2-3 (sequence information of PpTpase2-5 and PpTpase2-3 are shown in Table 1, withNot IAndEcoR Venzyme cutting site), PCR amplification is carried out, and recovery is carried out to obtainPpmar2The transposase amplification product isPpmar2A 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 1.3Ppmar2The transposase nucleotide sequence is connected to pMD18-T vector clone, and sequencing is confirmed,Ppmar2the transposase nucleotide sequence and the corresponding amino acid sequence are shown as SEQ ID number 2 and SEQ ID number 3, respectively.
To containPpmar2pMD18-T vector of transposon full-length sequence is used as template, primers MiniPmar 2-1F and MiniPmar 2-1R are used for amplificationPpmar2TIR at the 5' end and flanking sequence to obtain a 226 bp fragment, and MiniPPmar2-1F bandXho ⅠCleavage site (C TCGAG).
The reaction conditions for PCR amplification are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s, denaturation at 58 ℃ for 30 s, elongation at 72 ℃ for 40 s, and 35 cycles; 40 s at 72 ℃ and 10 min at 4 ℃.
Ppmar 23' end TIR and flanking sequence are amplified by primers MiniPPmar2-2F and MiniPPmar2-2R to obtain 664bp fragment, and MiniPPmar2-1R carriesXho ⅠCleavage site (C TCGAG).
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; 40 s at 72 ℃ and 10 min at 4 ℃.
After the two fragments were recovered, they were mixed at a molecular number molar ratio of 1:1, and the mixture was used as a template to obtain a shortened Phmar2NA transposon of 830 bp in length by primers MiniPPmar2-1F and MiniPPmar2-2R,Ppmar2NAthe sequence of (A) is shown in SEQ ID NO. 5.
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 1 min and 4 ℃ for 10 min.
Second, construction of yeast transposition expression vector
In the step 2.1, the method comprises the following steps of,Ppmar2construction of transposase expression vector
To step 1.3Ppmar2Transposase nucleotide sequenceNot IAndEcoR Vdouble digestion and recoveryPpmar2Large fragments of transposase enzyme digestion products; the pAG413-gal-ccdB vector is subjected toNot IAndEcoR Vdouble enzyme digestion, recovering the large fragment of the enzyme digestion product of the pAG413-gal-ccdB vector; and isPpmar2The 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 I (1U/µl), 1µl EcoR V (1U/. mu.l), 1. mu.g plasmid (c) (1U/. mu.l)Ppmar2Transposase coreNucleotide 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 withPpmar2The 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 20ngPpmar2Adding 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 usingPpmar2Replacing 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 pAG413-gal-Tpase isPpmar2Transposase expression vector carrying the codePpmar2A gene of transposase. The expression vector has a His (histidine) selection marker, so that a host introduced into the pAG 413-gal-Tpass vector can grow on a deletion medium which is lack of His.
In the step 2.2, the step of the method,Ppmar2construction of non-autonomous transposon Donor vectors
In step 1.4Ppmar2Nonautonomous transposon ofPpmar2NAIs used as a template and is used as a template,XhoⅠand (6) enzyme digestion and recovery.
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/ul), 1 ug of carrier pWL89a, and adding sterile water to fill 50 ul, and carrying out warm bath at 37 ℃ for 6 hours.
Then using T4 ligase to mixPpmar2NAInsertion of amplification product into vector pWL89a backboneADE2Among the genes, resulting in a reporter geneADE2Insertional inactivation to obtain pWL89a-Ppmar2NARecombinant plasmid is thatPpmar2A non-autonomous transposon donor vector. If it isPpmar2NATo make a transposition fromADE2Leaves on the gene, thenADE2The gene reading frame is restored. The vector has a URA3 selection marker for introduction pWL89a-Ppmar2NAA sinkThe master is able to grow on deletion medium lacking Ura (uracil).
To exclude false positives, selection and validation by sequencingPpmar2NAExperiments were performed with recombinant plasmids reverse inserted into pWL89a vector.
III,Ppmar2NAObtaining of transposon G25A-C27A mutant
Will be provided withPpmar2NAPerforming homology alignment between the TIR nucleotide sequence of the transposon and the TIR nucleotide sequence of MLE transposons of other plants, and selectingPpmar2NABoth G (guanine) and C (cytosine) at positions 669 and 671 of the transposon nucleotide sequence are mutated to A (adenine) (G25A-C27A).
Step 3.1, designing Site-Directed Mutagenesis primers G25A-C27A-F and G25A-C27A-R (sequence information of G25A-C27A-F and G25A-C27A-R is shown in Table 1) according to the QuikChange Site-Directed Mutagenesis Kit (Stratagene, USA) instruction, and using recombinant plasmid pWL89a-Ppmar2NA of step 2.2 as a template and using recombinant plasmid 3689 a-Ppmar2NA of step 2.2 as a template according to the QuikChange Site-Directed Mutagenesis Kit methodPfuTurboResynthesis of the mutant DNA polymerasePpmar2NAPlasmid DNA of transposon G25A-C27A mutant;
step 3.2, then 2. mu.L ofDpn IAnd (3) reacting the restriction enzyme for 5 min at 37 ℃ to completely degrade the original template sequence. Sequencing and confirming newly synthesized plasmid DNAPpmar2NATransposon G25A-C27A mutant;
Ppmar2NAthe nucleotide sequence of the transposon G25A-C27A mutant is shown as SEQ ID NO. 1.
Fourth, detection of transposon Activity
The experimental group is that the pAG 413-gal-Tpass recombinant plasmid of step 2.1 and the plasmid containing step 3.2Ppmar2NAPlasmid DNA of transposon G25A-C27A mutant was co-transformed into yeast by PEG/LiAc method and cultured in His/Ura double-deleted solid medium for selection. Galactose is used for inducing the expression of transposase, so that the transposition of the non-autonomous transposon is promoted.
In the wild typePpmar2NATransposon as control, step 2.1 with wild typePpmar2Recombinant plasmid pAG 413-gal-Tpass for transposase and pWL89a-Ppmar2NARecombinant plasmids were co-transformed into yeast by PEG/LiAc method, and selectively cultured on His/Ura double-deficient solid medium. Galactose is used to induce transposase expression, so as to promote transposition of the non-autonomous transposon.
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-Ppmar2NAThe ADE2 gene on the recombinant plasmid is expressed so that positive yeast strains can be grown on adenine-deficient media.
In the wild typePpmar2NATransposon as control, comparative transformation withPpmar2NAThe results of screening for transposon mutants with higher activity by the number of yeast colonies of transposon G25A-C27A are shown in Table 2.
As can be seen from Table 2, the wild typePpmar2NAThe number of positive yeast colonies of the transposon is obviously less thanPpmar2NATransposon G25A-C27A mutant, andPpmar2NAthe transposability of the transposon G25A-C27A mutant was increased to 188% of the original. This high activity is artificially modifiedPpmar2NAThe transposon G25A-C27A mutant will be utilizedPpmar2NAThe transposon lays an important foundation for developing gene labels.
TABLE 1 primer sequences for use in the invention
TABLE 2 number of Positive Yeast colonies induced by different transposases and catalytic Activity
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
<110> civilian soldier
<120> a Ppmar2NA transposon G25A-C27A mutant with high activity and application thereof
<141> 2021-11-22
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 830
<212> DNA
<213> Phyllostachys pubescens (Phyllostachys pubescens)
<400> 1
tactccctcc gtcccagtat aacgagagta taaaaaaatt tctgctgtcc cacaatacag 60
ggcgtccctt caattttgca ccgctttctc ccattttgcc ctcagcaatc tgcatgcatg 120
catgcatgcg tgacgcttag agttctgctt aaccgcgtga tgcattcaac tgctctgaga 180
agtgccagcg tgatgcatgc cactgcttag aaaagtgcga ggatccggtg acacgaccat 240
catagtcctc aatctcttcc caatggatag gttgatttag gtccaaattc gccattgatt 300
tcgagcaagc aactggacga gagaagagga gagagtggca gcagacctgt tgtgttcatg 360
tctatttata ggaggaaaac gaatttgaat ttctgggggc aatcttctca atccatatgc 420
actagctcaa tttctaggcg ccatcttcta aatgctgtgt tcaaattttc gctataatcc 480
gctatagact tctaaatgct gggcgccatc ttctaaatgc atgcgtgcga gcgagtacga 540
gcctacgagc gcgcgaatct gcatgcatcg ctcaaatctg catgccatgc ttctctcgcg 600
tgtgaatctg catgccatgc ttctctcgcg cacgaatctg catgctgtgc tttaaatctg 660
catgccgtgc tttaaatctg catgcatgca tggtcattga ttatagcaaa ctgcacgcat 720
gcatctcatc aatttaatta ggggtagatg aggaagaatc gctagctcgc gcgccctcct 780
tggtctctga aattttttta tactctcgtt atactgggac ggagggagta 830
<210> 2
<211> 1377
<212> DNA
<213> Phyllostachys pubescens (Phyllostachys pubescens)
<400> 2
atggcgaatt tggacctaaa tcaacctatc cattgggaag agattgagga ctatgatggc 60
cctgtcatcg accttaattt tgatcttgtg tttcatgata gcgatgaagg tgatggcggc 120
ccaacccatg gcgaagagga cggtggtgca ccttcccatg gagaagagga tggcggccca 180
acccatggcg aagaggatgg tggtgcacct tcccatggag aagaggacgg tgccccatcc 240
catggcgaag aggatggcac ccctgcacct aacgcctatg agacgatctc taccaacaag 300
gcaaagaatt gtggctggaa acgagtagcc ttcgatccgg aagccatcaa agatgtgcct 360
ttgagtagcc gaacgacaat ccgggatcta gcaggcgctc tgaatatttc aaagagcaca 420
ttgtttaggc agatgaaaga agggaagttt agacggcaca caaatgacat taagtttaca 480
ttgactgaag ataacaagaa agcatgtgtt aagttttgcc tctcaatgct agaaaaatta 540
agcatgccgc aagaaccaac ttttgagggt atgtacaaca tcgtgtacat agacgaaaag 600
tggttctatc ggatgaggaa atttcaaaac tactacttgg cgccagatga ggacaagcca 660
gaaagaacca caaagagtaa aaatttcata gagaaggtga tgttgctcgc agaaattgcg 720
agacctaaat ttgattggga tggaaatgtt acattttctg gaaagatagg cataattcct 780
ttcactttcg tagagctagc aaagcgaagt agtgcgaata ggcctgctgg tacattggtg 840
accaaggcaa tgacatcggt aaccaaggaa acaagccgtg agtaccttgt aaataaggta 900
ttgcccgcga tcaagcaaaa atggctagcg gaggaagttg gtacccccat attcatccag 960
caggataatg ctaggacgca tattgcaatc aatgatgacg agttttgtcg tgcggcatcc 1020
gcagatggtt ttgacataag tttgatgtgc cagccaccca actctcctga tctcaatgta 1080
ttagatcttg gtttttttgc ggccattcaa tccatgtttc aaaagtcgtc tccaagcaac 1140
attgaagaca ttgttgccaa ggtaatccaa gcttttgacg agtatccagt tgataggagt 1200
aaccgtattt tcctcactca ccaatcatgc atgagagaaa ttttgcgtca aaaaggaggg 1260
caacactatg caatcccaca cttgaagaag caatcacttg agaggaatgg tgttctttcc 1320
attagattac aatgtgacct agtagttgtg aatgaagcaa ttgtgtacat caattag 1377
<210> 3
<211> 458
<212> PRT
<213> Phyllostachys pubescens (Phyllostachys pubescens)
<400> 3
Met Ala Asn Leu Asp Leu Asn Gln Pro Ile His Trp Glu Glu Ile Glu
1 5 10 15
Asp Tyr Asp Gly Pro Val Ile Asp Leu Asn Phe Asp Leu Val Phe His
20 25 30
Asp Ser Asp Glu Gly Asp Gly Gly Pro Thr His Gly Glu Glu Asp Gly
35 40 45
Gly Ala Pro Ser His Gly Glu Glu Asp Gly Gly Pro Thr His Gly Glu
50 55 60
Glu Asp Gly Gly Ala Pro Ser His Gly Glu Glu Asp Gly Ala Pro Ser
65 70 75 80
His Gly Glu Glu Asp Gly Thr Pro Ala Pro Asn Ala Tyr Glu Thr Ile
85 90 95
Ser Thr Asn Lys Ala Lys Asn Cys Gly Trp Lys Arg Val Ala Phe Asp
100 105 110
Pro Glu Ala Ile Lys Asp Val Pro Leu Ser Ser Arg Thr Thr Ile Arg
115 120 125
Asp Leu Ala Gly Ala Leu Asn Ile Ser Lys Ser Thr Leu Phe Arg Gln
130 135 140
Met Lys Glu Gly Lys Phe Arg Arg His Thr Asn Asp Ile Lys Phe Thr
145 150 155 160
Leu Thr Glu Asp Asn Lys Lys Ala Cys Val Lys Phe Cys Leu Ser Met
165 170 175
Leu Glu Lys Leu Ser Met Pro Gln Glu Pro Thr Phe Glu Gly Met Tyr
180 185 190
Asn Ile Val Tyr Ile Asp Glu Lys Trp Phe Tyr Arg Met Arg Lys Phe
195 200 205
Gln Asn Tyr Tyr Leu Ala Pro Asp Glu Asp Lys Pro Glu Arg Thr Thr
210 215 220
Lys Ser Lys Asn Phe Ile Glu Lys Val Met Leu Leu Ala Glu Ile Ala
225 230 235 240
Arg Pro Lys Phe Asp Trp Asp Gly Asn Val Thr Phe Ser Gly Lys Ile
245 250 255
Gly Ile Ile Pro Phe Thr Phe Val Glu Leu Ala Lys Arg Ser Ser Ala
260 265 270
Asn Arg Pro Ala Gly Thr Leu Val Thr Lys Ala Met Thr Ser Val Thr
275 280 285
Lys Glu Thr Ser Arg Glu Tyr Leu Val Asn Lys Val Leu Pro Ala Ile
290 295 300
Lys Gln Lys Trp Leu Ala Glu Glu Val Gly Thr Pro Ile Phe Ile Gln
305 310 315 320
Gln Asp Asn Ala Arg Thr His Ile Ala Ile Asn Asp Asp Glu Phe Cys
325 330 335
Arg Ala Ala Ser Ala Asp Gly Phe Asp Ile Ser Leu Met Cys Gln Pro
340 345 350
Pro Asn Ser Pro Asp Leu Asn Val Leu Asp Leu Gly Phe Phe Ala Ala
355 360 365
Ile Gln Ser Met Phe Gln Lys Ser Ser Pro Ser Asn Ile Glu Asp Ile
370 375 380
Val Ala Lys Val Ile Gln Ala Phe Asp Glu Tyr Pro Val Asp Arg Ser
385 390 395 400
Asn Arg Ile Phe Leu Thr His Gln Ser Cys Met Arg Glu Ile Leu Arg
405 410 415
Gln Lys Gly Gly Gln His Tyr Ala Ile Pro His Leu Lys Lys Gln Ser
420 425 430
Leu Glu Arg Asn Gly Val Leu Ser Ile Arg Leu Gln Cys Asp Leu Val
435 440 445
Val Val Asn Glu Ala Ile Val Tyr Ile Asn
450 455
<210> 4
<211> 830
<212> DNA
<213> Phyllostachys pubescens (Phyllostachys pubescens)
<400> 4
tactccctcc gtcccagtat aacgggcgta taaaaaaatt tctgctgtcc cacaatacag 60
ggcgtccctt caattttgca ccgctttctc ccattttgcc ctcagcaatc tgcatgcatg 120
catgcatgcg tgacgcttag agttctgctt aaccgcgtga tgcattcaac tgctctgaga 180
agtgccagcg tgatgcatgc cactgcttag aaaagtgcga ggatccggtg acacgaccat 240
catagtcctc aatctcttcc caatggatag gttgatttag gtccaaattc gccattgatt 300
tcgagcaagc aactggacga gagaagagga gagagtggca gcagacctgt tgtgttcatg 360
tctatttata ggaggaaaac gaatttgaat ttctgggggc aatcttctca atccatatgc 420
actagctcaa tttctaggcg ccatcttcta aatgctgtgt tcaaattttc gctataatcc 480
gctatagact tctaaatgct gggcgccatc ttctaaatgc atgcgtgcga gcgagtacga 540
gcctacgagc gcgcgaatct gcatgcatcg ctcaaatctg catgccatgc ttctctcgcg 600
tgtgaatctg catgccatgc ttctctcgcg cacgaatctg catgctgtgc tttaaatctg 660
catgccgtgc tttaaatctg catgcatgca tggtcattga ttatagcaaa ctgcacgcat 720
gcatctcatc aatttaatta ggggtagatg aggaagaatc gctagctcgc gcgccctcct 780
tggtctctga aattttttta tacgcccgtt atactgggac ggagggagta 830
Claims (4)
1. A composition with high activityPpmar2NATransposonThe G25A-C27A mutant is characterized in that the amino acid sequence of the transposase I441A mutant is shown as SEQ ID NO. 1.
2. A recombinant plasmid carrying the code of claim 1Ppmar2NASequences of the transposon G25A-C27A mutant.
3. An engineered strain carrying the recombinant plasmid of claim 3.
4. The composition of claim 1 having high activityPpmar2NAUse of transposon G25A-C27A mutant in the construction of yeast mutants.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103602654A (en) * | 2013-11-20 | 2014-02-26 | 浙江农林大学 | Artificially modified high-activity Mariner-Like transposase |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103602654A (en) * | 2013-11-20 | 2014-02-26 | 浙江农林大学 | Artificially modified high-activity Mariner-Like transposase |
Non-Patent Citations (2)
| Title |
|---|
| MUTHUSAMY RAMAKRISHNAN等: "Affinities of Terminal Inverted Repeats to DNA Binding Domain of Transposase Affect the Transposition Activity of Bamboo Ppmar2 Mariner-Like Element", INT. J. MOL. SCI., vol. 20, pages 1 - 16 * |
| MUTHUSAMY RAMAKRISHNAN等: "Nuclear export signal (NES) of transposases affects the transposition activity of marinerlike elements Ppmar1 and Ppmar2 of moso bamboo", MOBILE DNA, vol. 10, pages 1 - 12 * |
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