CN103627684B - The high activity Mariner-Like transposase of artificial optimization - Google Patents
The high activity Mariner-Like transposase of artificial optimization Download PDFInfo
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Abstract
The invention discloses a kind of Mariner-Like transposase, the present invention obtains the plant MLE transposase of greater activity after carrying out molecule optimization by the active transposase being cloned in Phyllostachys pubescens Mazei ex H.de Lebaie or to it, for utilizing MLE transposon exploitation gene label to lay a good foundation, separate on a large scale for the genome times afterwards comprehensively and provide powerful guarantee with marker gene function.
Description
Technical field
The invention belongs to technical field of molecular biology, in particular to several high activity Mariner-Like transposases.
Background technology
Transposon (transposon) refers to the section of DNA sequence that can transfer to another site on genome from a site.From the forties in 20th century American Genetic man McClintock in Semen Maydis, first found that since transposon (Ac/Ds), scientists is found that polytype transposon, and they are widely present in antibacterial, yeast and high animals and plants.Along with people's deepening constantly transposon 26S Proteasome Structure and Function understanding on a molecular scale, some transposon have been transformed into gene label and have been applied to gene analysis, and one of are increasingly becoming the important means of extensive separating plant gene.
Mariner-Like transposon (Mariner-LikeElements, MLE) it is an important family in transposon, is find when a unstable mutation of research Mauritania fruit bat (Drosophilamauristiana) supercilious look gene the earliest.Hereafter in other animals and Plant Genome, have also discovered the existence of a large amount of MLE transposon.Compared with other transposon, MLE transposon have simple in construction, allos swivel base rate high, at genomic insertion site close to the feature such as random so that it is at exploitation gene label, isolated genes, on research gene function, be far superior to other transposon.
MLE transposon is by two ends inverted repeat (TerminalInvertedRepeats, and the genomic constitution of encoding transposase TIRs), transposase is responsible for catalysis transposon, and therefore the activity of transposase is the principal element of the transposition frequencies affecting transposon.MLE transposon has that simple in construction, allos swivel base rate are high, make it at exploitation gene label, isolated genes at genomic insertion site close to the feature such as random, on research gene function, is far superior to other transposon.But the MLE transposase that nature separates have accumulated sudden change more or less owing to " vertically inactivating " effect during evolution, partly or entirely lose catalysis swivel base ability, become low activity or inactive transposase, having had a strong impact on the application of MLE transposon, therefore artificial constructed highly active transposase just seems particularly significant.
Summary of the invention
It is an object of the invention to provide several high activity Mariner-Like transposase, in order to realize the purpose of the present invention, intend adopting the following technical scheme that
One aspect of the present invention relates to a kind of Mariner-Like transposase, it is characterised in that the aminoacid sequence of described Mariner-Like transposase is the sequence that SEQIDNO.3 or SEQIDNO.3 rite-directed mutagenesis is obtained, and described rite-directed mutagenesis refers to N58D, V85I, S107A, N131G, V135D, N138S, R143N, S144C, A148K, L151I, E168R, R228I, Y242N, C263V and/or R271K, it is preferred to N131G, the rite-directed mutagenesis aminoacid sequence of S144C, E168R, R228I or Y242N.
In a preferred embodiment of the present invention, the aminoacid sequence of described Mariner-Like transposase is the aminoacid sequence at the rite-directed mutagenesis of 242 Y242N of the aminoacid sequence represented by SEQIDNO.5, and the aminoacid sequence represented by SEQIDNO.6 is at the aminoacid sequence of the rite-directed mutagenesis of 228 R228I.
In another aspect of this invention, the invention still further relates to the nucleotide corresponding to above-mentioned Mariner-Like transposase.
In another aspect of this invention, the invention still further relates to a kind of Mariner-Like transposon, it is characterised in that described Mariner-Like transposon includes above-mentioned nucleotide sequence.
In one aspect of the invention, the gene order of described Mariner-Like transposon is SEQIDNO.1.
The present invention obtains the plant MLE transposase of greater activity after carrying out molecule optimization by the high activity transposase being cloned in Phyllostachys pubescens Mazei ex H.de Lebaie or to it, for utilizing MLE transposon exploitation gene label to lay a good foundation, separate on a large scale for the genome times afterwards comprehensively and provide powerful guarantee with marker gene function.
Accompanying drawing explanation
The structure flow chart of Fig. 1 MLE transposase expression vector: MLE transposase is inserted in expression vector pAG413-gal-ccdB between two restriction enzyme sites of NotI and EcoRV, obtains recombiant plasmid pAG413-gal-Tpase(Tpase and represents transposase).MLE transposase is controlled by GAL1 promoter.Carrier contains antibiotic-screening marker gene ammonia benzyl mycin (Ampicillin) and histidine riddled basins simultaneously；
The structure flow chart of the non-autonomous transposon donor vehicle of Fig. 2 MLE: the nonautonomy transposon of Ppmar1 is inserted on the HpaI restriction enzyme site of pWL89a carrier, obtains non-autonomous transposon donor vehicle pWL89a-Tn(Tn and represents nonautonomy transposon).Carrier contains antibiotic-screening marker gene ammonia benzyl mycin (Ampicillin) and carbamide (Ura) riddled basins simultaneously, is started by corresponding promoter respectively.Possibly together with adenine (ADE2) marker gene on carrier, nonautonomy transposon is just inserted on the HpaI restriction enzyme site of ADE gene internal and makes this gene inactivation, ADE2 gene can be made to recover to express if not autonomy transposon leaves.
Detailed description of the invention
The present invention is expanded on further below in conjunction with being embodied as step.Should be understood that these examples are merely to illustrate the present invention rather than restriction the scope of the present invention.Unless otherwise indicated, the experimental technique of unreceipted actual conditions in following Examples, generally conventionally condition, condition as described in " the Molecular Cloning: A Laboratory guide " edited such as Sambrook., or be operated according to the step of test kit statement.
One, the acquisition of the nonautonomy transposon of wild type MLE transposase and removal transposase
With fresh Leaves of Bamboo Phyllostachys pubescens (Phyllostachyspubescens, it is collected in Zhejiang A & F University botanical garden, north latitude N30 ° 15 ' 14.67 " east longitude E119 ° 43 ' 33.47 ") for material, CTAB method is adopted to extract Phyllostachys pubescens Mazei ex H.de Lebaie genomic DNA, primer Ppmar1-5-3(sequence details are designed in Table 1) according to MLE transposon TIR conserved sequence, Phyllostachys pubescens Mazei ex H.de Lebaie genome expands the total length Ppmar1(Ppmar1 sequence obtaining a MLE transposon and sees SEQ.NO.1), it is connected to pGEM-TVector(Promega company) cloning and sequencing.Predict intron and the exon of transposase through NetGene2 and GenScan bioinformatics software, by exon splicing, obtain rejecting the transposase coding sequence of intron.Ppmar1 transposase nucleotide sequence and corresponding aminoacid sequence are shown in SEQ.NO.2 and SEQ.NO.3 respectively.
PGEM-T carrier BseRI containing Phyllostachys pubescens Mazei ex H.de Lebaie Ppmar1 full length sequence is excised most of sequence of transposase in Ppmar1, then carrier is obtained after connecting the nonautonomy transposon (pGEM-Ppmar1-Tn) of Ppmar1.The nonautonomy transposon sequence of Ppmar1 is shown in SEQ.NO.4.
Two, the structure of yeast swivel base expression vector
The structure of 1.MLE transposase expression vector
The primer (PpTpase1-5 and PpTpase1-3, sequence details are in Table 1) modified with restriction enzyme site expands spliced Ppmar1 transposase.By Ppmar1 transposase amplified fragments and pAG3-gal-ccdB carrier after NotI and EcoRV double digestion, transposase digestion products is connected with pAG413-gal-ccdB carrier large fragment, namely transposase replaces the ccdB in pAG413-gal-ccdB carrier, obtain recombinant vector pAG413-gal-Tpase(Tpase and represent transposase), flow chart is shown in Fig. 1.This carrier has His (histidine) selection markers, enables the host of importing pAG413-gal-Tpase carrier to lack and grows in the disappearance culture medium of His.
The structure of the non-autonomous transposon donor vehicle of 2.MLE
With pGEM-Ppmar1-Tn for template, utilize the nonautonomy transposon of Ppmar1-5-3 primer amplification Ppmar2, simultaneously by carrier pWL89a HpaI enzyme action (restriction enzyme site is positioned at ADE2 gene), reclaim carrier framework.Then In-FusionAdvantagePCRCloningKit(TaKaRa company is used, Japan) nonautonomy transposon is inserted in the ADE2 gene of carrier pWL89a, cause that reporter gene ADE2 inserts inactivation, obtain pWL89a-Tn(Tn and represent nonautonomy transposon), vector construction flow process asks for an interview Fig. 2.If transposon generation swivel base leaves from ADE2 gene, then ADE2 gene reading frame is replied.This carrier has URA3 selection markers, enables importing pWL89a-Tn host growth in the disappearance culture medium lacking Ura (carbamide).
Three, cotransformation yeast and inducing transposition
By pAG413-gal-Tpase recombiant plasmid and pWL89a-Tn recombiant plasmid, with, in PEG/LiAc method cotransformation to yeast, carrying out selecting to cultivate with on the double; two scarce solid medium of His/Ura.With galactose inducing transposition expression of enzymes, promote non-autonomous transposon generation swivel base.
Four, rite-directed mutagenesis MLE transposase
Carrying out sequence analysis according to Ppmar1 transposase and other plant MLE transposase, the aminoacid choosing transposase critical sites suddenlys change.Ppmar1 transposase determines 15 amino acid mutation sites, they respectively: N58D, V85I, S107A, N131G, V135D, N138S, R143N, S144C, A148K, L151I, E168R, R228I, Y242N, C263V, R271K.
According to QuikChangeTMSite-DirectedMutagenesisKit(Stratagene company, the U.S.) test kit description, design rite-directed mutagenesis primer (sequence details are in Table 1), with pAG413-gal-Tpase for template, utilize PfuTurboTMDNApolymerase is synthetic DNA again.Then in synthetic product, add the DpnI restricted enzyme of 2 μ L, under 37 DEG C of conditions, react 5min, primary template sequence is thoroughly degraded.Synthetic product after enzyme action being converted bacillus coli DH 5 alpha, transformant carries out bacterium colony PCR checking, and extracts plasmid order-checking, the plasmid preservation that order-checking is correct is standby.
Five, the screening of the detection of transposase activity and high activity Phyllostachys pubescens Mazei ex H.de Lebaie MLE transposon
Cultivate at the disappearance enterprising row filter of His/Ura/Ade solid medium through the yeast of inducing culture, calculate the yeast bacterial plaque grown in culture medium.If swivel base occurs, yeast carries the ADE2 gene on plasmid pWL89a-Tn and just can express, therefore positive yeast strain can grow in the culture medium lacking adenine, just can be calculated the transposition frequencies of MLE transposon by formula (transposition frequencies=positive clump count/total bacteria count).With wild type MLE transposase for comparison, relatively the yeast colony number of each transposase mutant, filters out the transposase mutant of greater activity.
Analyze in 15 mutational sites of Ppmar1 transposase, at N131G, S144C, E168R, the transposase of R228I and Y242N sudden change all has raising in various degree in catalysis swivel base ability, the wherein transposase of Y242N sudden change, the highest 2.2 times (tables 2) bringing up to wild type of its transposition activity, two mutational sites of V85I and N138S are combined, transposase brings up to original 106% in catalysis swivel base ability, being combined in two mutational sites of Y242N and C263V, transposase brings up to 144% original activity in catalysis swivel base ability.The engineered transposase of these high activities will for utilizing MLE transposon exploitation gene label to establish important foundation.
The above, be only the specific embodiment of the present invention, but protection scope of the present invention be not limited thereto, any change expected without creative work or replacement, all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with claims protection defined.
Claims (2)
1. a Mariner-Like transposase, it is characterized in that, the aminoacid sequence of described Mariner-Like transposase is the aminoacid sequence at the rite-directed mutagenesis of 242 Y242N of the aminoacid sequence represented by SEQIDNO.3, and the aminoacid sequence of this sudden change is such as shown in SEQIDNO.5.
2. the nucleotide corresponding to Mariner-Like transposase described in claim 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106701710B (en) * | 2017-01-20 | 2020-08-28 | 浙江农林大学 | Ppmar1 transposase F302Q mutant with high catalytic activity and application thereof |
| CN106754815B (en) * | 2017-01-20 | 2020-09-11 | 浙江农林大学 | Ppmar1 transposase C296I mutant with high catalytic activity and application thereof |
| CN106811447B (en) * | 2017-01-20 | 2020-09-11 | 浙江农林大学 | Ppmar1 transposase V376A mutant with high catalytic activity and application thereof |
| CN106916799B (en) * | 2017-01-20 | 2020-09-11 | 浙江农林大学 | Ppmar1 transposase D332S mutant with high catalytic activity and application thereof |
| CN106701711B (en) * | 2017-01-20 | 2020-08-28 | 浙江农林大学 | Ppmar1 transposase S171A mutant with high catalytic activity and application thereof |
| CN114958798A (en) * | 2021-11-22 | 2022-08-30 | 浙江农林大学 | High catalytic activityPpmar1Transposase L477A-P478E-L479I mutant and application thereof |
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| FR2850395B1 (en) * | 2003-01-28 | 2007-12-14 | Centre Nat Rech Scient | TRANSPOSASES OF MUTANT, NON-PHOSPHORYLABLES AND HYPERACTIVE MARINER GENETIC ELEMENTS |
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| CN1300827A (en) * | 1999-12-22 | 2001-06-27 | 上海博德基因开发有限公司 | Polypeptide-human sailor transposase 19 and polynucleotide for coding this polypeptide |
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| Distribution and polymorphism of Mariner-like elements in the Bambusoideae subfamily;Ming-Bing Zhou，et al;《Plant Syst Evol》;20100814;第289卷;1－11 * |
| Isolation and characterization of seventy-nine full-length mariner-like transposase genes in the Bambusoideae subfamily;Ming-Bing Zhou,Hao Zhong,Ding-Qin Tang;《J Plant Res》;20101217;第124卷;607–617 * |
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