CN103627684A

CN103627684A - Manually optimized high-activity Mariner-Like transposase

Info

Publication number: CN103627684A
Application number: CN201310589076.8A
Authority: CN
Inventors: 周明兵; 汤定钦; 郑丽娜
Original assignee: Zhejiang A&F University ZAFU
Current assignee: Zhejiang A&F University ZAFU
Priority date: 2013-11-20
Filing date: 2013-11-20
Publication date: 2014-03-12
Anticipated expiration: 2033-11-20
Also published as: CN103627684B

Abstract

The invention discloses a Mariner-Like transposase. The transposase is a plant MLE (Mariner-Like Enzyme) transposase with high activity obtained by the active transposase cloned from moso bamboo or the active transposase obtained after moso bamboo is subjected to molecular optimization, thereby laying a foundation for developing genetic labels by using the MLE transposase and providing powerful guarantee for large-scale separating and marking gene functions in the post-genome era.

Description

Artificial optimization's high reactivity Mariner-Like transposase

Technical field

The invention belongs to technical field of molecular biology, in particular to several high reactivity Mariner-Like transposases.

Background technology

Transposon (transposon) refers on genome can transfer to from a site section of DNA sequence in another site.Since the forties in 20th century, first American Genetic man McClintock found transposon (Ac/Ds) in corn, scientists has been found polytype transposon, and they are extensively present in bacterium, yeast and high animals and plants.Along with deepening constantly that people are familiar with transposon structure and function on molecular level, some transposons have been transformed into gene label and have been applied to genetic analysis, and become gradually one of important means of extensive separating plant gene.

Mariner-Like transposon (Mariner-LikeElements, MLE) being an important family in transposon, is to find when a unstable mutation of research Mauritanian fruit bat (Drosophila mauristiana) supercilious look gene the earliest.After this in other animals and Plant Genome, also found the existence of a large amount of MLE transposons.Compare with other transposon, that MLE transposon has is simple in structure, allos swivel base rate is high, in genome insertion point, approach the features such as random, makes it at exploitation gene label, and isolated genes, on research gene function, is far superior to other transposons.

MLE transposon is by two ends inverted repeats (Terminal Inverted Repeats, TIRs) and coding transposase genomic constitution, transposase is responsible for catalysis transposon swivel base, so the activity of transposase is the principal element that affects the swivel base frequency of transposon.That MLE transposon has is simple in structure, allos swivel base rate is high, in genome insertion point, approach the feature such as random makes it at exploitation gene label, and isolated genes, on research gene function, is far superior to other transposons.Yet the MLE transposase of nature separation is because " vertical inactivation " effect has during evolution accumulated sudden change more or less, partly or entirely lost catalysis swivel base ability, become low activity or inactive transposase, had a strong impact on the application of MLE transposon, therefore artificial constructed highly active transposase just seems very important.

Summary of the invention

The object of this invention is to provide several high reactivity Mariner-Like transposases, in order to realize object of the present invention, intend adopting following technical scheme:

One aspect of the present invention relates to a kind of Mariner-Like transposase, the aminoacid sequence that it is characterized in that described Mariner-Like transposase is SEQ ID NO.3 or the resulting sequence of SEQ ID NO.3 rite-directed mutagenesis, and described rite-directed mutagenesis refers to N58D, V85I, S107A, N131G, V135D, N138S, R143N, S144C, A148K, L151I, E168R, R228I, Y242N, C263V and/or R271K, be preferably N131G, S144C, E168R, the rite-directed mutagenesis aminoacid sequence of R228I or Y242N.

In a preferred embodiment of the present invention, the aminoacid sequence of described Mariner-Like transposase be the represented aminoacid sequence of SEQ ID NO.5 at the aminoacid sequence of the rite-directed mutagenesis of 242 Y242N, and the represented aminoacid sequence of SEQ ID NO.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 corresponding Nucleotide of 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 characterized in that described Mariner-Like transposon comprises above-mentioned nucleotide sequence.

In one aspect of the invention, the gene order of described Mariner-Like transposon is SEQ ID NO.1.

The present invention carries out by the high reactivity transposase being cloned in mao bamboon or to it plant MLE transposase that molecule optimization obtains greater activity afterwards, for utilizing MLE transposon exploitation gene label to lay a good foundation, for extensive separated and marker gene function of genome times afterwards comprehensively provides powerful guarantee.

Accompanying drawing explanation

The structure schema of Fig. 1 MLE transposase expression vector: MLE transposase is inserted in expression vector pAG413-gal-ccdB between Not I and two restriction enzyme sites of EcoR V, obtains recombinant plasmid pAG413-gal-Tpase(Tpase and represents transposase).MLE transposase is controlled by GAL 1 promotor.On carrier, contain antibiotic-screening marker gene ammonia benzyl mycin (Ampicillin) and histidine selection markers gene simultaneously;

The structure schema of the non-autonomous transposon donor of Fig. 2 MLE carrier: the nonautonomy transposon of Ppmar1 is inserted on the Hpa I restriction enzyme site of pWL89a carrier, obtains non-autonomous transposon donor carrier pWL89a-Tn(Tn and represents nonautonomy transposon).On carrier, contain antibiotic-screening marker gene ammonia benzyl mycin (Ampicillin) and urea (Ura) selection markers gene simultaneously, by corresponding promotor, started respectively.On carrier, also contain VITAMIN B4 (ADE2) marker gene, nonautonomy transposon is just inserted on the Hpa I restriction enzyme site of ADE gene inside and makes this gene inactivation, if not autonomy transposon swivel base leaves, can make ADE2 gene recover to express.

Embodiment

Below in conjunction with concrete implementation step, further set forth the present invention.Should be understood that these examples are only not used in and limit the scope of the invention for the present invention is described.Unless otherwise indicated, the experimental technique of unreceipted actual conditions in following example, conventionally according to normal condition, condition described in the chief editors' such as Sambrook. < < molecular cloning experiment guide > >, or operate 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 (Phyllostachys pubescens, be collected in Zhejiang A & F University Botanical gardens, north latitude N30 ° 15 ' 14.67 " east longitude E119 ° 43 ' 33.47 ") be material, adopt CTAB method to extract mao bamboon genomic dna, according to MLE transposon TIR conserved sequence, design primer Ppmar1-5-3(sequence details in Table 1), the total length Ppmar1(Ppmar1 sequence that amplification obtains a MLE transposon in mao bamboon genome is shown in SEQ.NO.1), be connected to pGEM-T Vector(Promega company) cloning and sequencing.Intron and exon through NetGene2 and GenScan bioinformatics software prediction transposase, by exon splicing, obtain rejecting the transposase encoding sequence of intron.Ppmar1 transposase nucleotide sequence and corresponding aminoacid sequence are shown in respectively SEQ.NO.2 and SEQ.NO.3.

Most of sequence by the pGEM-T carrier that contains mao bamboon Ppmar1 full length sequence with transposase in BseRI excision Ppmar1, then carrier is obtained after connecting to 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

With the primer (PpTpase1-5 and PpTpase1-3, sequence details are in Table 1) that restriction enzyme site the is modified spliced Ppmar1 transposase that increases.By Ppmar1 transposase amplified fragments and pAG3-gal-ccdB carrier after Not I and EcoR V double digestion, transposase enzyme is cut to product to be connected with pAG413-gal-ccdB carrier large fragment, be that transposase is replaced the ccdB in pAG413-gal-ccdB carrier, obtain recombinant vectors pAG413-gal-Tpase(Tpase and represent transposase), schema is shown in Fig. 1.This carrier has His (Histidine) selection markers, and the host who makes to import pAG413-gal-Tpase carrier can lack on the disappearance substratum of His and grows.

The structure of the non-autonomous transposon donor of 2.MLE carrier

Take pGEM-Ppmar1-Tn as template, utilize the nonautonomy transposon of Ppmar1-5-3 primer amplification Ppmar2, carrier pWL89a is cut to (restriction enzyme site is positioned at ADE2 gene) with HpaI enzyme simultaneously, reclaim carrier framework.Then use In-Fusion Advantage PCR CloningKit(TaKaRa company, Japan) nonautonomy transposon is inserted in the ADE2 gene of carrier pWL89a, cause reporter gene ADE2 to insert inactivation, obtain pWL89a-Tn(Tn and represent nonautonomy transposon), vector construction flow process is asked for an interview Fig. 2.If transposon generation swivel base leaves from ADE2 gene, ADE2 gene reading frame is replied so.This carrier has URA3 selection markers, and making to import pWL89a-Tn host can grow on the disappearance substratum that lacks Ura (urea).

Three, cotransformation yeast and induction swivel base

By pAG413-gal-Tpase recombinant plasmid and pWL89a-Tn recombinant plasmid, by PEG/LiAc method, be jointly transformed in yeast, with selecting to cultivate on the two scarce solid mediums of His/Ura.With semi-lactosi induction transposase, express, impel non-autonomous transposon generation swivel base.

Four, rite-directed mutagenesis MLE transposase

According to Ppmar1 transposase and other plant MLE transposase, carry out sequence analysis, the amino acid of choosing transposase critical sites suddenlys change.Ppmar1 transposase has been determined 15 amino acid mutation sites, they respectively: N58D, V85I, S107A, N131G, V135D, N138S, R143N, S144C, A148K, L151I, E168R, R228I, Y242N, C263V, R271K.

According to QuikChange ^tMsite-Directed Mutagenesis Kit(Stratagene company, the U.S.) test kit specification sheets, design rite-directed mutagenesis primer (sequence details are in Table 1), take pAG413-gal-Tpase as template, utilizes PfuTurbo ^tMdNA polymerase is synthetic DNA again.Then the Dpn I restriction enzyme that adds 2 μ L in synthetic product reacts 5min under 37 ℃ of conditions, and primary template sequence is thoroughly degraded.Synthetic product after enzyme is cut transforms bacillus coli DH 5 alpha, and transformant is carried out to bacterium colony PCR checking, and extracts plasmid order-checking, standby to the plasmid preservation of checking order correct.

Five, the screening of the detection of transposase activity and high reactivity mao bamboon MLE transposon

Yeast through inducing culture is cultivated at the enterprising row filter of disappearance His/Ura/Ade solid medium, calculates the yeast bacterial plaque growing on substratum.If swivel base occurs, the ADE2 gene that yeast carries on plasmid pWL89a-Tn just can be expressed, therefore positive yeast strains can be grown on the substratum that lacks VITAMIN B4, just can calculate the swivel base frequency of MLE transposon by formula (swivel base frequency=positive colony number/total count).The wild-type MLE transposase of take is contrast, and the yeast colony number of each transposase mutant strain relatively filters out the transposase mutant strain 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 transposase that wherein Y242N suddenlys change, the highest 2.2 times (table 2) bringing up to wild-type of its transposition activity, V85I and two mutational sites of N138S are combined, transposase brings up to original 106% in catalysis swivel base ability, Y242N and two mutational sites of C263V are combined, and transposase is brought up to 144% original activity in catalysis swivel base ability.The engineered transposase of these high reactivities will be 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 is not limited to this, and any variation of expecting without creative work or replacement, within all should being encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claims were limited.

Claims

1. a Mariner-Like transposase, is characterized in that the aminoacid sequence of described Mariner-Like transposase is SEQ ID NO.3 or the resulting sequence of SEQ ID NO.3 rite-directed mutagenesis, and described rite-directed mutagenesis refers to N58D, V85I, S107A, N131G, V135D, N138S, R143N, S144C, A148K, L151I, E168R, R228I, Y242N, C263V and/or R271K, be preferably N131G, S144C, E168R, the rite-directed mutagenesis aminoacid sequence of R228I or Y242N.

2. Mariner-Like transposase according to claim 1, the aminoacid sequence of described Mariner-Like transposase is the represented aminoacid sequence of SEQ ID NO.3 at the aminoacid sequence of the rite-directed mutagenesis of 242 Y242N or at the aminoacid sequence of the rite-directed mutagenesis of 228 R228I, and its concrete sequence is SEQ ID NO.5 or SEQ ID NO.6.

3. the corresponding Nucleotide of Mariner-Like transposase described in claim 1 or 2.

4. a Mariner-Like transposon, is characterized in that described Mariner-Like transposon comprises nucleotide sequence claimed in claim 3.

5.Mariner-Like transposon, its gene order is SEQIDNO.1.