CN103710342A - Artificially induced MLE (Mariner-Like Element) transposon kit and application thereof - Google Patents

Abstract

The invention discloses an artificially induced MLE (Mariner-Like Element) transposon transposition kit and application thereof in plant gene mutation. The transposase of the the MLE transposon is regulated by an inducible promoter, and the expression of the transposase is artificially regulated to control the jumping and rejumping of the transposon; and thus, the problem of mutation stability is solved, the reverse mutation becomes controllable, and purposeful induced transpositional mutation can be performed on different tissues and organs of plants in any plant growth and development period, thereby implementing overall regulation on mutagenesis in the aspects of space-time quantities. Besides, the green fluorescence protein gene is utilized to report the transposition conditions of the MLE transposon so as to simply and effectively screen the plant with variable transposition and track the transposon insertion site.

Description

Artificial induction's MLE transposon test kit and application thereof

Technical field

The invention belongs to biology field, in particular to a kind of artificial induction's Mariner-Like Element (MLE) transposon test kit and the application in plant gene mutation thereof.

Background technology

Development along with new technologies, the method of Analysis and Identification gene function is more and more, broad scale research gene function mainly depends on structure mutant library, but directly effective means is to build saturated gene mutants population, by Analysis of Mutants identified gene function.Therefore the structure of mutagenized populations is the basis of functional genomics.Constantly perfect along with agriculture bacillus mediated plant transgenic technology, the method for setting up T-DNA mutant library by creating a large amount of T-DNA independence transformants has become the main method in rapid build insertion mutation body storehouse.T-DNA insertion mutation have insert stable, simple to operate, but easily produce directly series connection, inverted repeat and produce disappearance on border, impact analysis of molecules subsequently.

Transposon refers on genome can transfer to the section of DNA sequence another item chromosome to another position or from item chromosome from the chromosomal position transfer of same, have widely distributed, copy number is high, insertion point is single-minded, produce sudden change stable, be not subject to the advantages such as the impact of floristics difference, allos swivel base rate are high, tissue culture induction activation.Can be in the situation that do not understand biochemical property and the expression pattern of gene product, separating clone plant gene, i.e. transposon tagging (transposontagging).Its principle is to utilize the insertion of plant transposon to cause transgenation, take transposon sequence as basis, from the gene library of mutant strain, filter out the clone with this transposon, it must contain the partial sequence of the mutator gene adjacent with transposon sequence, recycles this part sequence and from wild type gene group, obtains complete gene.

With transposon, make label isolated genes and have following advantage: the sudden change meeting that the insertion of (1) transposon causes is because cutting off of transposon replied.(2), because transposon is always along its contiguous site swivel base, therefore, after as long as transposon locate on karyomit(e), be just easy to the relative position of definite mutator gene chain with it.(3) due to the continuous jump of transposon, once transposon is imported to target plant, by selfing, the means such as hybridize, backcross just can obtain a larger transposon colony containing different insertion points.(4) transposon is imported to the restriction that allos plant is not subject to conversion condition.Utilize at present transposon tagging success clone plant gene to only limit at present Ac/Ds and this two classes transposon of Spm/dSpm, it is not high that this two classes transposon all exists transposition activity, undesirable and the preferential swivel base of mutation efficiency arrives the problem of phase adjacent locations with it, is to utilize at present transposon to build random saturation mutant group's major obstacle.Therefore further studying, excavate new transposon, improve test kit, reach optimum label condition, is the main task of current transposon tagging research.

Mariner-Like transposon (Mariner-LikeElements, MLE) being an important family in DNA transposable element, is to find when a unstable mutation of research Mauritanian fruit bat (Drosophilamauristiana) supercilious look gene the earliest.After this in other animals and Plant Genome, also found the existence of a large amount of MLE transposons.MLE transposon is realized swivel base by excision/pasting mechanism of DNA.Compare with other transposon, MLE transposon has following three distinguishing features: (1) is simple in structure, by the genomic constitution of two ends inverted repeats (TerminalInvertedRepeats, TIRs) and coding transposase, inverted repeats length is generally 10-40bp.Transposase encoding sequence length is generally 1000-1500bp, DNA, in conjunction with territory and swivel base catalytic domain, consists of.Can catalysis be as short as 200bp, the DNA fragmentation swivel base that only comprises TIRs and flanking sequence, also can catalysis carry goal gene (as resistant gene), grow to the swivel base of 6000bpDNA fragment, therefore MLE transposon both can be developed to the sudden change instrument of Mutagenesis, also can be developed to the transgenosis instrument of transporter gene.(2) MLE transposon approaches random in genome insertion point, target sequence is TA, statistics shows, MLE transposon 80% insertion point is positioned at gene 5, and ' downstream and the gene coded sequence of end UTR, this feature makes MLE transposon at the transposon that is applied to more be superior on gene label other types.(3) allos swivel base rate is high, overwhelming majority MLE transposon swivel base does not rely on host's factor, as long as have active transposase and the TIRs that can be identified by transposase to exist, just can realize swivel base, therefore from host clone's transposon, import nonhost postgenome, still can keep transposition activity.Therefore develop the efficient test kit based on superactivity MLE transposon, by for the extensive plant mutant storehouse that builds provides new instrument with research gene function, make the separation of plant gene and identify more simple.Inquiry previous patent, not yet has the report that utilizes plant MLE transposon to build transposon test kit.

Summary of the invention

Utilize at present transposon tagging success clone plant gene to only limit at present Ac/Ds and this two classes transposon of Spm/dSpm, it is not high that this two classes transposon all exists transposition activity, undesirable and the preferential swivel base of mutation efficiency arrives the problem of phase adjacent locations with it, is to utilize at present transposon to build random saturation mutant group's major obstacle.Therefore further studying, excavate new transposon, improve test kit, reach optimum label condition, is the main task of current transposon tagging research.

One aspect of the present invention relates to a kind of MLE transposon test kit, it is characterized in that comprising contain nucleotides sequence classify as SEQIDNO.2 miniPpMLE21 carrier and contain the carrier that nucleotides sequence is classified the transposase sequence of SEQIDNO.3 as.

In a preferred embodiment of the present invention, wherein containing nucleotides sequence, to classify the carrier of the miniPpMLE21 of SEQIDNO.2 as be that miniPpMLE21 transposon is building up on pBINm-gfp5-ER carrier, obtains transposon donor carrier pBINm-gfp5-ER-miniPpMLE21.

In another preferred embodiment of the present invention, described contain nucleotides sequence to classify the carrier of the transposase sequence of SEQIDNO.3 as be that transposase sequence construct is to pCAMBIA1301 carrier.

The present invention also relates to the application of above-mentioned MLE transposon test kit in mutagenesis transgenic plant on the other hand.

In a preferred implementation, described application comprises the steps:

(1) miniPpMLE21 transposon is building up on pBINm-gfp5-ER carrier, obtains transposon donor carrier pBINm-gfp5-ER-miniPpMLE21, arabidopsis thaliana transformation, screens positive plant by kantlex;

(2) transposase sequence (SEQIDNO.3) is building up to pCAMBIA1301 carrier, obtains transposase expression vector pCAMBIA1301-Tp, transforms the Arabidopis thaliana that carries PpMLE21 transposon, by kantlex and Totomycin, jointly screens positive plant;

(3) by improving the temperature of culture environment, induction transposase is expressed and swivel base occurs.

MLE transposon of the present invention, transposase is regulated and controled by inducible promoters, the artificial swivel base expression of enzymes that regulates, controlling transposon jumps and jumps, solved sudden change stability problem on the one hand, reverse mutation also becomes controlled, can be in any period of growth and development of plants, the different tissues organ of plant is carried out to autotelic induction swivel base sudden change, really realized to mutagenesis the comprehensive regulation and control in space-time amount.In addition, utilize green fluorescence protein gene to report the swivel base situation of MLE transposon, can be easy, the plant that effectively swivel base occurred be screened and transposon insertion site is followed the tracks of.

Accompanying drawing explanation

Fig. 1: green fluorescence indication swivel base occurs, a wherein, b, c is that different transgenic line green fluorescences are observed.WT is wild-type.

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 mao bamboon MLE transposon

According to MLE transposon two ends inverted repeats, utilize primer MLE21 (primer details are in Table 1), from mao bamboon genome amplification, go out the full length sequence of transposon PpMLE21, sequence is SEQIDNO.11.

Two, transformation of Arabidopsis thaliana vector construction

1. the structure of transposon donor carrier pBINm-gfp5-ER-miniPpMLE21

(1) by the sequence of BseRI(identification, be GAGGAG (N) 10) most of sequence of transposase in excision mao bamboon PpMLE21 transposon full length sequence, with ligase enzyme, the sequence of PpMLE21 transposon both sides is connected again again, called after miniPpMLE21, (sequence is SEQIDNO.2);

(2) XbaI enzyme cutting pBINm-gfp5-ER carrier, by the linearizing of pBINm-gfp5-ER carrier;

(3) by in-fusion technology, miniPpMLE21 transposon is inserted into the XbaI enzyme cutting site of pBINm-gfp5-ER carrier (being obtained commercially), obtains recombinant vectors pBINm-gfp5-ER-miniPpMLE21.When miniPpMLE21 transposon swivel base leaves, green fluorescence protein gene below can normal expression, can be by the whether convenient swivel base situation of analyzing of Green Fluorescent Protein Gene Expression.

2. the structure of transposase expression vector pCAMBIA1301-hspTp

(1) the genetic expression unit on PART7 carrier is scaled off from carrier by sacI and PstI; Utilize sacI and PstI by pCAMBIA1301 carrier (being obtained commercially) linearizing simultaneously; By ligase enzyme, the genetic expression unit that derives from PART7 carrier is inserted between pCAMBIA1301 carrier sacI and two restriction enzyme sites of PstI and obtains recombinant vectors pCAMBIA1301-ex;

(2) utilize primer MLE21-TP-5 and MLE21-TP-3, by round pcr, Sma I and BamH I restriction enzyme site are added in PpMLE21 transposase sequence (sequence is SEQIDNO.3) two ends, after pcr amplification, with Sma I and BamH I double digestion PCR product, utilize SmaI and BamHI enzyme to cut pCAMBIA1301-ex carrier simultaneously, by ligase enzyme, the cDNA sequence of PpMLE21 transposase is introduced between pCAMBIA1301-ex carrier S maI and two restriction enzyme sites of BamHI, obtained recombinant vectors pCAMBIA1301-Tp;

(3) by round pcr by soybean heat-inducible promoter--Gmhsp17.5C promotor (sequence SEQIDNO.4) is from the genome amplification of soybean out (primer is Gmhsp-5 and Gmhsp-3), and introduce sacI and two restriction enzyme sites of KpnI, after sequence verification, by Gmhsp17.5C promoter sequence and pCAMBIA1301-Tp carrier by sacI and KpnI double digestion, by Gmhsp17.5C promotor, replace the 35S promoter before pCAMBIA1301-Tp carrier transposase, obtain recombinant vectors pCAMBIA1301-hspTp.The expression of transposase can regulate and control by envrionment temperature.

Three, the conversion of Arabidopis thaliana

The Arabidopis thaliana seed 6-8min that sterilizes in 8%NaClO ethanolic soln, with absolute ethanol washing seed to be sowed more than 5 times, with blotting dehydrated alcohol without cotton paper, on Bechtop, dry up, seed is evenly sowed on 1/2MS substratum, and 4 ℃ of depositions 3 days, are then placed under Arabidopis thaliana cultivating chamber light (the cultivating chamber condition: 23 ℃ of temperature of normally cultivating, humidity 60-70%, light intensity 150umols-1m-2).After one week, grow 1 pair of true leaf, Arabidopsis thaliana Seedlings is moved to and in matrix, cultivates 3 weeks left and right boltings and bloom, remove main a kind of sedge, after about one week, the lotus throne leaf side shoot of extraction is bloomed in a large number, cuts the fruit pod of having born pods, and prepares to transform.

Getting 5mL Agrobacterium (EHA105) adds 100mL to contain 50 μ g/mL Rifampin (Rif)+50 μ g/mL cards to receive penicillin (Kan) liquid YEP(formula: yeast extract, 10g/L; Peptone, 10g/L; NaCl, 5g/L) in, 28 ℃, 180rpm, shake bacterium and spend the night and make its OD600 reach 1.0-1.2,4000rpm, centrifugal 15min, abandons supernatant, add 100mL soaking method substratum (1/2MS+5% sucrose), and add tensio-active agent Silwet-77, make its final concentration reach 0.02%.Resuspended Agrobacterium bacterium liquid is placed in to culture dish, Arabidopis thaliana inflorescence is immersed in solution completely, keep 30s-60s.Use preservative film moisturizing, in all switch cabinets, add water, cultivate after 24h in darkroom, under light, normally cultivates.Every about 5 days, can again contaminate contaminating plant, improve transformation efficiency.After seed maturity, suitably reduce irrigation times, when kind of a pod jaundice is ftractureed, gather in the crops seed, for follow-on screening.

Four, transgenic positive plant screening and identification

The Arabidopis thaliana seed of importing transposon donor carrier pBINm-gfp5-ER-miniPpMLE21 is sowed containing in the 1/2MS substratum of 50mg/L kantlex and is screened, resistant plant is verified (primer is MLE21) by PCR, can amplify the positive plant of object band of 1.2kb left and right

The above-mentioned positive Arabidopis thaliana plant of take is acceptor, continue to transform transposase expression vector pCAMBIA1301-hspTp, the Arabidopis thaliana seed of results is sowed containing in the 1/2MS substratum of 50mg/L kantlex+50mg/L Totomycin and screened, transgenosis resistant plant is verified (primer is MLETP21-5 and MLE21TP-3) by PCR, can amplify the positive plant of object band of 1.5kb left and right, the two positive plants of this positive transfer-gen plant called after.

Five, the evaluation of swivel base induction and swivel base event

(1) swivel base of two positive plants induction

The seedling of twice pair of positive plant of 42 ℃ of continuous heat shocks, can induce the highest transposition activity in each 2 hours.Then be placed on 21 ℃ of renewal cultivations, increase the humidity of culture environment simultaneously, two days later at fluorescence microscopy Microscopic observation fluorescence.

(2) green fluorescence is observed

Get the blade that Arabidopis thaliana is fresh, be torn into 0.1-1.0mm thin slice by hand, the optical source wavelength of fluorescent microscope is transferred to 477nm place, observe the position of green fluorescence.There is the cell of green fluorescence point to show to have swivel base to occur.As can be seen from Figure 1 compare with wild-type, after high temperature induction, can see the cell (part of green fluorescence) of a large amount of generation swivel bases.This explanation, the Mariner-LikeElement of structure (MLE) transposon test kit can be at swivel base under artificial induction, and keeps higher swivel base efficiency.

Table 1 primer table

The above is the preferred embodiments of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of principle of the present invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (5)

1. a MLE transposon test kit, it is characterized in that comprising contain nucleotides sequence classify as SEQIDNO.2 miniPpMLE21 carrier and contain the carrier that nucleotides sequence is classified the transposase sequence of SEQIDNO.3 as.

2. test kit according to claim 1, wherein containing nucleotides sequence, to classify the carrier of the miniPpMLE21 of SEQIDNO.2 as be that miniPpMLE21 transposon is building up on pBINm-gfp5-ER carrier, obtains transposon donor carrier pBINm-gfp5-ER-miniPpMLE21.

3. test kit according to claim 1, described contain nucleotides sequence to classify the carrier of the transposase sequence of SEQIDNO.3 as is that transposase sequence construct is to pCAMBIA1301 carrier.

4. the application of above-mentioned MLE transposon test kit in mutagenesis transgenic plant.

5. application according to claim 1, described application comprises the steps:

(1) miniPpMLE21 transposon is building up on pBINm-gfp5-ER carrier, obtains transposon donor carrier pBINm-gfp5-ER-miniPpMLE21, arabidopsis thaliana transformation, screens positive plant by kantlex;

(2) transposase sequence (SEQIDNO.3) is building up to pCAMBIA1301 carrier, obtains transposase expression vector pCAMBIA1301-Tp, transforms the Arabidopis thaliana that carries PpMLE21 transposon, by kantlex and Totomycin, jointly screens positive plant;

(3) by improving the temperature of culture environment, induction transposase is expressed and swivel base occurs.

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