CN104673825A - Plant expression vector containing encoded hpaXm protein gene and construction method of plant expression vector - Google Patents

Abstract

The invention relates to the field of gene engineering, and discloses a plant expression vector containing an encoded hpaXm protein gene and a construction method of the plant expression vector. According to the plant expression vector containing the encoded hpaXm protein gene, target genes hpaXm and hpaXm delta LP in a transformation model plant or other crops are integrated to a plant genome, and expression, location and secretion conditions of the target genes in the plants are detected with methods of immune colloidal gold and the like. The construction method is simple, easy to implement and convenient to popularize.

Description

A kind of plant expression vector and construction process thereof containing coding hpaXm protein gene

Technical field

The present invention relates to genetically engineered field, particularly a kind of containing the plant expression vector of hpaXm protein gene and the construction process of described plant expression vector of encoding.

Background technology

Harpin albumen, be a kind of produced by Gram-negative plant pathogenetic bacteria can excite HR(hypersensitive response on non-host plant) albumen that reacts, be the proteinous elicitor of a class energy induced resistance.Research over 20 years shows, harpin albumen plays multiple player in pathogenetic bacteria and plant do mutually, but current is not still fully aware of to the precise mechanism at infection process period harpin albumen and plant interaction, whether smooth the performance of and its function of harpin Protein transport process be inseparable.

In early-stage Study, contriver is from cotton angular leaf spot fungus x. citrisubsp. malvacearum(a synonym of xanthomonas campestris pv. malvacearum(Smith 1901) Dye 1978, Xcm), new harpin the proteinoid------hpaXm of first identified.HpaXm has the denominator of harpin, and is positioned hrpregion.HpaXm can excite HR, excites tobacco to the resistance of TMV.Especially, secondary structure prediction display hpaXm1-15 amino acid of N end is signal peptide similar sequence (signal peptide), and, deleted signal peptide similar sequence hpaXmcan not be secreted born of the same parents in escherichia expression system outer but HR after purifying, still can be excited to react.

In order to 1-15 amino acid before probing into N end is for hpaXm albumen making binding site mutually and secreting the impact of artificial situation in transgenic plant, and the endogenous expression of hpaXm albumen is on the impact of transgenic plant resistance, the plant expression vector that must build this goal gene just can carry out a series of probing into.

Summary of the invention

An object of the present invention is to provide a kind of plant expression vector containing coding hpaXm protein gene, has finishing operations quick and easy, is convenient to the features such as observation.

Another object of the present invention is to the construction process proposing a kind of plant expression vector, step is reasonable, is convenient to promote.

The technical solution used in the present invention is:

A plant expression vector containing coding hpaXm protein gene, comprises pB121 carrier and is connected on described pB121 carrier, the gene fragment of hpaXm albumen of can encoding.

Further, described plant expression vector comprises pBI121- hpaXmand pBI121 -hpaXm △ LP.

The construction process of described plant expression vector, comprises the following steps:

A) goal gene total length hpaXm, or the mutant of deleted signal peptide similar sequence hpaXm △ LPamplification;

B) fusion gene fragment is cloned into pMD ^tMin 18-T carrier;

C) fusion gene is transferred on pBI121 carrier.

Further, in step c, with on pBI121 carrier xmaIwith xbalas restriction enzyme site.

In order to the expression of the hpaXm △ LP from multi-angle observation hpaXm and deleted signal peptide similar sequence thereof in plant materials and location, hpaXm and the hpaXm △ LP making interpolation and do not add gfp (green fluorescent protein) compares as parallel laboratory test and verifies, obtain science and the more convictive experimental result of tool more, facilitate the implementation of subsequent experimental and detection, inventors performed further creation.

The described plant expression vector containing coding hpaXm protein gene, except comprising pBI121 carrier and being connected on described pBI121 carrier, can encode outside the gene fragment of hpaXm albumen, also comprise the gene of expressing green fluorescent protein gfp.

Further, described in gfpwith described hpaXmmutual fusion.

Further, described plant expression vector comprises pBI121- hpaXm::gfpand pBI121- hpaXm △ LP::gfp.

Describedly to contain gfpthe construction process of plant expression vector, comprise the following steps:

(1) according to goal gene hpaXm::gfp or hpaXm △ LP::gfpsequence, and carrier pBI121 sequence selection restriction enzyme site xbaIwith xmaIand design primer.

(2) fusion gene hpaXm::gfp or hpaXm △ LP::gfpamplification;

(3) be that template overlap-extension PCR obtains fusion gene fragment with PCR primer;

(4) fusion gene fragment is cloned into pMD ^tMin 18-T carrier;

(5) fusion gene is connected on pBI121 carrier.

Further, in step b, reclaim to cut glue the gene fragment that purifying has overlapping chain hpaXm/hpaXm △ LPgene and gfpgene, as template, carries out PCR overlap-extension PCR, and this process is divided into two steps, is respectively:

(2a) do not add primer in PCR reaction system to increase;

(2b) in the reaction system of the first step, add two ends primer to increase;

In described step (3), comprise following steps:

(3a) the gene fragment 3' end obtained increasing does not add A base, carries out adding A process before being connected with carrier T;

(3b) fusion gene and pMD ^tM18-T carrier connects, and is transformed in competent escherichia coli cell;

(3c) qualification of positive recombinant and order-checking.

Further, in described step (4), with on pBI121 carrier xmaIwith xbalas restriction enzyme site.

The invention has the beneficial effects as follows:

1) plant expression vector containing coding hpaXm protein gene in the present invention, by transformation mode plant or other crops by goal gene hpaXmand hpaXm △ LPbe incorporated in Plant Genome, utilize the expression of the method testing goal genes such as immune colloid gold in plant, locate and secrete artificial situation.

2) further, will encode in the present invention target protein and green fluorescent protein gfpafter the fusion gene of (Green Fluorescent Protein) is incorporated into Plant Genome, will make us can expression more intuitively from basis of microscopic observation to fusion gene and expressed target protein making binding site mutually and secreting artificial situation in vegetable cell.

3) construction process of the present invention is reasonable in design, and convenient operation is promoted.

Accompanying drawing explanation

In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.

Fig. 1 is fusion gene over-lap PCR schematic diagram;

Fig. 2: pBI121- hpaXm::gfpfor template amplification goal gene;

Fig. 3: pBI121- hpaXm △ LP::gfpfor template amplification goal gene;

Fig. 4: pBI121- hpaXm::gfpfor template amplification goal gene;

Fig. 5: pBI121- hpaXm △ LP::gfpfor template amplification goal gene.

Embodiment

Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.

According to method disclosed by the invention, proceed as follows:

Embodiment 1

Build pBI121- hpaXmplant expression vector

1. goal gene fragment xmaI- hpaXm-XbaI(420bp)amplification and the mutant of deleted signal peptide similar sequence xmaI- hpaXm △ LP-XbaI(360bp)amplification

Restriction enzyme site is chosen according to goal gene sequence and pBI121 sequence xbaIwith xmaI, purpose of design gene amplification primer.Goal gene fragment PCR Amplification is as described below.

PCR reaction system (50 μ L):

Template (plasmid) 2 μ L

Upstream primer 1 μ L

Downstream primer 1 μ L

10×Buffer 5μL

2.5mMdNTP 4μL

（Trans）Taq-T DNA polymerase 1μL

Supply ultrapure water to 50 μ L

Wherein template uses and clone carrying out from cotton angular leaf spot fungus (X.) hpaXmthe plasmid pHM1-of full length sequence hpaXm(being preserved by this laboratory), amplification hpaXmfull-length gene use primer for HpaXm-F (5'-GCTCTAGAATGAATTCTTTGAACACACAGA-3') and

HpaXm-R（5'-TCCCCCCGGGTTATTTGTAGAGCTCATCCATGCC-3'）；

Amplification hpaXm △ LPthe primer that gene uses for downstream primer be HpaXm △ LP-F(5'-GCTCTAGAATGCAGGTCGACCCGAGCCAGA-3') and

HpaXm-R（5'-TCCCCCCGGGTTATTTGTAGAGCTCATCCATGCC-3'）

PCR reaction conditions is:

94 DEG C of denaturation 4min;

94 DEG C of sex change 30sec;

58 DEG C of renaturation 30sec; 30 circulations

72 DEG C extend 1min;

Last 72 DEG C extend 10min.

The PCR primer that takes a morsel carries out agarose gel electrophoresis detection according to preceding method, and amplified fragments size is consistent with expection clip size, then the glue of remaining PCR primer macropore is carried out electrophoresis, and reclaims DNA fragmentation.Obtain according to the method described above hpaXm andfor hpaXm △ LP gene fragment agarose gel electrophoresis figure (see Fig. 1 and Fig. 2).

2. by goal gene fragment and pMD ^tM18-T carrier connects and is transformed in competent escherichia coli cell

Reaction system 10 μ L

pMD18-T simple vector 0.5μL

Control Insert 0.5μL

Cut glue and reclaim product 4 μ L

Solution Buffer 5μL

Reaction conditions 16 DEG C of connections are spent the night

(1) in micro centrifugal pipe, add the fusion gene fragment of 1uL carrier T and 4uL.

(2) mix gently, room temperature (20 DEG C-37 DEG C) reaction 15 minutes, is placed in centrifuge tube on ice.

(3) add connection product (to add when competent cell just thaws and connect product) in 50 μ L Trans1-T1 competent cells, flick mixing, ice bath 20-30 minute.

(4) 42 DEG C of heat shocks 30 seconds, are placed in 2 minutes immediately on ice.

(5) add 250 μ L balances to the LB of room temperature, 200 turns, hatch 1 hour for 37 DEG C.

(6) get 8 μ L, 500 mM IPTG, 40 μ L, 20 mg/mL X-gal mix, and are applied to ready containing on penbritin (100ug/mL) LB flat board equably, place 30 minutes at 37 DEG C.

(7) treat IPTG, after X-gal is absorbed, get 200 μ L bacterium liquid bed boards, overnight incubation (for obtaining comparatively polyclone, centrifugal 1 min of 4000 rpm, discards part supernatant, retains 100-150 μ L, flicks suspension thalline, get whole bacterium liquid coated plate, overnight incubation).

3 .the qualification of positive recombinant and order-checking

(1) select white colonies to containing 1uL liquid LB(Amp, 100ug/mL) 1.5mL centrifuge tube in, 37 DEG C of concussions are cultivated.

(2) 1uL bacterium liquid is got as template, pcr amplification of entering.

(3) PCR primer is carried out agargel electrophoresis detection, by amplified fragments size, and the assorted LB bacterium liquid liquid-transfering gun be with consistent with imagination is transferred to containing 10mL liquid LB(Amp, 100ug/mL) triangular flask in, 37 DEG C shake incubated overnight.

Get 1mL bacterium liquid and send to order-checking, and remaining bacterium liquid is preserved.

4 .goal gene is cloned on carrier pBI121

Extract the plasmid of positive transformant, use xmaIwith xbalrestriction enzyme is to the plasmid pMD carrying goal gene ^tM18- hpaXmand pMD ^tM18- hpaXm △ LPcarry out double digestion and reclaim.

4a) mini-scale plasmid extracting method is as follows:

(1) that preserves-80 DEG C is sprinkled upon LB+Amp(100 ug/mL uniformly containing the bacterium liquid of plasmid) or+Km (25 ug/mL) flat board on, cultivate 10 ~ 12 hours for 37 DEG C.

(2) bacterium colony that activated of picking, in the flat lining out dilution of LB+Km (25 ug/mL) or+Amp (100ug/mL), 37 DEG C of overnight incubation.

(3) the well-grown single bacterium colony of picking, be inoculated in the triangular flask containing LB+Km (25 ug/mL) or+Amp (100 ug/mL) liquid nutrient medium, 37 DEG C of shaking culture are spent the night.

(4) draw the LB bacterium liquid of 1mL in the centrifuge tube of 1.5mL, centrifugal one minute of 13000rpm, removes waste liquid, repeats four times.

(5) in centrifuge tube, add 250uLbufferA1, shake abundant suspended bacterial cell with liquid-transfering gun or swirling flow.

(6) add 250BufferB1, reverse 10 times lightly to mix, then leave standstill and clarify to solution thickness for 5 minutes.

(7) add 350uL BufferN1, reverse repeatedly immediately, fully mix to solution, now occur white flock precipitate.

(8) centrifuge tube is gone to supercentrifuge, at room temperature centrifugal 10 minutes of 13000rpm (if adularescent precipitation in supernatant, can recentrifuge).

(9) careful draw centrifugal after supernatant liquor to (avoiding picking up precipitation) with in the DNA post of collection tube, under room temperature, centrifugal 1 minute of 13000rpm, outwells the waste liquid in collection, is placed back in collection tube by centrifugal column.

(10) in centrifugal column, add 500uL DNA Wash Buffer, under room temperature, centrifugal 1 minute of 13000rpm, outwells the waste liquid in collection tube, is placed back in collection tube by centrifugal column.

(11) repeating step " 7 ".

(12) centrifugal column is put back in supercentrifuge, uncap under 13000rpm room temperature centrifugal 2 minutes, thoroughly to remove residual ethanol.

(13) gone to by centrifugal column in a new 1.5mL centrifuge tube, the middle to DNA post adds the ddH of 50-100uL ₂o or Eloution Buffer, room temperature places 2 minutes, centrifugal 1 minute of 13000rpm, wash-out plasmid DNA.Elutriant is joined in post again, at 13000rpm centrifugal 1 minute, collect elutriant.

)agarose gel electrophoresis is analyzed

After plasmid extraction terminates, detect concentration and the quality of institute's upgrading grain with agarose gel electrophoresis.Concrete operation step is as follows:

(1) in the triangular flask filling a certain amount of 1 × TAE, add agarose by 1%, in microwave oven, be heated to agarose melt completely.

(2) after solution is cooled to 60 DEG C, Golden view solution is added by 5 μ L/100mL solution, mixing.

(3) agarose solution of low-grade fever is poured in rubber moulding, and places comb, thickness about 3 to 5 mm of gel, check comb tooth under or between cog whether have bubble, if having bubble should manage removing.

(4), after gel solidifies completely, carefully comb is removed.Gel is put into electrophoresis chamber, adds 1 enough × TAE, make its dipped glue face 1 mm.

(5) the plasmid 5 μ L extracted is mixed with 6 × damping fluid 1 μ L, added in well with micropipet.

(6) switch on power, voltage is adjusted to 120V, electrophoresis migrates to suitable distance to tetrabromophenol sulfonphthalein in gel.

(7) cut off the electricity supply, take out gel, gel is carried out observing and photographing in ultraviolet gel imaging instrument, preserve image.

4c) plasmid enzyme restriction

During design primer, introduce at the two ends of fusion gene xbai and xmathe restriction enzyme site of I, the multiple clone site of pBI121 also has xbaIwith xmthe restriction enzyme site of aI.Therefore, with xbai He xmai as restriction enzyme site, by object fusion gene hpaXmwith hpaXm △ LPbe connected on pBI121 and be built into restructuring transgene carrier pBI121:: hpaXmwith pBI121 ∷ hpa _xm △ LP.

Endonuclease reaction system (20 μ L):

NEBuffer 4 2 uL

Plasmid DNA ≤1 μg

100×BSA 0.2uL

XmaI 0.5uL

XbaⅠ 0.5uL

ultrapure water complements to 20 μ L

Centrifugal after mixing, be placed in 37 DEG C of enzymes and cut 2 ~ 3 h.For the plasmid that demand is large, multitube can be divided to carry out amount that enzyme cut or strengthened reaction system and enzyme is carried out enzyme and is cut, and then concentrates.After enzyme cuts end, by preceding method take a morsel digestion products carry out agarose gel electrophoresis detect enzyme cut effect.If fail to cut completely, add a small amount of enzyme continuation enzyme and cut; As complete degestion, sepharose reclaims enzyme and cuts the object fragment obtained.

4d) sepharose DNA fragment reclaims

Test kit (purchased from BIOMIGA company) is reclaimed, to warp with glue xmai and xbathe pBI121 plasmid that I double digestion is crossed, and xmaI-hpaXm-XbaIwith xmaI-hpa _xm △ LP-XbaIsmall segment object electrophoretic band reclaim.Experimental procedure is carried out according to test kit specification sheets.

4f) under the effect of T4 ligase enzyme (purchased from Takara company), with same use xmaIwith xbaIthe pBI121 carrier that restriction enzyme cuts connects.Operation steps is as follows:

Be sequentially added in a new centrifuge tube:

Exogenous sequences DNA/ xbai -Xmai 5 uL

pBI121 / XbaI -XmaI 5 uL

10×Ligase buffer 2 uL

T ₄DNA ligase 1 uL

sterilizing redistilled water to 20 uL

22 DEG C are incubated 1 hour, get 5 uL reaction mixtures and be transformed into 50 μ L transin 5a competent cell (purchased from transcompany) (add when competent cell just thaws and connect product), flick mixing, ice bath 20 ~ 30 minutes.42 DEG C of heat shocks 30 seconds, are placed in 2 minutes immediately on ice.Add 250 μ L balances to the LB substratum of room temperature, 200 turns, hatch 1 hour for 37 DEG C.Get 200uL bacterium liquid and coat LB+Km(25ug/mL uniformly) on flat board.

Recombinant plasmid is imported in competent escherichia coli cell, in with the flat board of kantlex carrying out blue hickie sieve, positive transformant uses goal gene Auele Specific Primer HapXm-F (5'-GCTCTAGAATGAATTCTTTGAACACACAGA-3') respectively, HpaXm △ LP-F(5'-GCTCTAGAATGCAGGTCGACCCGAGCCAGA-3') and HpaXm-R(5'-TCCCCCCGGGTTATTTGTAGAGCTCATCCATGCC-3') amplification.After carrying out PCR checking, the positive transformant that can amplify fragment consistent with goal gene clip size send genome company to check order, sequencing result use NCBI-BLAST software with hpaXm(GenBank Accession No.DQ643828) original series is compared, filter out with hpaXmthe positive transformant of original gene 100% homology.Extract positive transformant plasmid, obtain recombinant plant expression vector pBI1121:: hpaXmand pBI1121:: hpaXm △ LP.

According to the carrier that above aspect builds, contriver carries out the detection of goal gene, and the result obtained is as figure

Fig. 1: plasmid pBI121- hpaXmfor template amplification goal gene

Fig. 2: plasmid pBI121- hpaXm △ LPfor template amplification goal gene.

Wherein, M: DL20000Marker using empty plasmid pBI121 for template is as negative control.

In addition, in the present embodiment, as long as the design of primer is based on above-mentioned principle, both acquisition can be designed according to the state of the art.

Embodiment 2

One, pBI121-is built hpaXm::gfpplant expression vector

1.fusion gene hpaXm::gfpamplification

1a) PCR acquisition has spacer end hpa _xm with gfpgene fragment.

Acquisition has spacer end hpa _xm gene fragment: add in PCR reaction system hpa _xm upstream primer HpaXm-F and with gfpthe downstream primer HpaXm::gfp-R of gene complementation end, template be with from cotton angular leaf spot fungus ( xanthomonas citrisubsp. malvacearum) in the total length that clones hpaXmthe plasmid pHM1-of gene hpaXm;

Acquisition has spacer end gfpgene fragment: add in PCR reaction system with hpaXmgene complementation end gfpupstream primer HpaXm::gfp-F and gfpthe respective upstream and downstream primer of downstream primer gfp-R gene, template is pMUTIN – gfp+ plasmid.

Amplification contains spacer end hapXm(hpa)with gfpgene fragment.

Wherein, PCR reaction system (50uL) is as follows:

DNA profiling 1uL

Primer 1 1uL

Primer 2 1uL

10×EasyPfu Buffer 5uL

2.5mMdNTPs 4uL

EasyPfu DNA polysaccharase 1uL

sterilizing ultrapure water complements to 50uL

PCR reaction conditions is:

94 DEG C of denaturation 4min;

94 DEG C of sex change 30sec;

58 DEG C of renaturation 30sec; 30 circulations

72 DEG C extend 1min;

Last 72 DEG C extend 10min.

Pcr amplification gfpthe reaction system of gene and expansion hpaXm (hpa)gene response system is identical.Amplification gfpgene extension time used is 1min 45sec, and other conditions are identical, and primer parameter is in table 1, and the goal gene be suitable for is in table 2:

Table 1:

Primer	Primer sequence
		HpaXm-F	5'-GCTCTAGAATCAATTCTTTGAACACACAGA-3'
HpaXm-R	5'-TCCCCCCGGGTTACTGCATCGATCCGGTGT-3'
		HpaXm△LP-F	5'-GCTCTAGAATGCAGGTCGACCCGAGCCAGA-3'
gfp-R	5'-TCCCCCCGGGTTATTTGTAGAGCTCATCCATGCC-3'
		HpaXm::gfp-R	5'-GTTCTTCTCCTTTGCTAGCCATCTGCATCGATCCGGTGTC-3'
HpaXm::gfp-F	5'-GACACCGGATCGATGCAGATGGCTAGCAAAGGAGAAGAAC -3'

Table 2:

The PCR primer that takes a morsel carries out agarose gel electrophoresis detection according to preceding method, and amplified fragments size is consistent with expection clip size, then the glue of remaining PCR primer macropore is carried out electrophoresis, and reclaims DNA fragmentation.Obtain according to the method described above hpaXm(hpa)gene fragment and gfpgene fragment agarose gel electrophoresis figure.

Be that template overlap-extension PCR obtains fusion gene fragment with PCR primer

The gene fragment that purifying has overlapping chain is reclaimed to cut glue hpaXm (hpa)gene and gfpgene, as template, carries out PCR overlap-extension PCR.This process is divided into two steps:

The first step: do not add primer in reaction system

PCR reaction system:

DNA profiling ( hpaXm (hpa)with gfp) each 1uL

10×Easy Pfu Buffer 5uL

2.5mMdNTPs 4uL

EasyPfu DNA polysaccharase 1uL

sterilizing ultrapure water complements to 50uLPCR reaction conditions:

94 DEG C of denaturation 4min;

94 DEG C of sex change 30sec;

58 DEG C of renaturation 30sec; 5 circulations

72 DEG C extend 2min30sec;

Last 72 DEG C extend 10min.

Second step: add two ends primer in the reaction system of the first step,

Due to hapXm ∷ gfpin fusion gene hpaXm(hpa)gene, in upstream, is increasing hpaXm::gfpsecond step reaction system in add hpaXmupstream primer HapXm-F (5'-GCTCTAGAATGAATTCTTTGAACACACAGA-3') and the downstream primer gfp-R (5'-TCCCCCCGGGTTATTTGTAGAGCTCATCCATGCC-3') of gfp, then to increase.

PCR reaction conditions:

94 DEG C of denaturation 4min;

94 DEG C of sex change 30sec;

58 DEG C of renaturation 30sec; 18 circulations

72 DEG C extend 2min30sec;

Last 72 DEG C extend 10min.

Carry out gel electrophoresis according to preceding method, and carry out cutting glue recovery, obtain fusion gene fragment, as shown in Figure 1.

3. fusion gene fragment is cloned into pMD ^tMin 18-T carrier

3.1 do not add A base with the gene fragment 3 ' end that EasyPfu polymeric enzymatic amplification obtains, and carry out adding A process before being connected with carrier T.

Add A system for handling (20uL) as follows:

Fusion gene fragment 14.5uL

10×TransFast Taq Buffer 2.5uL

2.5mM dNTPs 2uL

TransFast Taq polysaccharase 1uL

72 degree of water-baths 25 minutes, reclaim gene fragment.

3.2 fusion genes and pMD ^tM18-T carrier connects, and is transformed in competent escherichia coli cell:

Reaction system 10 μ L

pMD18-T simple vector 0.5μL

Control Insert 0.5μL

Cut glue and reclaim product 4 μ L

Solution Buffer 5μL

Reaction conditions 16 DEG C of connections are spent the night

(1) in micro centrifugal pipe, add the fusion gene fragment of 1uL carrier T and 4uL.

(2) mix gently, room temperature (20 DEG C-37 DEG C) reaction 15 minutes, is placed in centrifuge tube on ice.

(3) add connection product (to add when competent cell just thaws and connect product) in 50 μ L Trans1-T1 competent cells, flick mixing, ice bath 20-30 minute.

(4) 42 DEG C of heat shocks 30 seconds, are placed in 2 minutes immediately on ice.

(5) add 250 μ L balances to the LB of room temperature, 200 turns, hatch 1 hour for 37 DEG C.

(6) get 8 μ L, 500 mM IPTG, 40 μ L, 20 mg/mL X-gal mix, and are applied to ready containing on penbritin (100ug/mL) LB flat board equably, place 30 minutes at 37 DEG C.

(7) treat IPTG, after X-gal is absorbed, get 200 μ L bacterium liquid bed boards, overnight incubation (for obtaining comparatively polyclone, centrifugal 1 min of 4000 rpm, discards part supernatant, retains 100-150 μ L, flicks suspension thalline, get whole bacterium liquid coated plate, overnight incubation).

The qualification of 3.3 positive recombinants and order-checking

(1) select white colonies to containing 1uL liquid LB(Amp, 100ug/mL) 1.5mL centrifuge tube in, 37 DEG C of concussions are cultivated.

(2) 1uL bacterium liquid is got as template, pcr amplification of entering.

(3) PCR primer is carried out agargel electrophoresis detection, by amplified fragments size, and the assorted LB bacterium liquid liquid-transfering gun be with consistent with imagination is transferred to containing 10mL liquid LB(Amp, 100ug/mL) triangular flask in, 37 DEG C shake incubated overnight.

Get 1mL bacterium liquid and send to order-checking, and remaining bacterium liquid is preserved.

4. by fusion gene cloning on carrier pBI121

Binary expression vector pBI121 total length 14758bp, wherein T-DNA region comprises a lot of restriction enzyme site, and according to the sequence alignment of goal gene, we select xmaIwith xbalas double enzyme site, during design primer, introduce at the two ends of fusion gene xbaIwith xmaIrestriction enzyme site, the multiple clone site of pBI121 also has xbaIwith xmathe restriction enzyme site of I.Goal gene with these two restriction enzyme sites is cloned with step according to the method described above, is connected respectively to after carrying out double digestion on binary expression vector pBI121.Fusion gene is used xbaIwith xmaIrestriction enzyme is from pMD ^tM18-T carrier scales off, and reclaims.Under the effect of T4 ligase enzyme, with same use xbaIwith xmaIthe pBI121 carrier that restriction enzyme cuts connects.Recombinant plasmid is imported in competent escherichia coli cell, in with the flat board of kantlex carrying out blue hickie sieve, positive transformant goal gene Auele Specific Primer HpaXm-F and gfp-R(primer sequence are in table 1) carry out PCR checking after, the positive transformant that can amplify fragment consistent with goal gene clip size send genome company to check order, sequencing result use NCBI-BLAST software with hpaXm(GenBank Accession No.DQ643828) original series is compared, filter out with hpaXmthe positive transformant of original gene 100% homology.Extract positive transformant plasmid, obtain recombinant plant expression vector pBI1121:: hpaXm::gfp.

Two, pBI121: is built: hpaXm △ LP::gfpplant expression vector.

1. goal gene fragment hpaXm △ LP::gfpamplification

PCR reaction system (50 μ L):

Template (plasmid) 2 μ L

Upstream primer 1 μ L

Downstream primer 1 μ L

10×Buffer 5μL

2.5mMdNTP 4μL

（Trans）Taq-T DNA polymerase 1μL

Supply ultrapure water to 50 μ L

Wherein template uses the recombinant plant expression vector obtained in first part

PBI1121:: hpaXm::gfpplasmid, upstream primer is

HpaXm △ LP-F(5'-GCTCTAGAATGCAGGTCGACCCGAGCCAGA-3'), downstream primer is gfp-R(5'-TCCCCCCGGGTTATTTGTAGAGCTCATCCATGCC-3').

PCR reaction conditions is:

94 DEG C of denaturation 4min;

94 DEG C of sex change 30sec;

58 DEG C of renaturation 30sec; 30 circulations

72 DEG C extend 1min;

Last 72 DEG C extend 10min.

The PCR primer that takes a morsel carries out agarose gel electrophoresis detection according to preceding method, and amplified fragments size is consistent with expection clip size, then the glue of remaining PCR primer macropore is carried out electrophoresis, and reclaims DNA fragmentation.Obtain according to the method described above hpaXm △:: gfpgene fragment agarose gel electrophoresis figure.

2. by goal gene fragment and pMD ^tM18-T carrier connects and is transformed in competent escherichia coli cell

Reaction system 10 μ L

pMD18-T simple vector 0.5μL

Control Insert 0.5μL

Cut glue and reclaim product 4 μ L

Solution Buffer 5μL

Reaction conditions 16 DEG C of connections are spent the night

(1) in micro centrifugal pipe, add the fusion gene fragment of 1uL carrier T and 4uL.

(2) mix gently, room temperature (20 DEG C-37 DEG C) reaction 15 minutes, is placed in centrifuge tube on ice.

(3) add connection product (to add when competent cell just thaws and connect product) in 50 μ L Trans1-T1 competent cells, flick mixing, ice bath 20-30 minute.

(4) 42 DEG C of heat shocks 30 seconds, are placed in 2 minutes immediately on ice.

(5) add 250 μ L balances to the LB of room temperature, 200 turns, hatch 1 hour for 37 DEG C.

(6) get 8 μ L, 500 mM IPTG, 40 μ L, 20 mg/mL X-gal mix, and are applied to ready containing on penbritin (100ug/mL) LB flat board equably, place 30 minutes at 37 DEG C.

(7) treat IPTG, after X-gal is absorbed, get 200 μ L bacterium liquid bed boards, overnight incubation (for obtaining comparatively polyclone, centrifugal 1 min of 4000 rpm, discards part supernatant, retains 100-150 μ L, flicks suspension thalline, get whole bacterium liquid coated plate, overnight incubation).

3 .the qualification of positive recombinant and order-checking

(1) select white colonies to containing 1uL liquid LB(Amp, 100ug/mL) 1.5mL centrifuge tube in, 37 DEG C of concussions are cultivated.

(2) 1uL bacterium liquid is got as template, pcr amplification of entering.

(3) PCR primer is carried out agargel electrophoresis detection, by amplified fragments size, and the assorted LB bacterium liquid liquid-transfering gun be with consistent with imagination is transferred to containing 10mL liquid LB(Amp, 100ug/mL) triangular flask in, 37 DEG C shake incubated overnight.

Get 1mL bacterium liquid and send to order-checking, and remaining bacterium liquid is preserved.

4 .goal gene is cloned on carrier pBI121

With xmaIwith xbalrestriction enzyme is to carrying goal gene hpaXm △ LP::gfpcarrier T pMD ^tM18-T carrier carries out double digestion and reclaims.Under the effect of T4 ligase enzyme, with same use xbaIwith xmaIthe pBI121 carrier that restriction enzyme cuts connects.Recombinant plasmid is imported in competent escherichia coli cell, in the flat board of kantlex carrying out blue hickie sieve, positive transformant goal gene Auele Specific Primer

HpaXm △ LP-F(5'-GCTCTAGAATGCAGGTCGACCCGAGCCAGA-3') and

gfp-R（5'-TCCCCCCGGGTTATTTGTAGAGCTCATCCATGCC-3'）。After carrying out PCR checking, the positive transformant that can amplify fragment consistent with goal gene clip size send genome company to check order, sequencing result use NCBI-BLAST software with hpaXm(GenBank Accession No. dQ643828) original series compares, filter out with hpaXmthe positive transformant of original gene 100% homology.Extract positive transformant plasmid, obtain recombinant plant expression vector pBI1121:: hpaXm △ LP::gfp.

According to the carrier that above aspect builds, contriver carries out the detection of goal gene, and the result obtained is as figure Fig. 4: plasmid pBI121- hpaXm::gfpfor template amplification goal gene and

Fig. 5: plasmid pBI121- hpaXm △ LP::gfpfor template amplification goal gene.

Wherein, M: DL20000Marker using empty plasmid pBI121 for template is as negative control.

In addition, in the present embodiment, as long as the design of primer is based on above-mentioned principle, both acquisition can be designed according to the state of the art.

The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (10)

1. containing coding hpaXm(SEQ NO:1) plant expression vector of protein gene, it is characterized in that: described plant expression vector comprises pBI121 carrier and is connected on described pBI121 carrier, the gene fragment of hpaXm albumen of can encoding.

2. plant expression vector as described in the appended claim 1, is characterized in that: described plant expression vector comprises pBI121- hpaXmand pBI121- hpaXm △ LP.

3., as the construction process of plant expression vector as described in arbitrary in claim 1 or 2, it is characterized in that, comprise the following steps:

A) goal gene total length hpaXm, or the mutant of deleted signal peptide similar sequence hpaXm △ LPthe amplification of (SEQ NO:2);

B) fusion gene fragment is cloned into pMD ^tMin 18-T carrier;

C) fusion gene is transferred on pBI121 carrier.

4. the construction process of plant expression vector as claimed in claim 3, is characterized in that: in step c, with on pBI121 carrier xmaIwith xbalas restriction enzyme site.

5. plant expression vector as described in the appended claim 1, is characterized in that: the gene also containing expressing green fluorescent protein in described plant expression vector gfp.

6., as claim removes plant expression vector described in 5, it is characterized in that: described in gfpwith described hpaXmmutual fusion.

7. plant expression vector as recited in claim 6, is characterized in that: comprise pBI121- hpaXm::gfpand pBI121- hpaXm △ LP::gfp.

8. contain as described in arbitrary in claim 5-7 gfpthe construction process of plant expression vector, it is characterized in that: comprise the following steps:

(1) according to goal gene hpaXm::gfp(SEQ NO:3) sequence or hpaXm △ LP::gfp(SEQ NO:4) and carrier pBI121 sequence selection restriction enzyme site xbaIwith xmaIand design primer;

(2) fusion gene hpaXm::gfp or hpaXm △ LP::gfpamplification;

(3) be that template overlap-extension PCR obtains fusion gene fragment with PCR primer;

(4) fusion gene fragment is cloned into pMD ^tMin 18-T carrier;

(5) fusion gene is connected on pBI121 carrier.

9. the construction process of plant expression vector as claimed in claim 8. containing gfp, is characterized in that: in described step b, to reclaim purifying have the gene fragment of overlapping chain to cut glue hpaXm/hpaXm △gene and gfpgene, as template, carries out PCR overlap-extension PCR, and this process is divided into two steps, is respectively:

(2a) do not add primer in PCR reaction system to increase;

(2b) in the reaction system of the first step, add two ends primer to increase;

In described step (3), comprise following steps:

(3a) the gene fragment 3' end obtained increasing does not add A base, carries out adding A process before being connected with carrier T;

(3b) fusion gene and pMD ^tM18-T carrier connects, and is transformed in competent escherichia coli cell;

(3c) qualification of positive recombinant and order-checking.

10. the construction process of plant expression vector as claimed in claim 8. containing gfp, is characterized in that: in described step (4), with on pBI121 carrier xmaIwith xbalas restriction enzyme site.