CN105274137A - Method of producing lactoferrin by using Artemisia apiacea - Google Patents

Abstract

The invention discloses a method of producing lactoferrin by using Artemisia apiacea. The method comprises following steps: a, performing modifying to obtain a carrier T202; b, performing modifying to amplify lactoferrin gene; c, building a recombinant plasmid T202-Blf expressing the lactoferrin gene; d, transforming Agrobacterium EHA105 cells by using the recombinant plasmid T202-Blf; e, screening positive Agrobacterium EHA105 strains, and performing enzyme digestion identification; f, transforming Artemisia apiacea to obtain positive Artemisia apiacea transformed plants containing lactoferrin; g, performing real-time quantitative PCR (polymerase chain reaction) (aRT-PCR) to identify an expression quantity of the lactoferrin gene. The method using an Artemisia apiacea expression system has the advantages that the nucleotide sequence of the lactoferrin gene is optimized according to codon preferences of Artemisia apiacea, thus ensuring efficient and stable mRNA transcription, quick and accurate translation and higher foreign protein accumulation; plant expression of lactoferrin is low in cost, safe and good in economic benefit; the expressed protein products are directly applicable to addition without purification.

Description

The method of lactoferrin produced by a kind of sweet wormwood

Technical field

The present invention relates to biotechnology and gene engineering technology field, be specifically related to a kind of method that lactoferrin produced by sweet wormwood.

Background technology

The healthy and orderly development of livestock industry, not only concerns the food safety of the people, also has tremendous influence to economy.And the generation of animal epidemic, have a strong impact on health and the output of herding, bring huge financial loss to numerous raisers.At present, raiser usually reaches the object of preventing and treating epidemic disease mainly through adding antibiosis in feed.But, use microbiotic in a large number, the microecological balance of meeting havoc animal digestive system, and can resistance be produced.It is residual that microbiotic has in various degree in animal body, and then affect the healthy of eater.Therefore, people start to appeal to completely forbid to add microbiotic in animal-feed.

Antibacterial peptide (Antibacterialpeptide, ABP) be the important component part of animal innate immunity, to pathogenic micro-organisms such as bacterium, fungi, mycobacterium, spirochete, togavirus, mycoplasma, chlamydozoan, spirochete and some malignant cells (as tumour cell) and hiv virus all have lethal effect.And antibacterial peptide molecule is by destroying the membrane structure of pathogenic micro-organism, causes water-soluble substances in born of the same parents to ooze out in a large number, and finally cause it dead, therefore pathogenic micro-organism is difficult to produce resistance to it.Antibacterial peptide has non-immunogenicity, has the advantages such as efficient, safety, noresidue, can immunity moderation effect, and not disturbing the characteristics such as intestinal beneficial bacterium, is a kind of Substitutes For Antibiotic of most DEVELOPMENT PROSPECT.

Lactoferrin (Lactoferrin, LF) is found when sepg whey albumen by people such as nineteen thirty-nine Sorensen, and is obtained in being separated from cow's milk in nineteen sixty by Groves.Bovine lactoferricin (BovineLactoferricin, be abbreviated as LfcinB) be 25 amino-acid residues that Bovinelactoferrin (bLF) N-holds (17 ~ 41), molecular weight is about 3.1kd, it is the anti-microbial activity center of bLF, bLF anti-microbial activity is played an important role (Kangetal, 1996), its fungistatic effect is 400 times of bLF, and plays a significant role in maintenance composition of gut flora.LfcinB have heat-resisting, be not easily degraded at digestive tube, no antigen, immunity moderation, broad-spectrum antimicrobial, do not disturb the characteristics such as intestinal beneficial bacterium.LfcinB has good anti-microbial effect, as intestinal bacteria, Salmonella enteritidis, Pseudomonas aeruginosa, streptococcus aureus, clostridium perfringens etc., but does not affect the beneficial bacterias such as bifidus bacillus, simultaneously can also the growth of irritation cell.Therefore, have broad application prospects in fields such as agricultural, herding, medical treatment.

Invented a kind of method of yeast production lactoferrin before our company, improved lactoferrin fungistatic effect is better; And yeast growth speed is fast, output is high, without the need to adopting methanol induction, is easy to high-density culture; The protein product of expressing is easy to separation and purification; There is multiple posttranslational modification characteristic, as folding in polypeptide, glycosylation, methylate, acetylize etc.; Genetic background is clear, is easy to operate expression vector.But when being extended to large-scale commercial process, there is output stable not, easily by problems such as living contaminantses, and relative to the plantation of plant, the cost of this kind of method and technical requirements are still higher.

And sweet wormwood (Artemisiacarvifolia) is annual herb, gas perfume (or spice) is special, mildly bitter flavor, has refrigerant sense, can play good health-care effect.Bud stage is gathered, and extracts over-ground part, chopping, after drying, can directly be used as feed and add.Sweet wormwood, as the plant producing lactoferrin, also has thick raw easily pipe, the advantages such as vegetative period is short, less investment, and income is fast.

Summary of the invention

The present invention is intended to overcome the deficiencies in the prior art, provides a kind of sweet wormwood to produce the method for lactoferrin.

In order to achieve the above object, technical scheme provided by the invention is:

The method that lactoferrin produced by described sweet wormwood comprises the steps:

(1) carrier T202 is transformed; Improved carrier T202 sequence as shown in SEQIDNO.1, total length 12259bp;

(2) the recombinant plasmid T202-Blf of the improved bovine lactoferrin gene of sweet wormwood cells is built; The sequence of recombinant plasmid T202-Blf is as shown in SEQIDNO.2;

(3) sweet wormwood is transformed with recombinant plasmid T202-Blf;

(4) screen positive transgenic sweet wormwood plant, obtain the sweet wormwood plant producing lactoferrin.

Preferably, the process of step (1) described transformation carrier T202 is as follows:

A () take pCambia1300 as template, carry out pcr amplification (product length is 441bp) by the pRB sequence of following primer pair TDNA, pRB sequence is as shown in SEQIDNO.6:

Primer pRB-F1:5 '- gAATTCaCTGGGAATTCCCTGGCGTTA-3 ' (SEQIDNO.7); EcoRI

Primer pRB-R1:5 '- gGTACCcAGCCTGTCGGGTACCTTAGGA-3 ' (SEQIDNO.8); KpnI

B pRB sequence qualification that pcr amplification obtains by (); Be connected on pCambia1301 by correct pRB sequence, obtain carrier pC13001T, carrier pC13001T sequence is as shown in SEQIDNO.3;

C () cuts Actin promotor with KpnI and SmaI enzyme, cut carrier pC13001T with KpnI and PmacI enzyme simultaneously, and the Actin promotor that carrier pC13001T and the enzyme cut by enzyme are cut is connected, and is built into improved carrier T202.

Preferably, the process of the recombinant plasmid T202-Blf of step (2) described structure sweet wormwood cells bovine lactoferrin gene is as follows:

A () synthesizes improved bovine lactoferrin gene sequence, and be stored in PMD19Simple plasmid, and improved bovine lactoferrin gene sequence is as shown in SEQIDNO.4;

B () is increased with the improved bovine lactoferrin gene that following primer pair is stored in PMD19Simple plasmid:

Primer BlfF1:5 '- cTGCAGtGGCATCTCTCAGCACATATG-3 ' (SEQIDNO.9); PstI

Primer BlfR1:5 '- tCTAGAcTTGTCGTCGTCTTAGTAACAC-3 ' (SEQIDNO.10); XbalI

C improved bovine lactoferrin gene order-checking qualification that pcr amplification obtains by (); Be connected with T-carrier by correct improved bovine lactoferrin gene, obtain carrier B lf-T, carrier B lf-T sequence is as shown in SEQIDNO.5;

(d) with PstI, XbaI respectively enzyme cut improved carrier T202 and carrier B lf-T, the improved carrier T202 cut by enzyme is connected with the carrier B lf-T that enzyme is cut, and is built into recombinant plasmid T202-Blf.

The invention will be further described below:

In the process of step (1) described transformation carrier T202, PCR reaction conditions: 94 DEG C of 5min denaturations, 94 DEG C 30s-59 DEG C 30s-72 DEG C of 70s, 30 circulations, 72 DEG C of 7min; After 2% agarose gel electrophoresis, reclaim purifying object fragment, be stored in-20 DEG C for subsequent use; The pRB fragment obtained is connected with T-carrier, obtains pRB-T carrier, transformation of E. coli DH5 α bacterial strain, at 37 DEG C, with HindIII and XbalI double digestion screening positive strain, order-checking qualification; Be connected on pCambia1301 by the pRB sequence be cloned into, restriction enzyme site is (EcoRI-KpnI, GAATTC---GGTACC), and novel vector is named: pC13001T.Cut Actin promotor with KpnI and SmaI enzyme (to be cloned by this seminar member in advance and connect into pMD18TVector, and order-checking display is correct), cut pC13001T with KpnI and PmacI enzyme simultaneously, be connected with Actin promotor, be built into improved carrier T202.Select EcoRI single endonuclease digestion, the band of 810bp should be occurred.

In step (2), the process of transformation bovine lactoferrin gene sequence is as follows:

A () utilizes SeqnConverter software analysis sweet wormwood genome albumen coded sequence.

The gene that expressing quantity is higher, annotation of gene function is clear and definite is screened from NCBI (http://www.ncbi.nlm.nih.gov/), 101 that use software SeqnConverter (http://www.cibj.com/seqnconverter.zip) Analysis and Screening to go out complete albumen coded sequences, calculate sweet wormwood relative synonymous codon usage degree (relativesynonymouscodonusage, RSCU): the ratio of the frequency that a certain codon uses and its expected frequence when unbiasedness uses.If relative synonymous codon usage degree (relativesynonymouscodonusage) RSCU=1 of a certain codon, shows that the use of this codon does not have Preference; If RSCU > is l, show that the usage bias of this codon is relatively high.

B () bovine lactoferrin gene is codon optimized

Keeping under the prerequisite that lactoferrin protein amino acid sequence is constant, the codon that wherein RSCU value is less than 0.5 is being replaced to the codon that sweet wormwood has a preference for, the codon that namely RSCU value is maximum in synonymous code subgroup; The codon of RSCU value between 0.5 ~ 1.0 just part replaces to the codon of sweet wormwood preference; And the codon of RSCU value more than 1 is not done to change.In synonym replacement process, reduce the ratio shared by codon ACC, AGC, GCC and CCC, to reduce, methylated site may occur.

C () Poly (A) tailing recognition site is analyzed

Bovine lactoferrin gene sequence after the optimization of sweet wormwood preference codon may contain Poly (A) tailing recognition sequence.Synonym Shift Method is adopted to eliminate Poly (A) tailing recognition sequence (AATAAA<SEQIDNO.17>, ATTAAA<SEQIDNO.18>, AACCAA<SEQIDNO.19> and AATTAA<SEQIDNO.20>), to ensure the integrity that bovine lactoferrin gene is transcribed.

The analysis of (d) intron cutting recognition sequence

Intron shear signal sequence may be there is in the bovine lactoferrin gene sequence after above-mentioned optimization.Potential intron cutting recognition sequence is doped with SoftBerry line server (http:/linux1.softberry.com/berry.phtml), intron cutting recognition sequence is eliminated, to ensure the integrity that bovine lactoferrin gene is transcribed again with synonym Shift Method.

When improved bovine lactoferrin gene is increased, PCR reaction conditions: 94 DEG C of 5min denaturations, 94 DEG C 30s-56 DEG C 30s-72 DEG C of 20s, 30 circulations, 72 DEG C of 7min; After 2% agarose gel electrophoresis, reclaim purifying object fragment, be stored in-20 DEG C for subsequent use; The bovine lactoferrin gene obtained is connected with T-carrier, obtains Blf-T carrier, transformation of E. coli DH5 α bacterial strain, at 37 DEG C, with PstI and XbalI double digestion screening positive strain, order-checking qualification.

The detailed process of the step (d) in step (2) is: with PstI, XbaI respectively enzyme cut improved carrier T202 and Blf-T carrier, product, after 1.5% agarose gel electrophoresis, reclaims bovine lactoferrin gene fragment and carrier T202 fragment; T4 ligase enzyme, 16 DEG C connect 2h and obtain recombinant plasmid T202-Blf, transformation of E. coli DH5 α bacterial strain; Conversion rear extraction plasmid DNA is carried out enzyme and is cut qualification.To detect correct plasmid, be transformed in Agrobacterium ,-70 DEG C save backup.

Step (3) infects sweet wormwood by Agrobacterium-medialed transformation method and obtains transgene abrotanum seedling; Screening positive plant, performing PCR of going forward side by side is identified, the method for described screening positive strain utilizes its expression amount of real-time quantitative RT-PCR augmentation detection.Not having lactoferrin inside wild sweet wormwood, as long as so have lactoferrin inside transgene abrotanum, is exactly positive.PCR identifies that positive rate, RT-PCR identify the expression amount of lactoferrin, is ordinary method.

The detailed process that recombinant plasmid T202-Blf transforms sweet wormwood is as follows:

1) three days in advance Agrobacteriums prepare: LB plate (is cooled to 60 DEG C after the sterilizing of LB solid medium, adds Kan100mg/L+CHL34mg/L+Rif100mg/L, be down flat plate;

2) coating of bacterium liquid;

3) cultivation 2-3 days is inverted for 28 DEG C;

4) NBM solid medium, is down flat plate after having added As, for subsequent use.NBM liquid nutrient medium.Filter paper, triangular flask, water, sterilizing in advance;

5) the dull and stereotyped bacterium of cultured LB is washed in the Dual culture base (NBM+As0.1mM) of 50ml liquid, and range estimation concentration, adjusts OD600=0.5;

6) agrobacterium mediation converted: sheet sweet wormwood seedling being cut into the square size of 1cm, transfers in Agrobacterium EHA105 bacterium liquid and soaks 30min, and shaking table shakes.With sterilizing filter paper suck dry moisture, dry up slightly.Sweet wormwood is transferred to Dual culture base (NBM+As0.1mM) on it and put a sterilizing filter paper, light culture 3 days at 25 ~ 26 DEG C;

7) screening and culturing: after 3 days, proceeds to sweet wormwood in sterilized triangular flask, with bacterium water rinse 5 times not muddy to liquid, then be added with 500mg/L head born of the same parents and 400mg/L carboxylic paleness mycin aqua sterilisa soak 30min, shaking table shake.With sterilizing filter paper suck dry moisture.Transfer to screening culture medium (MS+500mg/L head born of the same parents+400mg/L carboxylic paleness mycin+50mg/L Totomycin), light culture at 25 ~ 26 DEG C, after 20 days, the callus entirety growing kanamycin-resistant callus tissue is proceeded in new screening culture medium;

8) differentiation culture: by growing the callus global transfer of kanamycin-resistant callus tissue in screening culture medium in division culture medium after terminating twice screening, be placed in 25 ~ 26 DEG C, 14h illumination cultivation, light intensity 1000 ~ 1500lx illumination cultivation, changes a subculture in every 20 days;

The qualification of the positive rate of transgene abrotanum

1) CTAB method extracts sweet wormwood genomic dna

Get the sweet wormwood Taibei 309 blade and be about 0.1g, shred, add liquid nitrogen and be ground into powder, be placed in rapidly the 1.5mL centrifuge tube that 800 μ LCTAB damping fluids are housed in advance, mixing of turning upside down, be placed in 65 DEG C of water-bath 30min, period, every 5min shook several times gently.Add isopyknic chloroform: primary isoamyl alcohol (24:1) shakes up gently, 4 DEG C, the centrifugal 10min of 12000g.Draw supernatant in new 1.5mL centrifuge tube, add the dehydrated alcohol of 2.5 times of volumes, rock evenly, room temperature places 10min.4 DEG C, 12000g, centrifugal 10min.Remove supernatant liquor, add 75% washing with alcohol of 1mL, precipitate in a moment, 4 DEG C, the centrifugal 5min of 12000g.Remove liquid, be inverted by centrifuge tube, room temperature leaves standstill 15 ~ 30min, makes DNA dry.Adding 40 μ L makes DNA dissolve completely containing the TE of RNase, is placed in 37 DEG C of incubation 30min and digests RNA.With 0.8% agarose through row electrophoresis experiment, after detecting the quality of DNA and concentration, be placed in 4 DEG C for subsequent use, or be placed in-20 DEG C of Long-term Cryopreservations.

2) transgene abrotanum T0 identifies for the PCR of plant

After obtaining transgene abrotanum strain by Agrobacterium-mediated transformation callus, pcr amplification technical evaluation gained strain is utilized whether to be positive transgenic plant.Adopt CTAB method to extract the DNA of each sweet wormwood strain blade respectively as template, carry out pcr amplification by Totomycin specificity amplification primer.Sequence is as follows:

Primer HygF1:5 '-CGACAGCGTCTCCGACCTGA-3 ' (SEQIDNO.11)

Primer HygR1:5 ’ – CGCCCAAGCTGCATCATCGAA-3 ' (SEQIDNO.12);

Reaction system is as follows:

Thermal cycling is set to: 95 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 57 DEG C of annealing 40s, 72 DEG C extend 30s, 35cycles; 72 DEG C extend 35min.Arrange feminine gender to contrast without Template Controls, negative wild type control and positive plasmid simultaneously.

The expression amount qualification of lactoferrin

1) plant Total RNAs extraction

After plant tissue materials is drawn materials, shred, be placed in rapidly liquid nitrogen and grind to form dry powder, get the dusty material of about 100mg, loading fills in the 1.5mL centrifuge tube of 1.0mLTRIzol extracting solution (Invitrogen) in advance, cover tightly pipe lid, shake, makes sample fully mix with TRIzol extracting solution.After low temperature marking pen numbering, be placed in-70 DEG C of Ultralow Temperature Freezers frozen for subsequent use.

After sample for subsequent use frozen in-70 DEG C of Ultralow Temperature Freezers is taken out, ice bath 5min, make the complete cracking of the nucleoprotein of sample, appropriate chloroform is added according to 200 μ l chloroform/mlTRIzol, firmly shake 15s (note: forbid vortex oscillation instrument herein, in order to avoid by disconnected for genomic dna shake) afterwards room temperature place 15min, then 4 DEG C, the centrifugal 15min of 12000g; Carefully draw out upper strata aqueous phase, proceed in another clean centrifuge tube; Add the Virahol of prior precooling according to 0.5ml Virahol/mlTRIzol, mixing of turning upside down, be placed in-20 DEG C of precipitation 10min; 4 DEG C, the centrifugal 10min of 12000g; Abandon supernatant, now RNA has been sunken at the bottom of pipe.In centrifuge tube, add 75% ethanol of the prior precooling of 1mL, turn upside down centrifuge tube, suspension washing of precipitate; 4 DEG C, the centrifugal 5min of 8000g; Abandon supernatant, after room temperature or vacuum-drying 5-10min (note: RNA sample is too not dry, otherwise is difficult to dissolve).Add the water of 50 μ lRNase-free, fully dissolve, 55-60 DEG C of process 5-10min.The institute RNA that carries, after DNA enzymatic (Fermentas), surveys the concentration of the quantitative RNA of OD value, as the template of quantitative fluorescent PCR and first chain of synthesis cDNA.

The reaction system of DNase digestion is:

2) cDNA first chain synthesis

In the centrifuge tube of aseptic 0.2ml, configure following primer/template ribonucleic acid mixed solution, after 70 DEG C of incubation 10min, be placed in rapidly 2 ~ 5min on ice, the quick centrifugal several seconds, the denaturing soln of primer/template ribonucleic acid is combined at the bottom of pipe.

The reaction system of cDNA first chain synthesis is:

Configure following inverse transcription reaction liquid in above-mentioned centrifuge tube after, 42 DEG C of incubation 1h; Subsequently again at 70 DEG C of incubation 15min, be placed in cooled on ice, obtain cDNA for pcr amplification afterwards.

3) real-time quantitative PCR (qRT-PCR) is analyzed

QRT-PCR uses SYBRGreenRT-PCROneStepKit (QIAGEN company), and instrument selection ABIPrism7900HT quantitative real time PCR Instrument, each sample repeats 3 times.

PCR reaction system is:

Thermal cycling is set to: 48 DEG C of 30min, 1cycle; 95 DEG C of 10min, 1cycle; 95 DEG C of 15s, 58 DEG C of 40s, 72 DEG C of 20s, 40cycles.Calculate by relative quantification method, goal gene relative expression quantity Rel.Exp=2-Δ Δ Ct, wherein Δ Δ Ct=(Calibrator Δ Ct)-(unknown sample Δ Ct), unknown sample Δ Ct=(reference gene Ct). (goal gene Ct), Calibrator Δ Ct=(reference reference gene Ct). (reference goal gene Ct).Reference gene is TFC1 and UBC6.

Primer sequence is as follows:

Primer TFC1F1:5 '-CATAACTGAAGAGCCAGACGAC-3 ' (SEQIDNO.13);

Primer TFC1R1:5 '-CAGAATTAGCATCCACAACGAC-3 ' (SEQIDNO.14);

Primer UBC6F1:5 ’ – GCGGGGATGGAGCATTGGTTCTTT-3 ' (SEQIDNO.15);

Primer UBC6R1:5 ’ – GACTGAACGGACAAGAGGCATTG-3 ' (SEQIDNO.16).

The present invention first transforms carrier T202; Synthesize and the bovine lactoferrin gene that increases; The carrier T202 of this gene and our company's transformation is utilized to build the recombinant plasmid can expressing this bovine lactoferrin gene in sweet wormwood; Then recombinant plasmid transformed sweet wormwood is used; Screening positive transgenic sweet wormwood seedling, finally obtains lactoferrin sweet wormwood.

Instant invention overcomes by shortcomings such as the technical requirements of yeast production lactoferrin are high, cost is high, expression amount is unstable; According to the codon preference of sweet wormwood, the nucleotide sequence of bovine lactoferrin gene is optimized, ensure that efficient and stable mRNA transcribes, translate fast and accurately and the accumulation of higher exogenous protein; And the transgene abrotanum seedling of the production lactoferrin obtained, expression amount is high, and growth velocity is fast; The protein product of expressing need not separation and purification, can directly add; There is multiple posttranslational modification characteristic, as folding in polypeptide, glycosylation, methylate, acetylize etc.; Sweet wormwood belongs to plant, safe fanout free region, can be directly used in feed and add.

Accompanying drawing explanation

Fig. 1 is the pcr amplification product electrophorogram of pRB;

Fig. 2 is that the plasmid DNA enzyme after pRB connects carrier T cuts qualification electrophorogram;

Fig. 3 is that the enzyme after insertion connects the pRB sequence of cloning and obtaining in pCambia1301 cuts qualification electrophorogram;

Fig. 4 is that the T202 enzyme built cuts qualification electrophorogram;

Fig. 5 is the pcr amplification product electrophorogram of bovine lactoferrin gene;

Fig. 6 is that the plasmid DNA enzyme after lactoferrin connects carrier T cuts qualification electrophorogram;

Fig. 7 is after carrier T202-Blf transformation of E. coli DH5 α, and the enzyme extracting plasmid cuts qualification electrophorogram;

The plasmid DNA of extraction, after T202-Blf is proceeded to Agrobacterium EHA105, is proceeded to the enzyme after bacillus coli DH 5 alpha and cuts qualification electrophorogram by Fig. 8 again;

Fig. 9 is the preparation of sweet wormwood aseptic seedling;

Figure 10 is the differentiation of antibacterial peptide sweet wormwood;

Figure 11 is resistant transgenic sweet wormwood seedling;

Figure 12 is that PCR screens positive transgenic sweet wormwood seedling;

Figure 13 is the expression that real-time fluorescence quantitative RT-PCR detects Blf.

In FIG, swimming lane M is 1kbplusmarker (10000bp, 8000bp, 6000bp, 5000bp, 4000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp), and swimming lane 3 is the pcr amplification product of pRB.

In fig. 2, swimming lane M is 1kbplusmarker (10000bp, 8000bp, 6000bp, 5000bp, 4000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp), and swimming lane 1 is the digestion products of pRB-T carrier.

In figure 3, swimming lane M is 1kbplusmarker (10000bp, 8000bp, 6000bp, 5000bp, 4000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp), and swimming lane 3 is the digestion products after the pRB sequence that in pCambia1301, insertion connection clone obtains.

In the diagram, swimming lane M is 1kbplusmarker (10000bp, 8000bp, 6000bp, 5000bp, 4000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp), and swimming lane 2 cuts qualification for the T202 enzyme built.

In Figure 5, swimming lane M is D2000marker (2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp), and swimming lane 2,3 is the PCR primer of bovine lactoferrin gene.

In figure 6, swimming lane M is D2000marker, and swimming lane 7-10 is the digestion products of Blf-T carrier.

In the figure 7, swimming lane M is D2000marker, and swimming lane 7,8 is after carrier T202-Blf transformation of E. coli DH5 α, extract plasmid digestion products.

In fig. 8, swimming lane M is D2000marker, and swimming lane 6,7 is, after T202-Blf is proceeded to Agrobacterium EHA105, the plasmid DNA of extraction is proceeded to the digestion products after bacillus coli DH 5 alpha again.

In fig .9, the sweet wormwood aseptic seedling for preparing.

In Fig. 10, be the differentiation of antibacterial peptide sweet wormwood.

In fig. 11, be transgene abrotanum seedling.

In fig. 12, swimming lane M is DL2000DNAmarker (2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp), swimming lane 1 is without Template Controls, swimming lane 2 is wild type control, and swimming lane 9,10,11,13,16,18 is transfer-gen plant PCR screening positive transgenic sweet wormwood seedling;

In fig. 13, for real-time fluorescence quantitative RT-PCR detects the expression of Blf.

Sequence used in embodiment embodiment is sequence mentioned above

The method of lactoferrin produced by sweet wormwood of the present invention, and it comprises the following steps:

A, transformation carrier T202;

The recombinant plasmid T202-Blf of b, structure sweet wormwood cells bovine lactoferrin gene;

C, use recombinant plasmid T202-Blf transform sweet wormwood;

D, screening positive transgenic sweet wormwood seedling, and carry out real-time RT-PCR qualification.

Specific operation process is:

1, carrier T202 is transformed

Clone pRB fragment:

With with pCambia1300 for template, design and synthesis primer amplification pRB (Fig. 1).PCR reaction conditions: 94 DEG C of 5min denaturations, 94 DEG C 30s-59 DEG C 30s-72 DEG C of 70s, 30 circulations, 72 DEG C of 7min.After 2% agarose gel electrophoresis, reclaim purifying object fragment, be stored in-20 DEG C for subsequent use.The pRB fragment obtained is connected with T-carrier (being purchased from precious biotechnology (Dalian) company limited), transformation of E. coli DH5 α bacterial strain, at 37 DEG C, with HindIII and XbalI double digestion screening positive strain (Fig. 2).Send the positive bacteria Ye Song company sequencing analysis checking pRB exactness of sequence.

Be connected on pCambia1301 by the pRB sequence be cloned into, restriction enzyme site is (EcoRI-KpnI, GAATTC---GGTACC), and novel vector is named: pC13001T (Fig. 3).Cut Actin promotor (cloned by this seminar member in advance and connect into pMD18TVector, and order-checking display is correct) with KpnI and SmaI enzyme, cut pC13001T with KpnI and PmacI enzyme simultaneously, be connected with Actin promotor, be built into T202.Select EcoRI single endonuclease digestion, the band (Fig. 4) of 810bp should be occurred.

Synthesis and clone's bovine lactoferrin gene:

According to the gene order of lactoferrin mature peptide in NCBIGenBank, the improved bovine lactoferrin gene of synthetic (hereinafter referred to as " bovine lactoferrin gene ") is also stored in PMD19Simple plasmid.Synthesize a pair special primer BlfF1 and BlfF2 (table 1) according to its gene order engineer again, with in PMD19Simple plasmid for template to be increased lactoferrin (LfcinB) gene fragment by pcr clone.PCR reaction conditions: 94 DEG C of 5min, 94 DEG C 30s-56 DEG C 30s-72 DEG C of 20s, 30 circulations, 72 DEG C of 7min.After 2% agarose gel electrophoresis, as shown in Figure 5, in figure 6, swimming lane M is D2000marker to result, and swimming lane 2-3 is the PCR primer of bovine lactoferrin gene.

Reclaim purifying object fragment, be stored in-20 DEG C for subsequent use.The bovine lactoferrin gene obtained is connected with carrier T, transformation of E. coli DH5 α bacterial strain, at 37 DEG C, with PstI and XbaI double digestion screening positive strain (Fig. 6), send the exactness of positive bacteria Ye Song company sequencing analysis checking bovine lactoferrin gene sequence.

Sequencing result shows: the carrier T being connected into LfcinB gene order, as shown in 1-105 position Nucleotide in sequence 4, is denoted as Blf-T carrier by the sequence of LfcinB gene.

Table 1 the primer sequence

2, construction of expression vector-recombinant plasmid T202-Blf

With PstI, XbaI respectively enzyme cut improved carrier T202 and carrier B lf-T, product, after 1.5% agarose gel electrophoresis, reclaims bovine lactoferrin gene fragment and T202 carrier segments.T4 ligase enzyme, 16 DEG C connect 2h and obtain recombinant plasmid T202-Blf, transformation of E. coli DH5 α bacterial strain.Conversion rear extraction plasmid DNA is carried out enzyme and is cut qualification (Fig. 7).

After recombinant plasmid T202-Blf after qualification is proceeded to Agrobacterium EHA105, the plasmid DNA of extraction is proceeded to again the enzyme after bacillus coli DH 5 alpha and cut qualification (Fig. 8).

3, recombinant plasmid T202-Blf transforms sweet wormwood

1) three days in advance Agrobacteriums prepare: LB plate (is cooled to 60 DEG C after the sterilizing of LB solid medium, adds Kan100mg/L+CHL34mg/L+Rif100mg/L, be down flat plate;

2) coating of bacterium liquid;

3) cultivation 2-3 days is inverted for 28 DEG C;

4) NBM solid medium, is down flat plate after having added As, for subsequent use.NBM liquid nutrient medium.Filter paper, triangular flask, water, sterilizing in advance;

5) the dull and stereotyped bacterium of cultured LB is washed in the Dual culture base (NBM+As0.1mM) of 50ml liquid, and range estimation concentration, adjusts OD600=0.5;

6) agrobacterium mediation converted: be cut into the sheet of the square size of 1cm by realizing accurate originally good sweet wormwood aseptic seedling (Fig. 9), transfer in bacterium liquid and soak 30min, shaking table shakes.With sterilizing filter paper suck dry moisture, dry up slightly.Sweet wormwood is transferred to Dual culture base (NBM+As0.1mM)

It is put a sterilizing filter paper, light culture 3 days at 25 ~ 26 DEG C;

7) screening and culturing: after 3 days, proceeds to sweet wormwood in sterilized triangular flask, with bacterium water rinse 5 times not muddy to liquid, then be added with 500mg/L head born of the same parents and 400mg/L carboxylic paleness mycin aqua sterilisa soak 30min, shaking table shake.With sterilizing filter paper suck dry moisture.Transfer to screening culture medium (MS+500mg/L head born of the same parents+400mg/L carboxylic paleness mycin+50mg/L Totomycin), light culture at 25 ~ 26 DEG C, after 20 days, the callus entirety growing kanamycin-resistant callus tissue is proceeded in new screening culture medium;

8) differentiation culture: the callus global transfer of kanamycin-resistant callus tissue will be grown in screening culture medium to (Figure 10) in division culture medium after terminating twice screening, be placed in 25 ~ 26 DEG C, 14h illumination cultivation, light intensity 1000 ~ 1500lx illumination cultivation, within every 20 days, change a subculture, to differentiating healthy and strong transgene abrotanum seedling (Figure 11);

9) with after the positive rate of PCR detection gained resistance sweet wormwood plant (Figure 12), real-time RT-PCR is utilized to detect the expression amount (Figure 13) of antibacterial peptide lactoferrin in sweet wormwood, result shows, in wild-type sweet wormwood, there is no lactoferrin, and in positive transgenic sweet wormwood, lactoferrin is all had to express in L1-L5, and with the expression amount in L1 for 1, the relative expression quantity of L3 and L5 is higher, be respectively 2.46 and 2.87, the relative expression quantity of L2 and L4 is the highest, be respectively 5.97 and 4.39, we will select the vegetative propagation (tissue cultured seedling) of L2 and L4, seedling used is produced as next step.

Claims (3)

1. produce a method for lactoferrin with sweet wormwood, it is characterized in that, described method comprises the steps:

(1) carrier T202 is transformed; Improved carrier T202 sequence is as shown in SEQIDNO.1;

(2) the recombinant plasmid T202-Blf of the improved bovine lactoferrin gene of sweet wormwood cells is built; The sequence of recombinant plasmid T202-Blf is as shown in SEQIDNO.2;

(3) sweet wormwood is transformed with recombinant plasmid T202-Blf;

(4) screen positive transgenic sweet wormwood plant, obtain the sweet wormwood plant producing lactoferrin.

2. the method for claim 1, is characterized in that, the process of step (1) described transformation carrier T202 is as follows:

A () take pCambia1300 as template, carry out pcr amplification by the pRB sequence of following primer pair TDNA:

Primer pRB-F1:5 '- gAATTCaCTGGGAATTCCCTGGCGTTA-3 ';

Primer pRB-R1:5 '- gGTACCcAGCCTGTCGGGTACCTTAGGA-3 ';

B pRB sequence qualification that pcr amplification obtains by (); Be connected on pCambia1301 by correct pRB sequence, obtain carrier pC13001T, carrier pC13001T sequence is as shown in SEQIDNO.3;

C () cuts Actin promotor with KpnI and SmaI enzyme, cut carrier pC13001T with KpnI and PmacI enzyme simultaneously, and the Actin promotor that carrier pC13001T and the enzyme cut by enzyme are cut is connected, and is built into improved carrier T202.

3. the method for claim 1, is characterized in that, the process of the recombinant plasmid T202-Blf of step (2) described structure sweet wormwood cells bovine lactoferrin gene is as follows:

A () synthesizes improved bovine lactoferrin gene sequence, and be stored in PMD19Simple plasmid, and improved bovine lactoferrin gene sequence is as shown in SEQIDNO.4;

B () is increased with the improved bovine lactoferrin gene that following primer pair is stored in PMD19Simple plasmid:

Primer BlfF1:5 ' cTGCAGtGGCATCTCTCAGCACATATG3 ';

Primer BlfR1:5 ' tCTAGAcTTGTCGTCGTCTTAGTAACAC3 ';

C improved bovine lactoferrin gene order-checking qualification that pcr amplification obtains by (); Be connected with T-carrier by correct improved bovine lactoferrin gene, obtain carrier B lf-T, carrier B lf-T sequence is as shown in SEQIDNO.5;

(d) with PstI, XbaI respectively enzyme cut improved carrier T202 and carrier B lf-T, the improved carrier T202 cut by enzyme is connected with the carrier B lf-T that enzyme is cut, and is built into recombinant plasmid T202-Blf.