CN105349568A - Yeast expression vector containing encoding Mnp protein gene and method of establishing and preparing Mnp protein and degrading lignin - Google Patents

Abstract

The invention discloses a yeast expression vector containing an encoding Mnp protein gene. The vector contains an Mnp gene and can produce extracellularly Mnp proteins in connection with Lactic acid yeast. The invention also discloses the establishment of the yeast expression vector containing an encoding Mnp protein gene and a method for preparing Mnp protein and degrading lignin in oilseed rape straws. Mnp is cloned by using the modern molecular technologies to successfully establish the expression vector. Extracellular secretion and expression of Mnp can be carried out using the vector in connection with Lactic acid yeast GG799, culturing time of an original strain is greatly shortened, Mnp activity is improved, and the Mnp produced is effective in degrading the lignin of rape stalks. Operating is simple, and the commercial production and application of Lentinus edodes Mnp are facilitated.

Description

A kind of Yeast expression carrier containing coding Mnp protein gene and structure, prepare the method for Mnp albumen and lignin degrading

Technical field

The invention belongs to the expression vector genetically engineered studying technological domain of saccharomyces lactis cell exocrine albumen, be specifically related to a kind of expression vector for saccharomyces lactis (KluyveromyceslactisGG799) cell exocrine mushroom manganese peroxidase (Mnp) albumen.The invention still further relates to the construction process of this expression vector.

Background technology

China's rapeseed cultivation area is large, rape stalk aboundresources.Containing a large amount of carbohydrate such as Mierocrystalline cellulose (stem reaches 53%), hemicellulose (18.34%) in rape stalk, the important feed source (Zhao Mengmeng etc. of ruminating animal, the composition analysis of several agricultural crop straw, material Leader, 2011(16): 122-125).But in producing, rape stalk is not widely used in livestock breeding industry, major part is directly burned (Cao Guoliang etc., the estimation of regional farmland Open Burning Crop Residue quantity discharged, Science Bulletin, 2007(15): 1826-183) or decomposition also field, not only waste resource, go back serious environment pollution.Trace it to its cause and be, rape stalk degree of lignification is high, stalk is thick and stiff, palatability is poor; In rape stalk, xylogen and Mierocrystalline cellulose form firm ester bond structure, suppress the degraded of rumen microorganism, reduce cud digestibility, if not processing treatment, animal feed intake can be affected, reduce production performance (Ramirez-BribiescaJE, WangY, JinL, etal.ChemicalcharacterizationandinvitrofermentationofBra ssicastrawtreatedwiththeaerobicfungus, Trametesversicolor.CanadianJournalofAnimalScience, 2011,91 (4): 695-702).Adopting the mode such as acidifying, alkalization process rape stalk more in the past, but its residual soda acid, can damage animal itself, and contaminate environment (Zhang Wenjie etc., the progress of stalk treatment process, Chinese animal and veterinary, 2011(07): 30-33).Compared with above-mentioned chemical process, utilize microbial fermentation technology process rape stalk, then green, safe, pollution-free, and palatability and the utilization ratio of feed can be improved.

White-rot fungi is the most effective microorganism of occurring in nature lignin degrading.Use white-rot fungi Treating straw, content of lignin can be reduced significantly, improve cud utilization ratio (SharmaRK, AroraDS.Productionoflignocellulolyticenzymesandenhanceme ntofinvitrodigestibilityduringsolidstatefermentationofwh eatstrawbyPhlebiafloridensis.Bioresourcetechnology, 2010,101 (23): 9248-9253; AroraDS, GillPK.ProductionofligninolyticenzymesbyPhlebiafloridens is.WorldJournalofMicrobiologyandBiotechnology, 2005,21 (6-7): 1021-1028).Applicant finds in early-stage Study, Lentinus Edodes fungus (L.edodes) can utilize manganese peroxidase (Mnp) lignin degrading of secretion in fermentation rape stalk process, improve the Effective Degradability of rape stalk and external organic substance disappearance rate, but simultaneously, the cellulase produced in fermenting process also can Mierocrystalline cellulose in severe attrition rape stalk, the cuds such as hemicellulose can utilize material, cause the waste (Zhaoetal. of fodder energy, Effectoffungaltreatmentsofrapestrawonchemicalcomposition andinvitrorumenfermentationcharacteristics.Bioresources, 2015, 10 (1): 622-637).How finding a kind of lignin degrading and the method for not degraded cellulose, is current problem demanding prompt solution.

Summary of the invention

The present invention utilizes molecular approach successfully to construct can at yeast vivoexpression Mnp albumen (Lentinulaedodesmnp2mRNAformanganeseperoxidase, completecds, GenBank:AB306943.1, http://www.ncbi.nlm.nih.gov/nuccore/AB306943.1) expression vector pKL-Mnp, utilize this carrier and saccharomyces lactis can prepare Mnp albumen, and then provide technical support for the application aborning of Mnp albumen.

The present invention is achieved through the following technical solutions foregoing invention content:

A kind of Yeast expression carrier containing coding mushroom manganese peroxidase (Mnp) albumen, is characterized in that, containing Mnp gene in this carrier, and can extracellular production Mnp albumen in conjunction with saccharomyces lactis bacterium.

The present invention also provides this to contain the construction process of the Yeast expression carrier of coding Mnp protein gene, the steps include:

(1) Lentinus Edodes fungus solid state fermentation on the rape stalk pulverized is cultivated 20d, solid state fermentation moisture content is 70%; Solid state fermentation terminates rear extraction mushroom mycelium RNA, utilizes round pcr amplification in vitro mushroom Mnp gene fragment;

(2) Mnp gene fragment is connected pMD19-T cloning vector, import competent escherichia coli cell DH5 α by thermal shock method, transformation of E. coli builds recombinant cloning vector pMD-Mnp;

(3) utilize restriction enzyme XhoI and BamHI to carry out double digestion to recombinant cloning vector pMD-Mnp, glue reclaims Mnp gene fragment;

(4) the restriction enzymes double zyme cutting expression vector pKLAC2 identical with step 3 is utilized;

(5) by step 3 glue reclaim Mnp gene fragment with cut in step 4 after pKLAC2 carrier be connected, competent escherichia coli cell DH5 α is imported by thermal shock method, transformation of E. coli builds recombinant expression vector pKL-Mnp, and recycling plasmid extraction kit extracts expression vector plasmid pKL-Mnp from recombination bacillus coli.

The present invention also provides a kind of Yeast expression carrier containing coding Mnp protein gene to prepare the method for Mnp albumen, and its step is,

(1) extract above-mentioned recombinant expression vector pKL-Mnp, utilize restriction enzyme SacII to carry out linearizing to pKL-Mnp carrier, reaction system is: 10 × QuickCutbuffer5 μ L, SacII1 μ L, plasmid 8 μ L, and aqua sterilisa 36 μ L, hatches 5min for 30 DEG C;

(2) linearizing pKL-Mnp carrier is proceeded to commercial yeast bacterium by electroporation apparatus, condition: voltage, 2.5kv; Time, 5ms; The YCB substratum being placed in acetamide-containing afterwards is again cultivated, and screening is containing the recombination microzyme bacterium colony of Mnp gene;

(3) join in YPD liquid culture medium by filtering out containing the recombination microzyme bacterium colony of Mnp gene, 30 DEG C of concussion cultivation 3 ~ 7d, Mnp albumen is secreted in nutrient solution, and the recombination microzyme bacterium liquid 12h containing Mnp gene is secreted into external manganese peroxidase enzymic activity and reaches 56.5U/L.

As optimization, the YCB substratum of described acetamide-containing consists of, 30mmol/LTris-HCl, 1.17% yeast carbon source basis, 2% agar powder, 5mmol/L ethanamide;

As optimization, described YPD liquid culture medium consists of, 1% yeast extract paste, 2% peptone, 2% glucose.

The present invention also provides a kind of method of the Yeast expression carrier lignin degrading containing coding Mnp protein gene, and its step is,

(1) get 250ml Erlenmeyer flask, add 50 ~ 100ml containing 2 ~ 4%(percent by volume) the above-mentioned YPD substratum having integrated the recombination microzyme bacterium liquid of Mnp gene;

(2) in Erlenmeyer flask, 0.5 ~ 1.0g rape stalk is added again;

Shake cultivation 3 ~ 7d in (3) 30 DEG C of shaking tables, degrade 10 ~ 20%(weight percentage) rape stalk xylogen.

Utilizing goal gene that is that in-vitro separation can arrive by recombinant DNA technology or synthesis, being connected by recombinating with carrier, import not containing the recipient cell of this gene, make recipient cell produce goal gene albumen.The Mnp enzyme produced in fermentation rape stalk process based on Lentinus Edodes fungus can effective lignin degrading, this patent is intended to build a kind of Yeast expression carrier containing Mnp gene, think Mnp albumen commercially produce and rape stalk fermentation and in other in application provide fundamental basis and technical support.

Use expression vector of the present invention, yeast 12h internal secretion can be made to reach 56.5U/L to external manganese peroxidase enzymic activity, meet or exceed the manganese peroxidase enzymic activity secreted by mushroom solid state fermentation rape stalk 30d.Illustrate that the present invention successfully constructs expression vector pKL-Mnp, and in yeast GG799, there is higher expression activity.Core of the present invention can effectively to be degraded rape stalk xylogen for mushroom manganese peroxidase, but mushroom cultivates the longer and realistic problem that manganese enzyme secretion is lower consuming time, molecular approach is utilized to be cloned by mushroom Mnp gene, success construction of expression vector, and carry out exocytosis and expression in conjunction with saccharomyces lactis bacterium, result reduces incubation time greatly, improves the activity of manganese enzyme.Simple to operate, be convenient to commercially producing and applying of mushroom Mnp.

Accompanying drawing explanation

Fig. 1 is pcr amplification product Mnp electrophorogram;

Fig. 2 is restructuring cloned plasmids transformation of E. coli;

The PCR electrophorogram of Fig. 3 cloned plasmids pMD-Mnp;

The double digestion electrophoresis of Fig. 4 cloned plasmids pMD-Mnp;

The PCR electrophorogram of Fig. 5 expression plasmid pKL-Mnp;

The PCR electrophorogram of Fig. 6 recombination microzyme;

The Mnp enzymic activity that Fig. 7 recombination microzyme 12h secretes;

Fig. 8 recombination microzyme is to rape stalk xylogen and cellulosic degraded.

Embodiment

Below in conjunction with accompanying drawing and embodiment, the present invention is described in further detail.

Embodiment 1

The preparation of bacterial strain, carrier, reagent: competent escherichia coli cell DH5 α is purchased from TIANGEN Biotech (Beijing) Co., Ltd. (CB101-01); Lentinus Edodes fungus (Lentinulaedodes50040) is purchased from Chinese agriculture Culture Collection.Saccharomyces lactis and expression vector pKLAC2 are purchased from NEB company, fungi total serum IgE extracting and purifying test kit (SK8655), DNA glue reclaims test kit (SK8743), PCR primer purification kit (SK8741), the little extraction reagent kit of plasmid (SK8791) is purchased from Sheng Gong biotechnology limited-liability company, cDNA synthetic agent box (6210A), T4-DNA ligase enzyme (2011A), PremixTaq (ExTaqVersion2.0) PCR mixed solution (RR003A), restriction enzyme XhoI(1635), BamHI(1605), SacII(1628), pMD19-T cloning vector kit (6013) is purchased from Dalian Bao Bio-Engineering Company.

Embodiment 2

Design of primers and synthesis: according to the gene order ((Lentinulaedodesmnp2mRNAformanganeseperoxidase of mushroom manganese peroxidase (Mnp) in GeneBank, completecds, GenBank:AB306943.1, http://www.ncbi.nlm.nih.gov/nuccore/AB306943.1), design primer (F:CGACTCGAGAAAAGAGCGGTTTGTTCTGACGG; R:TCGGATCCTTAAGAAGTGCAAGTGGCTTC), restriction enzyme XhoI and BamHI site is added at the two ends of primer respectively during design; The Mnp clip size of expection amplification is that 1079bp(contains restriction enzyme site).

Embodiment 3

The extraction of mushroom total serum IgE: get the Lentinus Edodes fungus of growth on solid medium, be inoculated in the culture dish containing rape stalk, gets mushroom mycelium after growth 20d, extracts mushroom total serum IgE according to fungi pillar total serum IgE test kit specification sheets.

Concrete grammar is as follows:

1, the Bufferrlysis-FG getting 450 μ L adds in the centrifuge tube without RNA enzyme of 1.5ml for subsequent use.

2, get 25-50mg fresh hypha,hyphae liquid nitrogen grinding powdered, join in the centrifuge tube of above-mentioned 1.5ml, shake mixing immediately, room temperature places 5min.

3, in 12000rpm rotating speed, centrifugal 3min at 4 DEG C, supernatant is transferred in the centrifuge tube of 1.5ml without RNA enzyme.

4, add the dehydrated alcohol of 1/2 volume, fully mix.

5, adsorption column is put into centrifuge tube, all added in adsorption column by solution with pipettor, centrifugal 1min under standing 1min, room temperature 12000rpm rotating speed, outwells waste liquid in collection tube.

6, adsorption column is put back in centrifuge tube, add 500 μ LGTsolution, leave standstill the centrifugal 1min of 1min, room temperature 10000rpm rotating speed, outwell waste liquid in collection tube.

7, put back in centrifuge tube by adsorption column, add 500 μ LNTsolution, centrifugal 1min under standing 1min, room temperature 10000rpm rotating speed, outwells waste liquid in collection tube.

8, adsorption column is put back in collection tube, centrifugal 2min under room temperature 12000rpm rotating speed.

9, adsorption column is put into the centrifuge tube without RNA enzyme, add 30-50 μ LDEPC-treatedddH2O in adsorption film central authorities, centrifugal 2min under standing 2min, room temperature 12000rpm rotating speed, is placed in-70 DEG C of preservations, for follow-up test by obtained RNA solution.

Embodiment 4

RT-PCR amplification Mnp gene, carries out in two steps:

1, reverse transcription is carried out according to cDNA synthetic agent box: Random6mers1 μ l, dNTPMixture1 μ l, RNA8 μ l, after 65 DEG C of insulation 5min, solid-state trash ice cools rapidly, add 5*PrimeScriptBuffer4 μ l, RNase inhibitor 0.5 μ l, PrimeScriptRTase1 μ l, water 4.5 μ l, hatch 45min for 45 DEG C afterwards, hatch 15min for last 70 DEG C;

2, RT-PCR amplification: reaction system: PremixEXTaq25 μ l, cDNA2 μ l, each 1 μ l of primer, aqua sterilisa 21 μ l.Loop parameter: 94 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 1min; Circulate 30 times, amplification rear electrophoresis detects (Fig. 1).Result display band is consistent with object clip size.Check order to PCR primer, result and mushroom manganese enzyme mRNA similarity reach 99%(and see pcr amplification mushroom Mnp gene product gene order table), show that this fragment is the gene fragment of manganese enzyme.

Embodiment 5

The structure of cloning vector pMD-Mnp:

(1) utilize glue to reclaim test kit (Sheng Gong biotechnology limited-liability company, model SK8743), according to specification sheets method, purifying and recovery are carried out to PCR primer;

Method: make sepharose with tbe buffer liquid, agarose gel electrophoresis is carried out to target DNA; Under ultraviolet lamp, cut the sepharose containing target DNA band, exhaust the liquid of gel surface with paper handkerchief; Chopping blob of viscose, weighs blob of viscose weight, and calculate blob of viscose volume (according to 1mg=1 μ L), in blob of viscose, add blob of viscose melting liquid DR-Ibuffer, add-on is 3 times of blob of viscose volume; The rear 75 DEG C of heating and melting blob of viscoses of mixing mixing, are interrupted concussion simultaneously, blob of viscose are fully melted (about 6-10min); In blob of viscose melting liquid, add DR-IIbuffer, add-on is 1/2 of DR-Ibuffer, mixing mixing.When separation is less than the DNA fragmentation of 400bp, the Virahol that final concentration is 20% should be added in this solution; SpinColumn in test kit is placed on CollectionTube; Be transferred to by solution in SpinColumn, the centrifugal 1min of 3600r/min, abandons filtrate; Add in SpinColumn by the RinseA of 500 μ L, the centrifugal 30s of 3600r/min, abandons filtrate; Add in SpinColumn by the RinseB of 700 μ L, the centrifugal 30s of 3600r/min, abandons filtrate; Repeat previous step, the centrifugal 1min of 12000r/min; Be placed in by SpinColumn on the centrifuge tube of new 1.5mL, add the smuggled goods elutriant of 25 μ L in the central authorities of SpinColumn film, room temperature leaves standstill 1min; The centrifugal 1min eluted dna of 12000r/min; Agarose gel electrophoresis detects;

(2) get control group (, with the PCR primer of A base, pMD19-T support agent box provides for the 3 ' end of 500bp) and Mnp object fragment 4 μ l respectively, connect damping fluid 5 μ l, pMD19-T carrier 1 μ l mixes, 16 DEG C of water-bath overnight incubation; Get 10 μ l Incubating Solutions to add in 200 μ l competent escherichia coli cell DH5 α, softly mix, ice bath 30min; 42 DEG C of accurate thermal shock 90s, ice bath 2min; Add LB liquid nutrient medium (1% peptone, 0.5% yeast extract, 1% sodium-chlor) 800 μ l, cultivate 1h for 37 DEG C; Coating is dull and stereotyped containing the solid-state LB substratum (1% peptone, 0.5% yeast extract, 1% sodium-chlor, 1.5% agar powder) of Amp, X-gal, IPTG, 37 DEG C of overnight incubation; Picking white colony (Fig. 2) joins in the LB liquid medium containing Amp, 37 DEG C of incubated overnight, after substratum muddiness, utilizes plasmid extraction kit to extract transfer vector plasmid.

The qualification of recombinant cloning vector: the recombinant plasmid utilizing Mnp primer pair to extract carries out pcr amplification, to identify whether object fragment successfully inserts pMD19-T vector plasmid.Result display from the plasmid proceeding to Mnp gene the object fragment (Fig. 3) having amplified 1079bp, and control group is without object fragment band, shows that Mnp gene has successfully proceeded in pMD19-T carrier;

Utilize XhoI and BamHI restriction enzyme to carry out double digestion to recombinant plasmid, the enzyme system of cutting is: 10 × QuickCutbuffer5 μ L, XhoI1 μ L, BamHI1 μ L, plasmid 8 μ L, aqua sterilisa 35 μ L, hatch 5min for 30 DEG C, then 37 DEG C hatches 5min.Electrophoresis result shows, and has occurred two bands (Fig. 4) in digestion products, and more than a band 1000bp wherein, with (1079bp) in the same size of object fragment; (2692bp) in the same size of another band and carrier.Again show that object fragment has inserted pMD19-T carrier, cloning vector pMD-Mnp successfully constructs.

Embodiment 6

The structure of recombinant expression vector pKL-Mnp: utilize XhoI and BamHI restriction enzyme to carry out double digestion (method is with embodiment 5) to restructuring cloned plasmids pMD-Mnp and expression plasmid pKLAC2 respectively, glue reclaims the Mnp fragment in plasmid pMD-Mnp; The pKLAC2 plasmid that purifying enzyme is cut; Get Mnp object fragment 4 μ l, T4 ligase enzyme and damping fluid 5 μ l, the pKLAC2 carrier 1 μ l of purifying mixes, 16 DEG C of water-bath overnight incubation.Transformation of E. coli competent cell DH5 α, screening positive clone (method is with embodiment 5) in the LB substratum containing Amp.Picking white colony joins in the LB liquid medium containing Amp, 37 DEG C of concussion incubated overnight, after substratum muddiness, utilizes plasmid extraction kit to extract recombinant expression vector plasmid.

The qualification of recombinant expression vector pKL-Mnp: carry out PCR checking (with embodiment 5).Result obtains the band (Fig. 5) of a 1079bp respectively, consistent with object fragment, shows expression vector establishment success.

Embodiment 7:

Transform saccharomyces lactis: utilize restriction enzyme SacII to contrast expression vector pKLAC2() and recombinant expression vector pKL-Mnp(treatment group) carry out linearization for enzyme restriction, the enzyme system of cutting is: 10 × QuickCutbuffer5 μ L, SacII1 μ L, plasmid 8 μ L, aqua sterilisa 36 μ L, hatches 5min for 30 DEG C.PCR primer purification kit is utilized to carry out purifying to digestion products; Utilize electroporation apparatus that linearizing expression vector pKLAC2 and pKL-Mnp is proceeded to saccharomyces lactis competent cell, in the YCB substratum (30mmol/LTris-HCl of acetamide-containing, 1.17% yeast carbon source basis, 2% agar powder, 5mmol/L ethanamide) middle cultivation, utilize PCR screening positive clone (Fig. 6).From control group picking 1 white colony with join in the test tube of 10mlYPD substratum (1% yeast extract paste, 2% peptone, 2% glucose) respectively from treatment group picking 10 white colonies, 30 DEG C of concussion incubated overnight.

The mensuration of manganese peroxidase enzymic activity: the enzyme assay of 12h manganese peroxidase is carried out to the bacterium liquid in each test tube.Result shows, control group is without manganese peroxidase enzymic activity, and proceed in 10 pipe recombination microzyme liquid of expression vector pKL-Mnp treatment group and all detect manganese peroxidase enzymic activity, wherein the manganese peroxidase enzymic activity of 2# bacterium liquid 12h reaches 56.5U/L(Fig. 7).

Embodiment 8:

Recombination microzyme is to the degraded of rape stalk: get 6 250ml Erlenmeyer flasks, the YPD nutrient solution (treatment group) of 50ml containing 2# recombination microzyme bacterium liquid (percent by volume) in 2% embodiment 7 is added wherein in 3 Erlenmeyer flasks, remain 3 and add 50ml containing the saccharomycetic YPD nutrient solution (control group) of non-recombinant, backward each Erlenmeyer flask add 0.5g rape stalk, in 30 DEG C of shaking tables, 5d is cultivated in concussion, analyzes rape stalk xylogen residual in bottle and cellulose amount.Result shows, through the cultivation of 5d, control group without Degradation, and degrades the rape stalk xylogen (Fig. 8) of 13%, 15%, 20% to xylogen respectively containing the treatment group of recombination microzyme.In addition, control group and treatment group also degrade the Mierocrystalline cellulose (0.1% and 0.05%) of trace respectively, and this may be because saccharomyces lactis itself creates trace or more SA cellulase.These results suggest that, the recombination microzyme built in the present invention can be used for effective degraded of rape stalk xylogen.

Be pMD19-T cloning vector gene order table, pcr amplification mushroom Mnp gene product gene order table and the sequence table of pKL-Mnp vector gene below.

1, pMD19-T cloning vector gene order table

Precious biotechnology (Dalian) company limited of <110>

<120>pMD19-T cloning vector

<130>-

<160>1

<170>PatentInversion3.5

<210>1

<211>2692

<212>DNA

<213>Escherichiacoli

<400>1

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca60

cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtg120

ttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgc180

accatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgcc240

attcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctat300

tacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggt360

tttcccagtcacgacgttgtaaaacgacggccagtgaattcgagctcggtacccggggat420

cctctagagatatcgtcgacctgcaggcatgcaagcttggcgtaatcatggtcatagctg480

tttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcata540

aagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctca600

ctgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgc660

gcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctg720

cgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtta780

tccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggcc840

aggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgag900

catcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagatac960

caggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttacc1020

ggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgt1080

aggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccccc1140

gttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaaga1200

cacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgta1260

ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagta1320

tttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga1380

tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacg1440

cgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcag1500

tggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacc1560

tagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaact1620

tggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctattt1680

cgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggctta1740

ccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagattta1800

tcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatcc1860

gcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaat1920

agtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggt1980

atggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttg2040

tgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgca2100

gtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgta2160

agatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcgg2220

cgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaact2280

ttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccg2340

ctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatctttt2400

actttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaaggga2460

ataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagc2520

atttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaa2580

caaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccatt2640

attatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc2692

2, pcr amplification mushroom Mnp gene product gene order table

<110> Agricultural University Of Jiangxi

<120> mushroom manganese peroxidase PCR primer checks order

<130>-

<160>1

<170>PatentInversion3.5

<210>1

<211>1010

<212>DNA

<213>Escherichiacoli

<220>

<221> and mushroom manganese peroxidase mRNA comparison Matching band

<222>(17)..(1009)

<400>1

ggaaaatgggtccgcttttgctgtgattttattccgcttgcccaagatcttaccgcaact60

ctattcgagaaccagtgtggcgaaactgcccatgaggtacttcgtctgagcttccacgat120

gctatcgctatctcccaatctcttggacctgcggctggaggaggtgctgacggctctatg180

ttgattttcccggacgtcgaacccaacttcgctgccaaccttggtatctccgacagtgtc240

aatgacctcgctccattccttgcctcaggaaaattcccgactatcactgctggagatatg300

atccagttcggtgctgccgtcgctgtcggcctttgccctggcgcacctcagctggagttt360

ttggctggccgacccaatgccactgcgcctgcagttgacggtttgatccctgaacctcaa420

gactcggtcgactcaatcttggctcgtttccaggatgcggcaaacttgacctctgaagac480

attgtctctctgctcgtatcgcacacagtcgcccgagcagaccatgtcgaccccactctg540

gatgctgcgcctttcgactcgactccattcacctttgacacacagttcttccttgaaact600

ctcttgactggtgtcggattcccgggaactcctaacaacactggtgaagtgtcctcccct660

cttcccctcactgtcggtgtaaacgttggagagctgcgacttcaatccgactttgagctc720

gctcgtgacaaccggacagcctgcttctggcaaagcatgatcaacgaagaatccttgatg780

gcatcgaggttccaggctgctatggccaaaatggctatcattggacacaaccgtgctgac840

ttgattgactgctccgctgttgttccgactcccgttccttctttgggcgtccccgcgact900

ttcccggcgacgaagagtttcgctgatgttcagcaagcgtgcccttcgcccttcccgagt960

ctcacctccgaccgtgctcccagggagactgagatccacacgcccagacc1010

3, pKL-Mnp vector gene sequence table

<110> Agricultural University Of Jiangxi

The Yeast expression carrier of <120> mono-kind containing coding Mnp protein gene

<130>HeterologousproteinproductionintheyeastKluyveromyceslactis

<160>1

<170>PatentInversion3.5

<210>1

<211>10109

<212>DNA

<213> Kluyveromyces lactis (Kluyveromyceslactis)

<220>

<221> mushroom manganese peroxidase coding region

<222>(269)..(1324)

<400>1

aagcttgaaaaaaatgaaattctctactatattagccgcatctactgctttaatttccgt60

tgttatggctgctccagtttctaccgaaactgacatcgacgatcttccaatatcggttcc120

agaagaagccttgattggattcattgacttaaccggggatgaagtttccttgttgcctgt180

taataacggaacccacactggtattctattcttaaacaccaccatcgctgaagctgcttt240

cgctgacaaggatgatctcgagaaaagagcggtttgttctgacggcactgttgtgcccga300

cagcgtttgctgtgattttatcccacttgcccaagatcttaccgcaactctattcgagaa360

ccagtgtggcgaaactgcccatgaggtacttcgtctgagcttccacgatgctatcgctat420

ctcccaatctcttggacctgcggctggaggaggtgctgacggctctatgttgattttccc480

ggacgtcgaacccaacttcgctgccaaccttggtatctccgacagtgtcaatgacctcgc540

tccattccttgcctcaggaaaattcccgactatcactgctggagatatgatccagttcgg600

tgctgccgtcgctgtcggcctttgccctggcgcacctcagctggagtttttggctggccg660

acccaatgccactgcgcctgcagttgacggtttgatccctgaacctcaagactcggtcga720

ctcaatcttggctcgtttccaggatgcggcaaacttgacctctgaagacattgtctctct780

gctcgtatcgcacacagtcgcccgagcagaccatgtcgaccccactctggatgctgcgcc840

tttcgactcgactccattcacctttgacacacagttcttccttgaaactctcttgactgg900

tgtcggattcccgggaactcctaacaacactggtgaagtgtcctcccctcttcccctcac960

tgtcggtgtaaacgttggagagctgcgacttcaatccgactttgagctcgctcgtgacaa1020

ccggacagcctgcttctggcaaagcatgatcaacgaagaatccttgatggcatcgaggtt1080

ccaggctgctatggccaaaatggctatcattggacacaaccgtgctgacttgattgactg1140

ctccgctgttgttccgactcccgttccttctttgggcgtccccgcgactttcccggcgac1200

gaagagtttcgctgatgttcagcaagcgtgcccttcgcccttcccgagtctcacctccga1260

ccgtgctcccagggagactgagattcctcactgccccgacaacgaagccacttgcacttc1320

ttaaggatccgaattccctgcaggtaattaaataaaggccttgaatcgagaatttatact1380

tagataagtatgtacttacaggtatatttctatgagatactgatgtatacatgcatgata1440

atatttaaacggttattagtgccgattgtcttgtgcgataatgacgttcctatcaaagca1500

atacacttaccacctattacatgggccaagaaaatattttcgaacttgtttagaatatta1560

gcacagagtatatgatgttatccgttagattatgcatgattcattcctacaactttttcg1620

tagcataaggattaattacttggatgccaataaaaaaaaaaaacatcgagaaaatttcag1680

catgctcagaaacaattgcagtgtatcaaagtaaaaaaaagattttcactacatgttcct1740

tttgaagaaagaaaatcatggaacattagatttacaaaaatttaaccaccgctgattaac1800

gattagaccgttaagcgcacaacaggttattagtacagagaaagcattctgtggtgttgc1860

cccggactttcttttgcgacataggtaaatcgaataccatcatactatcttttccaatga1920

ctccctaaagaaagactcttcttcgatgttgtatacgttggagcatagggcaagaattgt1980

ggcttgagatcatccttttgttgtttccgggtgtacaatatggacttcctcttttctggc2040

aaccaaacccatacatcgggattcctataataccttcgttggtctccctaacatgtaggt2100

ggcggaggggagatatacaatagaacagataccagacaagacataatgggctaaacaaga2160

ctacaccaattacactgcctcattgatggtggtacataacgaactaatactgtagcccta2220

gacttgatagccatcatcatatcgaagtttcactaccctttttccatttgccatctattg2280

aagtaataataggcgcatgcaacttcttttctttttttttcttttctctctcccccgttg2340

ttgtctcaccatatccgcaatgacaaaaaaatgatggaagacactaaaggaaaaaattaa2400

cgacaaagacagcaccaacagatgtcgttgttccagagctgatgaggggtatctcgaagc2460

acacgaaactttttccttccttcattcacgcacactactctctaatgagcaacggtatac2520

ggccttccttccagttacttgaatttgaaataaaaaaaagtttgctgtcttgctatcaag2580

tataaatagacctgcaattattaatcttttgtttcctcgtcattgttctcgttccctttc2640

ttccttgtttctttttctgcacaatatttcaagctataccaagcatacaatcaagcaatt2700

ccagatctgccaccatgcctcaatcctgggaagaactggccgctgataagcgcgcccgcc2760

tcgcaaaaaccatccctgatgaatggaaagtccagacgctgcctgcggaagacagcgtta2820

ttgatttcccaaagaaatcgggcatcctttcagaggccgaactgaagatcacagaggctt2880

ccgctgcggatcttgtgtccaagctggcggccggagagttgacctcggtggaagttacgc2940

tagcattctgtaaacgggcagcaatcgcccagcagttaacaaactgcgcccacgagttct3000

tccctgacgccgctctcgcgcaggcaagggaactcgatgaatactacgcaaagcacaaga3060

gacccgttggtccacttcatggcctccccatctctctcaaagaccagcttcgagtcaagg3120

gctacgaaacatcaatgggctacatctcatggctaaacaagtacgacgaaggggactcgg3180

ttctgacaaccatgctccgcaaagccggtgccgtcttctacgtcaagacctctgtcccgc3240

agaccctgatggtctgcgagacagtcaacaacatcatcgggcgcaccgtcaacccacgca3300

acaagaactggtcgtgcggcggcagttctggtggtgagggtgcgatcgttgggattcgtg3360

gtggcgtcatcggtgtaggaacggacatcggtggctcgattcgagtgccggccgcgttca3420

acttcctgtacggtctaaggccgagtcatgggcggctgccgtatgcaaagatggcgaaca3480

gcatggagggtcaggagacggtgcacagcgttgtcgggccgattacgcactctgttgagg3540

acctccgcctcttcaccaaatccgtcctcggtcaggagccttggaaatacgactccaagg3600

tcatccccatgccctggcgccagtccgagtcggacattattgcctccaagatcaagaacg3660

gcgggctcaatatcggctactacaacttcgacggcaatgtccttccacaccctcctatcc3720

tgcgcggcgtggaaactaccgtcgccgcactcgccaaagccggtcacaccgtgaccccgt3780

ggacgccatacaagcacgatttcggccacgatctcatctcccatatctacgcggctgacg3840

gcagcgccgacgtaatgcgcgacatcagtgcatccggcgagccggcgattccaaatatca3900

aagacctactgaacccgaacatcaaagctgttaacatgaacgagctctgggacacgcatc3960

tccagaagtggaattaccagatggagtaccttgagaaatggcgggaggctgaagaaaagg4020

ccgggaaggaactggacgccatcatcgcgccgattacgcctaccgctgcggtacggcatg4080

accagttccggtactatgggtatgcctctgtgatcaacctgctggatttcacgagcgtgg4140

ttgttccggttacctttgcggataagaacatcgataagaagaatgagagtttcaaggcgg4200

ttagtgagcttgatgccctcgtgcaggaagagtatgatccggaggcgtatcatggggcac4260

cggttgcagtgcaggttatcggacggagactcagtgaagagaggacgttggcgattgcag4320

aggaagtggggaagttgctgggaaatgtggtgactccatagcccggggggggctcgatcc4380

cctcgcgagttggttcagctgctgcctgaggctggacgacctcgcggagttctaccggca4440

gtgcaaatccgtcggcatccaggaaaccagcagcggctatccgcgcatccatgcccccga4500

actgcaggagtggggaggcacgatggccgctttggtcgatctagattacgtggaagaaag4560

gtagtaaaagtagtagtataagtagtaaaaagaggtaaaaagagaaaaccggctacatac4620

tagagaagcacgtacacaaaaactcataggcacttcatcatacgacagtttcttgatgca4680

ttataatagtgtattagatattttcagaaatatgcatagaacctcctcttgcctttactt4740

tttatacatagaacattggcagatttacttacactactttgtttctacgccatttctttt4800

gttttcaacacttagacaagttgttgagaaccggactactaaaaagcaatgttcccactg4860

aaaatcatgtacctgcagcataataaccccctaattctgcatcgatccagtatgtttttt4920

tttctctactcatttttacctgaagatagagcttctaaaacaaaaaaaatcagtgattac4980

atgcatattgtgtgttctagtaaccaaaggaaaggaacagatagataaaattccgagact5040

gtcaaattaggtttttttctttttttttggcgggagtcagtgggccgaaatatgttcttg5100

gcctagaacttaatctggtttgatcatgccaatacttgcctgagtgcccgactttttgcc5160

caccctcttgccttctgtcatccttcaaaacccacctgttttccagccgtatcttcgctc5220

gcatctacacatactgtgccatatcttgtgtgtagccggacgtgactatgaccaaaaaca5280

aacaaggagaactgttcgccgatttgtaacactcctgcatccatccaagtgggtatgcgc5340

tatgcaatgttaagctaggtcaggtcagaccaggtccaaggacagcaacttgactgtatg5400

caacctttaccatctttgcacagaacatacttgtagctagctagttacacttatggaccg5460

aaaaggcaccccaccatgtctgtccggctttagagtacggccgcagaccgctgatttgcc5520

ttgccaagcagtagtcacaatgcatcgcatgagcacacgggcacgggcacgggcacagga5580

accattggcaaaaataccagatacactataccgacgtatatcaagcccaagtttaaaatt5640

cctaaatttccgcggctacttttcaattccctatagtgagtcgtattaaattcgtaatca5700

tgtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgag5760

ccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattg5820

cgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaa5880

tcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctca5940

ctgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcgg6000

taatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggcc6060

agcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcc6120

cccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggac6180

tataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccc6240

tgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcata6300

gctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgc6360

acgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtcca6420

acccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagag6480

cgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacacta6540

gaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttg6600

gtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagc6660

agcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggt6720

ctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaa6780

ggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatat6840

atgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcga6900

tctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatac6960

gggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccgg7020

ctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctg7080

caactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagtt7140

cgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgct7200

cgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgat7260

cccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagta7320

agttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtca7380

tgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaat7440

agtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccac7500

atagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaa7560

ggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatctt7620

cagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccg7680

caaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaat7740

attattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtattt7800

agaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgc7860

cctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacac7920

ttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcg7980

ccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctt8040

tacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgc8100

cctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactct8160

tgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataaggga8220

ttttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcga8280

attttaacaaaatattaacgcttacaatttccattcgccattcaggctgcgcaactgttg8340

ggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgc8400

tgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgac8460

ggccagtgccaagctcccgcggggatcgactcataaaatagtaaccttctaatgcgtatc8520

tattgactaccaaccattagtgtggttgcagaaggcggaattttcccttcttcgaattta8580

gcttgctttttcattttttattttccatttttcagtttttgtttgtgtcgaatttagcca8640

gttgcttctccaagatgaaaaaaacccctgcgcagtttctgtgctgcaagatcctaatcg8700

acttttccaccccccacaaaagtaaatgttcttttgttacattcgcgtgggtagctagct8760

ccccgaatcttcaaaggacttagggactgcactacatcagagtgtgttcacctggtttgc8820

tgcctggtttgaaagaaaagagcagggaactcgcgggttcccggcgaataatcatgcgat8880

agtcctttggccttccaagtcgcatgtagagtagacaacagacagggagggcaggaagga8940

tctttcactgagatcctgtatcttgttgggtaagtcggatgaaaggggaatcgtatgaga9000

ttggagaggatgcggaagaggtaacgccttttgttaacttgtttaattattatggggcag9060

gcgagagggggaggaatgtatgtgtgtgaggcgggcgagacggagccatccaggccaggt9120

agaaatagagaaagccgaatgttagacaatatggcagcgtagtagagtaggtaggtaggc9180

aagtactgctagcaaagaggagaagggtaagctcactcttcgcattccacaccgttagtg9240

tgtcagtttgaacaaaaaaacaatcatcataccaattgatggactgtggactggcttttg9300

gaacggcttttcggactgcgattattcgtgaggaatcaaggtaggaatttggtcatattt9360

acggacaacagtgggtgattcccatatcgagtaggaaaacgagatcatggtatcctcaga9420

tatgttgcggaaatctgttcaccgcaaagttcagggtgctctggtgggtttcggttggtc9480

tttgctttgcttctcccttgtcttgcatgttaataatagcctagcctgtgagccgaaact9540

tagggtaggcttagtgttggaacgtacatatctatcacgttgacttggtttaaccaggcg9600

acctggtagccagccatacccacacacgttttttgtatcttcagtatagttgtgaaaagt9660

gtagcggaaatttgtggtccgagcaacagcgtctttttctagtagtgcggtcggttactt9720

ggttgacattggtatttggactttgttgctacaccattcactacttgaagtcgagtgtga9780

agggtatgatttctagtggtgaacacctttagttacgtaatgttttcattgctgttttac9840

ttgagatttcgattgagaaaaaggtatttaatagctcgaatcaatgtgagaacagagaga9900

agatgttcttccctaactcgaaaggtatatgaggcttgtgtttcttaggagaattattat9960

tcttttgttatgttgcgcttgtagttggaaaaggtgaagagacaaaagctggaattgtga10020

gcggataacaagctcaacacttgaaatttaggaaagagcagaatttggcaaaaaaaataa10080

aaaaaaaataaacacacatactcatcgag10109

Precious biotechnology (Dalian) company limited of <110>

<120>pMD19-T cloning vector

<130>-

<160>1

<170>PatentInversion3.5

<210>1

<211>2692

<212>DNA

<213>Escherichiacoli

<400>1

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca60

cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtg120

ttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgc180

accatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgcc240

attcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctat300

tacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggt360

tttcccagtcacgacgttgtaaaacgacggccagtgaattcgagctcggtacccggggat420

cctctagagatatcgtcgacctgcaggcatgcaagcttggcgtaatcatggtcatagctg480

tttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcata540

aagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctca600

ctgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgc660

gcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctg720

cgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtta780

tccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggcc840

aggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgag900

catcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagatac960

caggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttacc1020

ggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgt1080

aggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccccc1140

gttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaaga1200

cacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgta1260

ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagta1320

tttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga1380

tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacg1440

cgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcag1500

tggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacc1560

tagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaact1620

tggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctattt1680

cgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggctta1740

ccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagattta1800

tcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatcc1860

gcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaat1920

agtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggt1980

atggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttg2040

tgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgca2100

gtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgta2160

agatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcgg2220

cgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaact2280

ttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccg2340

ctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatctttt2400

actttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaaggga2460

ataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagc2520

atttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaa2580

caaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccatt2640

attatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc2692

<110> Agricultural University Of Jiangxi

<120> mushroom manganese peroxidase PCR primer checks order

<130>-

<160>1

<170>PatentInversion3.5

<210>1

<211>1010

<212>DNA

<213>Escherichiacoli

<220>

<221> and mushroom manganese peroxidase mRNA comparison Matching band

<222>(17)..(1009)

<400>1

ggaaaatgggtccgcttttgctgtgattttattccgcttgcccaagatcttaccgcaact60

ctattcgagaaccagtgtggcgaaactgcccatgaggtacttcgtctgagcttccacgat120

gctatcgctatctcccaatctcttggacctgcggctggaggaggtgctgacggctctatg180

ttgattttcccggacgtcgaacccaacttcgctgccaaccttggtatctccgacagtgtc240

aatgacctcgctccattccttgcctcaggaaaattcccgactatcactgctggagatatg300

atccagttcggtgctgccgtcgctgtcggcctttgccctggcgcacctcagctggagttt360

ttggctggccgacccaatgccactgcgcctgcagttgacggtttgatccctgaacctcaa420

gactcggtcgactcaatcttggctcgtttccaggatgcggcaaacttgacctctgaagac480

attgtctctctgctcgtatcgcacacagtcgcccgagcagaccatgtcgaccccactctg540

gatgctgcgcctttcgactcgactccattcacctttgacacacagttcttccttgaaact600

ctcttgactggtgtcggattcccgggaactcctaacaacactggtgaagtgtcctcccct660

cttcccctcactgtcggtgtaaacgttggagagctgcgacttcaatccgactttgagctc720

gctcgtgacaaccggacagcctgcttctggcaaagcatgatcaacgaagaatccttgatg780

gcatcgaggttccaggctgctatggccaaaatggctatcattggacacaaccgtgctgac840

ttgattgactgctccgctgttgttccgactcccgttccttctttgggcgtccccgcgact900

ttcccggcgacgaagagtttcgctgatgttcagcaagcgtgcccttcgcccttcccgagt960

ctcacctccgaccgtgctcccagggagactgagatccacacgcccagacc1010

<110> Agricultural University Of Jiangxi

The Yeast expression carrier of <120> mono-kind containing coding Mnp protein gene

<130>HeterologousproteinproductionintheyeastKluyveromyceslactis

<160>1

<170>PatentInversion3.5

<210>1

<211>10109

<212>DNA

<213> Kluyveromyces lactis (Kluyveromyceslactis)

<220>

<221> mushroom manganese peroxidase coding region

<222>(269)..(1324)

<400>1

aagcttgaaaaaaatgaaattctctactatattagccgcatctactgctttaatttccgt60

tgttatggctgctccagtttctaccgaaactgacatcgacgatcttccaatatcggttcc120

agaagaagccttgattggattcattgacttaaccggggatgaagtttccttgttgcctgt180

taataacggaacccacactggtattctattcttaaacaccaccatcgctgaagctgcttt240

cgctgacaaggatgatctcgagaaaagagcggtttgttctgacggcactgttgtgcccga300

cagcgtttgctgtgattttatcccacttgcccaagatcttaccgcaactctattcgagaa360

ccagtgtggcgaaactgcccatgaggtacttcgtctgagcttccacgatgctatcgctat420

ctcccaatctcttggacctgcggctggaggaggtgctgacggctctatgttgattttccc480

ggacgtcgaacccaacttcgctgccaaccttggtatctccgacagtgtcaatgacctcgc540

tccattccttgcctcaggaaaattcccgactatcactgctggagatatgatccagttcgg600

tgctgccgtcgctgtcggcctttgccctggcgcacctcagctggagtttttggctggccg660

acccaatgccactgcgcctgcagttgacggtttgatccctgaacctcaagactcggtcga720

ctcaatcttggctcgtttccaggatgcggcaaacttgacctctgaagacattgtctctct780

gctcgtatcgcacacagtcgcccgagcagaccatgtcgaccccactctggatgctgcgcc840

tttcgactcgactccattcacctttgacacacagttcttccttgaaactctcttgactgg900

tgtcggattcccgggaactcctaacaacactggtgaagtgtcctcccctcttcccctcac960

tgtcggtgtaaacgttggagagctgcgacttcaatccgactttgagctcgctcgtgacaa1020

ccggacagcctgcttctggcaaagcatgatcaacgaagaatccttgatggcatcgaggtt1080

ccaggctgctatggccaaaatggctatcattggacacaaccgtgctgacttgattgactg1140

ctccgctgttgttccgactcccgttccttctttgggcgtccccgcgactttcccggcgac1200

gaagagtttcgctgatgttcagcaagcgtgcccttcgcccttcccgagtctcacctccga1260

ccgtgctcccagggagactgagattcctcactgccccgacaacgaagccacttgcacttc1320

ttaaggatccgaattccctgcaggtaattaaataaaggccttgaatcgagaatttatact1380

tagataagtatgtacttacaggtatatttctatgagatactgatgtatacatgcatgata1440

atatttaaacggttattagtgccgattgtcttgtgcgataatgacgttcctatcaaagca1500

atacacttaccacctattacatgggccaagaaaatattttcgaacttgtttagaatatta1560

gcacagagtatatgatgttatccgttagattatgcatgattcattcctacaactttttcg1620

tagcataaggattaattacttggatgccaataaaaaaaaaaaacatcgagaaaatttcag1680

catgctcagaaacaattgcagtgtatcaaagtaaaaaaaagattttcactacatgttcct1740

tttgaagaaagaaaatcatggaacattagatttacaaaaatttaaccaccgctgattaac1800

gattagaccgttaagcgcacaacaggttattagtacagagaaagcattctgtggtgttgc1860

cccggactttcttttgcgacataggtaaatcgaataccatcatactatcttttccaatga1920

ctccctaaagaaagactcttcttcgatgttgtatacgttggagcatagggcaagaattgt1980

ggcttgagatcatccttttgttgtttccgggtgtacaatatggacttcctcttttctggc2040

aaccaaacccatacatcgggattcctataataccttcgttggtctccctaacatgtaggt2100

ggcggaggggagatatacaatagaacagataccagacaagacataatgggctaaacaaga2160

ctacaccaattacactgcctcattgatggtggtacataacgaactaatactgtagcccta2220

gacttgatagccatcatcatatcgaagtttcactaccctttttccatttgccatctattg2280

aagtaataataggcgcatgcaacttcttttctttttttttcttttctctctcccccgttg2340

ttgtctcaccatatccgcaatgacaaaaaaatgatggaagacactaaaggaaaaaattaa2400

cgacaaagacagcaccaacagatgtcgttgttccagagctgatgaggggtatctcgaagc2460

acacgaaactttttccttccttcattcacgcacactactctctaatgagcaacggtatac2520

ggccttccttccagttacttgaatttgaaataaaaaaaagtttgctgtcttgctatcaag2580

tataaatagacctgcaattattaatcttttgtttcctcgtcattgttctcgttccctttc2640

ttccttgtttctttttctgcacaatatttcaagctataccaagcatacaatcaagcaatt2700

ccagatctgccaccatgcctcaatcctgggaagaactggccgctgataagcgcgcccgcc2760

tcgcaaaaaccatccctgatgaatggaaagtccagacgctgcctgcggaagacagcgtta2820

ttgatttcccaaagaaatcgggcatcctttcagaggccgaactgaagatcacagaggctt2880

ccgctgcggatcttgtgtccaagctggcggccggagagttgacctcggtggaagttacgc2940

tagcattctgtaaacgggcagcaatcgcccagcagttaacaaactgcgcccacgagttct3000

tccctgacgccgctctcgcgcaggcaagggaactcgatgaatactacgcaaagcacaaga3060

gacccgttggtccacttcatggcctccccatctctctcaaagaccagcttcgagtcaagg3120

gctacgaaacatcaatgggctacatctcatggctaaacaagtacgacgaaggggactcgg3180

ttctgacaaccatgctccgcaaagccggtgccgtcttctacgtcaagacctctgtcccgc3240

agaccctgatggtctgcgagacagtcaacaacatcatcgggcgcaccgtcaacccacgca3300

acaagaactggtcgtgcggcggcagttctggtggtgagggtgcgatcgttgggattcgtg3360

gtggcgtcatcggtgtaggaacggacatcggtggctcgattcgagtgccggccgcgttca3420

acttcctgtacggtctaaggccgagtcatgggcggctgccgtatgcaaagatggcgaaca3480

gcatggagggtcaggagacggtgcacagcgttgtcgggccgattacgcactctgttgagg3540

acctccgcctcttcaccaaatccgtcctcggtcaggagccttggaaatacgactccaagg3600

tcatccccatgccctggcgccagtccgagtcggacattattgcctccaagatcaagaacg3660

gcgggctcaatatcggctactacaacttcgacggcaatgtccttccacaccctcctatcc3720

tgcgcggcgtggaaactaccgtcgccgcactcgccaaagccggtcacaccgtgaccccgt3780

ggacgccatacaagcacgatttcggccacgatctcatctcccatatctacgcggctgacg3840

gcagcgccgacgtaatgcgcgacatcagtgcatccggcgagccggcgattccaaatatca3900

aagacctactgaacccgaacatcaaagctgttaacatgaacgagctctgggacacgcatc3960

tccagaagtggaattaccagatggagtaccttgagaaatggcgggaggctgaagaaaagg4020

ccgggaaggaactggacgccatcatcgcgccgattacgcctaccgctgcggtacggcatg4080

accagttccggtactatgggtatgcctctgtgatcaacctgctggatttcacgagcgtgg4140

ttgttccggttacctttgcggataagaacatcgataagaagaatgagagtttcaaggcgg4200

ttagtgagcttgatgccctcgtgcaggaagagtatgatccggaggcgtatcatggggcac4260

cggttgcagtgcaggttatcggacggagactcagtgaagagaggacgttggcgattgcag4320

aggaagtggggaagttgctgggaaatgtggtgactccatagcccggggggggctcgatcc4380

cctcgcgagttggttcagctgctgcctgaggctggacgacctcgcggagttctaccggca4440

gtgcaaatccgtcggcatccaggaaaccagcagcggctatccgcgcatccatgcccccga4500

actgcaggagtggggaggcacgatggccgctttggtcgatctagattacgtggaagaaag4560

gtagtaaaagtagtagtataagtagtaaaaagaggtaaaaagagaaaaccggctacatac4620

tagagaagcacgtacacaaaaactcataggcacttcatcatacgacagtttcttgatgca4680

ttataatagtgtattagatattttcagaaatatgcatagaacctcctcttgcctttactt4740

tttatacatagaacattggcagatttacttacactactttgtttctacgccatttctttt4800

gttttcaacacttagacaagttgttgagaaccggactactaaaaagcaatgttcccactg4860

aaaatcatgtacctgcagcataataaccccctaattctgcatcgatccagtatgtttttt4920

tttctctactcatttttacctgaagatagagcttctaaaacaaaaaaaatcagtgattac4980

atgcatattgtgtgttctagtaaccaaaggaaaggaacagatagataaaattccgagact5040

gtcaaattaggtttttttctttttttttggcgggagtcagtgggccgaaatatgttcttg5100

gcctagaacttaatctggtttgatcatgccaatacttgcctgagtgcccgactttttgcc5160

caccctcttgccttctgtcatccttcaaaacccacctgttttccagccgtatcttcgctc5220

gcatctacacatactgtgccatatcttgtgtgtagccggacgtgactatgaccaaaaaca5280

aacaaggagaactgttcgccgatttgtaacactcctgcatccatccaagtgggtatgcgc5340

tatgcaatgttaagctaggtcaggtcagaccaggtccaaggacagcaacttgactgtatg5400

caacctttaccatctttgcacagaacatacttgtagctagctagttacacttatggaccg5460

aaaaggcaccccaccatgtctgtccggctttagagtacggccgcagaccgctgatttgcc5520

ttgccaagcagtagtcacaatgcatcgcatgagcacacgggcacgggcacgggcacagga5580

accattggcaaaaataccagatacactataccgacgtatatcaagcccaagtttaaaatt5640

cctaaatttccgcggctacttttcaattccctatagtgagtcgtattaaattcgtaatca5700

tgtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgag5760

ccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattg5820

cgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaa5880

tcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctca5940

ctgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcgg6000

taatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggcc6060

agcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcc6120

cccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggac6180

tataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccc6240

tgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcata6300

gctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgc6360

acgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtcca6420

acccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagag6480

cgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacacta6540

gaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttg6600

gtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagc6660

agcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggt6720

ctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaa6780

ggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatat6840

atgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcga6900

tctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatac6960

gggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccgg7020

ctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctg7080

caactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagtt7140

cgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgct7200

cgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgat7260

cccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagta7320

agttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtca7380

tgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaat7440

agtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccac7500

atagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaa7560

ggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatctt7620

cagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccg7680

caaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaat7740

attattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtattt7800

agaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgc7860

cctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacac7920

ttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcg7980

ccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctt8040

tacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgc8100

cctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactct8160

tgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataaggga8220

ttttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcga8280

attttaacaaaatattaacgcttacaatttccattcgccattcaggctgcgcaactgttg8340

ggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgc8400

tgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgac8460

ggccagtgccaagctcccgcggggatcgactcataaaatagtaaccttctaatgcgtatc8520

tattgactaccaaccattagtgtggttgcagaaggcggaattttcccttcttcgaattta8580

gcttgctttttcattttttattttccatttttcagtttttgtttgtgtcgaatttagcca8640

gttgcttctccaagatgaaaaaaacccctgcgcagtttctgtgctgcaagatcctaatcg8700

acttttccaccccccacaaaagtaaatgttcttttgttacattcgcgtgggtagctagct8760

ccccgaatcttcaaaggacttagggactgcactacatcagagtgtgttcacctggtttgc8820

tgcctggtttgaaagaaaagagcagggaactcgcgggttcccggcgaataatcatgcgat8880

agtcctttggccttccaagtcgcatgtagagtagacaacagacagggagggcaggaagga8940

tctttcactgagatcctgtatcttgttgggtaagtcggatgaaaggggaatcgtatgaga9000

ttggagaggatgcggaagaggtaacgccttttgttaacttgtttaattattatggggcag9060

gcgagagggggaggaatgtatgtgtgtgaggcgggcgagacggagccatccaggccaggt9120

agaaatagagaaagccgaatgttagacaatatggcagcgtagtagagtaggtaggtaggc9180

aagtactgctagcaaagaggagaagggtaagctcactcttcgcattccacaccgttagtg9240

tgtcagtttgaacaaaaaaacaatcatcataccaattgatggactgtggactggcttttg9300

gaacggcttttcggactgcgattattcgtgaggaatcaaggtaggaatttggtcatattt9360

acggacaacagtgggtgattcccatatcgagtaggaaaacgagatcatggtatcctcaga9420

tatgttgcggaaatctgttcaccgcaaagttcagggtgctctggtgggtttcggttggtc9480

tttgctttgcttctcccttgtcttgcatgttaataatagcctagcctgtgagccgaaact9540

tagggtaggcttagtgttggaacgtacatatctatcacgttgacttggtttaaccaggcg9600

acctggtagccagccatacccacacacgttttttgtatcttcagtatagttgtgaaaagt9660

gtagcggaaatttgtggtccgagcaacagcgtctttttctagtagtgcggtcggttactt9720

ggttgacattggtatttggactttgttgctacaccattcactacttgaagtcgagtgtga9780

agggtatgatttctagtggtgaacacctttagttacgtaatgttttcattgctgttttac9840

ttgagatttcgattgagaaaaaggtatttaatagctcgaatcaatgtgagaacagagaga9900

agatgttcttccctaactcgaaaggtatatgaggcttgtgtttcttaggagaattattat9960

tcttttgttatgttgcgcttgtagttggaaaaggtgaagagacaaaagctggaattgtga10020

gcggataacaagctcaacacttgaaatttaggaaagagcagaatttggcaaaaaaaataa10080

aaaaaaaataaacacacatactcatcgag10109

Claims (6)

1. the Yeast expression carrier containing coding Mnp protein gene, is characterized in that, containing Mnp gene in this carrier, and can extracellular production Mnp albumen in conjunction with saccharomyces lactis bacterium.

2. the construction process of the Yeast expression carrier as claimed in claim 1 containing coding Mnp protein gene, the steps include:

(1) Lentinus Edodes fungus solid state fermentation on the rape stalk pulverized is cultivated 20d, solid state fermentation moisture content is 70%; Solid state fermentation terminates rear extraction mushroom mycelium RNA, utilizes round pcr amplification in vitro mushroom Mnp gene fragment;

(2) Mnp gene fragment is connected pMD19-T cloning vector, import competent escherichia coli cell DH5 α by thermal shock method, transformation of E. coli builds recombinant cloning vector pMD-Mnp;

(3) utilize restriction enzyme XhoI and BamHI to carry out double digestion to recombinant cloning vector pMD-Mnp, glue reclaims Mnp gene fragment;

(4) the restriction enzymes double zyme cutting expression vector pKLAC2 identical with step 3 is utilized;

(5) by step 3 glue reclaim Mnp gene fragment with cut in step 4 after pKLAC2 carrier be connected, competent escherichia coli cell DH5 α is imported by thermal shock method, transformation of E. coli builds recombinant expression vector pKL-Mnp, and recycling plasmid extraction kit extracts expression vector plasmid pKL-Mnp from recombination bacillus coli.

3. the Yeast expression carrier as claimed in claim 1 containing coding Mnp protein gene prepares the method for Mnp albumen, and its step is,

(1) extract above-mentioned recombinant expression vector pKL-Mnp, utilize restriction enzyme SacII to carry out linearizing to pKL-Mnp carrier, reaction system is: 10 × QuickCutbuffer5 μ L, SacII1 μ L, plasmid 8 μ L, and aqua sterilisa 36 μ L, hatches 5min for 30 DEG C;

(2) linearizing pKL-Mnp carrier is proceeded to commercial yeast bacterium by electroporation apparatus, condition: voltage, 2.5kv; Time, 5ms; The YCB substratum being placed in acetamide-containing afterwards is again cultivated, and screening is containing the recombination microzyme bacterium colony of Mnp gene;

(3) join in YPD liquid culture medium by filtering out containing the recombination microzyme bacterium colony of Mnp gene, 30 DEG C of concussion cultivation 3 ~ 7d, Mnp albumen is secreted in nutrient solution, and the bacterium liquid 12h containing the recombination microzyme of Mnp gene is secreted into external manganese peroxidase enzymic activity and reaches 56.5U/L.

4. the Yeast expression carrier as claimed in claim 3 containing coding Mnp protein gene prepares the method for Mnp albumen, and it is characterized in that, the YCB substratum of described acetamide-containing consists of, 30mmol/LTris-HCl, 1.17% yeast carbon source basis, 2% agar powder, 5mmol/L ethanamide.

5. the Yeast expression carrier containing coding Mnp protein gene as described in claim 3 or 4 prepares the method for Mnp albumen, and it is characterized in that, described YPD liquid culture medium consists of, 1% yeast extract paste, 2% peptone, 2% glucose.

6. the method for the Yeast expression carrier lignin degrading as claimed in claim 1 containing coding Mnp protein gene, its step is,

(1) get 250ml Erlenmeyer flask, add 50 ~ 100ml containing 2 ~ 4%(percent by volume) the above-mentioned YPD substratum having integrated the recombination microzyme bacterium liquid of Mnp gene;

(2) in Erlenmeyer flask, 0.5 ~ 1.0g rape stalk is added again;

Shake cultivation 3 ~ 7d in (3) 30 DEG C of shaking tables, degrade 10 ~ 20%(weight percentage) rape stalk xylogen.