CN103773748B - Diaphania saccharase, encoding gene, genophore, bacterial strain and application - Google Patents
Diaphania saccharase, encoding gene, genophore, bacterial strain and application Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2431—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01026—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
Abstract
The invention discloses a kind of saccharase from diaphania, encoding gene, genophore, bacterial strain and application. Does is diaphania β-FFase provided by the invention SEQ in sequence table? ID? the protein being formed by 488 amino acid residues shown in NO.1. The present invention clones and obtains in intestines transcription product from mulberry tree important pests diaphania<i>DpSuc1a</i>This gene is expressed in the middle intestines a large amount of diaphania, pET-24b is connected with expression vector, be transformed into BL21(DE3) competent cell, through abduction delivering, obtain soluble fusion protein DpSUC1a, identify through enzymatic property, restructuring DpSUC1a has the activity of β-FFase, illustrates that β-FFase is the diaphania mulberry tree of causing harm, and avoids the important enzyme albumen of mulberry tree alkaloid toxic action. Gene of the present invention and encoding proteins thereof are to the alkaloidal Study on Molecular Mechanism of food injurious insect of mulberry tree opposing mulberry tree, and further utilize the novel pest-resistant targets of technological development such as plant transgene RNAi to there is important theory and practical significance, by playing an important role in the research of Mulberry Pests control and enhancing mulberry tree insect resistace, have a extensive future.
Description
Technical field
The invention belongs to insect molecular biology and photobiochemistry field, be specifically related to diaphania β-FFase geneVivoexpression and the enzyme activity assay of the amplification of DpSuc1a, the cloning and identification of full-length cDNA and encoding proteins thereof.
Background technology
Various plants is oozed out emulsion or other secretion at the position of mechanical injuries or insect's food-taking. Large quantity research tableBright these emulsions are rich in number of chemical protective substance, and as terpene, alkaloid, flavonoids, steroids etc., it is by the middle intestines of insectHave an effect, hinder ingesting or growing up of insect, and then playing an important role aspect defence plant-feed insect food evil. Emulsion thereforeBe considered to closely related with the pest-resistant defense function of plant.
On the other hand, the plant-feed insect complicated adaptability to plant of evolving out, supports from molecule, physiology, behavior aspectThe infringement of imperial various emulsions. Mulberry tree is to be found up to now to contain the carbohydrates such as DNJ (DNJ) like alkaloidal severalPlant one of higher plant, in the emulsion of mulberry leaf, contain the secondary metabolite alkaloid of high concentration. The people's such as Konno research is sent outExisting, in mulberry leaf emulsion contained alkaloid to some lepidopterous insects as oligophagous castor silkworm and polyphagous lopper worm childrenWorm has strong toxic action.
Cane sugar hydrolytic enzyme has two kinds: a kind of is the alpha-glucosidase of catalytic action from glucosyl group one side, another kindThat (β-fructofuranosidase, β-FFase, in animal for saccharase from fructosyl one side sucrose hydrolysisRarely has report). Research shows, alkaloid is the potent inhibitor of alpha-glucosidase, and β-FFase is not had to inhibitory action.Silkworm (Bombyxmori) is the economic insects taking mulberry leaf as unique food and sugared source of nutrition, in the genome of silkworm, existsβ-FFase gene (BmSuc1), its in larva in intestines specifically expressing being proved there is the enzymatic activity that not suppressed by alkaloidMatter. Research thinks that silkworm utilizes the function of β-FFase fully to decompose the sucrose nutrition absorbing in mulberry leaf just, thereby successfullyAvoid the toxic action of Folium Mori alkaloid.
For pluralistic trend and the requirement of current sericulture there, mulberry tree not only can be used as the feed resource of silkworm,More wide, mulberry tree can also be served as ecological mulberry, for administering stony desertification, play that sand prevention is protected a forest and protection of the environment etc. in many waysThe effect of face. Diaphania (Diaphaniapyloalis) is one of mulberry tree important pests, to many provinces of China mulberry tree of summer and autumnCause harm quite serious. We find the enzymatic activity that contains β-FFase in the midgut tissue albumen of diaphania, same in the genome of diaphaniaThere is the homologous gene (DpSuc1a) of β-FFase in sample, this gene is at a large amount of transcriptional expressions of middle intestines quilt of diaphania. The present invention is to mulberryThe cDNA of β-FFase gene of snout moth's larva increases, clone and sequence analysis, by the vivoexpression DpSUC1a zymoprotein of recombinating, mirrorDetermine the active function of enzyme. Gene of the present invention and encoding proteins thereof grind the alkaloidal molecular mechanism of food injurious insect of mulberry tree opposing mulberry treeStudy carefully, and further utilize the novel pest-resistant targets of technological development such as plant transgene RNAi to there is important theoretical and actual meaningJustice, by playing an important role in the research of Mulberry Pests control and enhancing mulberry tree insect resistace, has a extensive future.
Summary of the invention
One of object of the present invention is to provide encoding gene, the amplification side of a kind of new diaphania β-FFase gene DpSuc1aMethod and amplimer group, the nucleotide sequence of its encoding gene DpSuc1a is as shown in SEQIDNO.2.
For achieving the above object, the invention provides following technical scheme:
The invention provides the primer sets for diaphania β-FFase gene that increases, formed by following primer:
Degenerate primer: forward primer SUCdgpF is as shown in SEQIDNO.3;
Reverse primer SUCdgpR is as shown in SEQIDNO.4.
RACE primer: 5 ' RACE outer primer DpSuc1a-5RAgsp, as shown in SEQIDNO.5;
5 ' RACE inner primer DpSuc1a-5RAngsp, as shown in SEQIDNO.6;
3 ' RACE outer primer DpSuc1a-3RAgsp, as shown in SEQIDNO.7;
3 ' RACE inner primer DpSuc1a-3RAngsp, as shown in SEQIDNO.8.
The present invention also provides the preparation method of diaphania β-FFase gene, comprises the steps:
Step 1: the total RNA that obtains intestines in diaphania;
Step 2: cDNA is synthesized in reverse transcription;
Step 3: taking cDNA as masterplate, be that primer carries out PCR with degenerate primer SUCdgpF and SUCdgpR, during amplification obtainsBetween sequence;
Step 4: 5 ' end and the 3 ' end fragment that obtains diaphania β-FFase genetic fragment with the amplification of RACE primer sets;
Step 5: by 5 ' end of the intermediate segment of described β-FFase gene cDNA sequence, described β-FFase gene and 3 ' endFragment assembly, obtains the full-length cDNA fragment of diaphania β-FFase gene.
As preferably, amplification is shell type two-wheeled PCR described in step 4.
Two of object of the present invention is to provide the recombinant plasmid pET-24b/ that contains new diaphania β-FFase geneDpSuc1a and construction method thereof.
For achieving the above object, the invention provides following technical scheme:
Primer DpSuc1aF and DpSuc1aR that utilization contains restriction enzyme site carry out pcr amplification to the ORF of DpSuc1a, expandIncrease primer DpSuc1aF and DpSuc1aR respectively as shown in SEQIDNO.9 and SEQIDNO.10; Pcr amplification reaction conditionFor: 94 DEG C of denaturation 5min, then 94 DEG C of sex change 40s, 58 DEG C of annealing 40s, 72 DEG C of extension 1min30s, totally 30 circulations,Latter 72 DEG C are extended 10min. Amplified production and carrier pET-24b are passed through respectively to BamH I and Xho I double digestion 6h, after connecting, transformIn bacillus coli DH 5 alpha competent cell, picking monoclonal extracts recombinant plasmid after cultivating, and is prokaryotic expression plasmid pET-24b/DpSuc1a。
Three of object of the present invention is to provide a kind of new β-FFase from diaphania, and its amino acid sequence is as SEQIDShown in NO.1, the nucleotides of its encoding gene DpSuc1a is as shown in SEQIDNO.2. It is by containing new diaphania β-FFaseThe recombinant plasmid pET-24b/DpSuc1a of gene transforms and obtains that engineering bacteria realizes after e. coli bl21.
For the technical scheme solving the problems of the technologies described above as follows:
Build a kind of recombinant plasmid pET-24b/DpSuc1a that contains diaphania β-FFase gene. By recombinant plasmid transformedTo e. coli bl21 (DE3) competent cell, picking monoclonal is in 10mlLB culture medium, and 37 DEG C, 220rpm, cultivates12h obtains the engineering bacteria of the recombinant plasmid pET-24b/DpSuc1a that contains new diaphania β-FFase gene. Get 4ml bacterium liquidBe inoculated in the LB fluid nutrient medium of 250ml and expand and cultivate, 37 DEG C, 220rpm, shaken cultivation is to OD600≈ 0.6-0.8, addsIPTG is to final concentration 0.5mM, 30 DEG C of abduction delivering 10h. By the bacterium liquid after induction, in 4 DEG C, the centrifugal 5min of 5000rpm, collects bacteriumBody, after ultrasonication, SDS-PAGE detects, and finds recombinant protein stably express and is present in a large number in the upper albumin of solubility.
The present invention also provides the application of the new β-FFase from diaphania, and it mainly depends on new from diaphaniaβ-FFase carry out the confirmation to enzymatic activity after enzymatic activity test, this enzymatic activity can be for to having β-FFase substrate knotThe material of structure transforms.
The present invention also provides the amino acid sequence of diaphania β-FFase and silkworm BmSUC1 amino acid sequence to compare,The homology of the two is up to 66%, and has the enzyme active sites of identical prediction, illustrate diaphania also can utilize β in body-FFase avoids the toxic action of the high-concentration biological alkali in mulberry tree emulsion, for utilizing β-FFase control Mulberry Pests to provideImportant theoretical foundation.
Brief description of the drawings
Fig. 1 is the DpSuc1a of diaphania saccharase and the coding protein sequence ratio of silkworm homologous gene BmSuc1Right. Black region is identical amino acid, and gray area is similarity amino acid, and the avtive spot of prediction is with downward arrow markGo out.
Fig. 2 be carry out with the primer DpSuc1aF that contains restriction enzyme site and DpSuc1aR that pcr amplification obtains containing DpSuc1aThe electrophoretogram of ORF.
Fig. 3 is that the recombinant plasmid pET-24b/DpSuc1a enzyme that contains diaphania β-FFase gene is cut proof diagram. Swimming lane 1:PET-24b empty carrier; Swimming lane 2:pET-24b empty carrier double digestion product; Swimming lane 3: recombinant plasmid; Swimming lane 4: the two enzymes of recombinant plasmidCut product; M:DNA molecular weight Marker.
Fig. 4 is the SDS-PAGE electrophoretogram of vivoexpression recombinant protein DpSUC1a. The unloaded plasmid of swimming lane 1:pET-24b turnsChange BL21(DE3) upper albumin after bacterium abduction delivering; Swimming lane 2: recombinant plasmid pET-24b/DpSuc1a transforms BL21(DE3)Upper albumin after bacterium abduction delivering; M: molecular weight of albumen Marker; Arrow is depicted as object recombinant protein.
Fig. 5 is the Westernblot figure of vivoexpression recombinant protein DpSUC1a. The unloaded plasmid of swimming lane 1:pET-24b turnsChange BL21(DE3) upper albumin after bacterium abduction delivering; Swimming lane 2: recombinant plasmid pET-24b/DpSuc1a transforms BL21(DE3)Unpurified upper albumin after bacterium abduction delivering; Swimming lane 3: the recombinant protein after purifying; M: molecular weight of albumen Marker. ArrowBe depicted as object recombinant protein.
Fig. 6 is the enzymatic activity of recombinant protein DpSUC1a under condition of different pH.
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described, and example is only for explaining the present invention, notLimit scope of the present invention.
In following embodiment, if no special instructions, be conventional method.
The extraction of the total RNA of embodiment 1. diaphania midgut tissue
Diaphania (Diaphaniapyloalis) gathers in Agricultural University Of Anhui agricultural mulberry field, garden (great Yang shop), and through shapeState is accredited as diaphania (Diaphaniapyloalis). Larva is dissected, got middle intestines, put into the mortar of precooling, be ground toPowdered, add the Trizol of 1ml, after mixing, transfer in 1.5mL centrifuge tube concuss. Add the chloroform of 200 μ l, playViolent shock swings 30s, and room temperature leaves standstill 5min; The centrifugal 15min of 12000 × g under 4 ° of C, gets honest and upright and thrifty 400 μ l and moves on to the new RNA enzyme pipe that goes,Add isopyknic isopropyl alcohol, turn upside down and mix several times, leave standstill 15-20min on ice; The centrifugal 15min of 12000 × g under 4 ° of C,4 ° of C after going to add after most supernatant 75% ethanol of 1ml precooling to clean 2 times, 12000 × g is centrifugal, and 5min is precipitated, make its fromAfter being so dried, add the water-soluble solution of appropriate DEPC RNA precipitation to obtain the total RNA of diaphania midgut tissue ,-80 DEG C of preservations.
Clone and the amino acid sequence analysis of embodiment 2. diaphania β-FFase encoding genes
The acquisition of intestines cDNA in 2.1 diaphanias
The total RNA of diaphania midgut tissue obtaining taking embodiment 1 is masterplate, utilizes reverse transcription to synthesize cDNA the first chain (followingEach reverse transcription agents useful for same all comes from kit TransScriptIIFirst-StrandcDNASynthesisSuperMix, purchased from Beijing Quanshijin Biotechnology Co., Ltd).
In microcentrifugal tube, configure following masterplate RNA/Primer reactant liquor:
Total RNA1 μ g
Oligo(dT)20Primer (l) 1 μ l of 0.5 μ g/ μ
2×TSIIReactionMIX1μl
TransScriptIIRT/RIEnzymeMix1μl
RNase-freeWaterto20μl
Mix latter 55 DEG C and hatch 30min, then 85 DEG C of heating 5min inactivation TransScriptIIRT.
The clone of 2.2 diaphania β-FFase genes
According to the conservative of cane sugar hydrolytic enzyme 32 families, SUCdgpF(is as SEQIDNO.3 institute for design pair of degenerate primersShow) and SUCdgpR(as shown in SEQIDNO.4) carry out pcr amplification.
With the cDNA of reverse transcription be masterplate, using degenerate primer SUCdgpF and SUCdgpR is primer, its reaction condition of PCRFor: 94 DEG C of denaturation 5min, then 94 DEG C of sex change 40s, 48 DEG C of annealing 1min, 72 DEG C of extension 1min30s, totally 30 circulations,Latter 72 DEG C are extended 10min. 1% agarose gel electrophoresis detects PCR product, and uses SanPrep pillar DNA glue to reclaim kit(Sangon) reclaim. This DNA fragmentation is connected to pMD19-T carrier (TaKaRa), is transformed into bacillus coli DH 5 alpha competence thinIn born of the same parents, picking positive colony is delivered to Shanghai Li Fei Bioisystech Co., Ltd order-checking, obtains 557bp diaphania β-FFase geneConserved sequence fragment.
According to the conservative fragments design gene-specific primer having obtained.
RACE primer: 5 ' RACE outer primer DpSuc1a-5RAgsp, as shown in SEQIDNO.5,
5 ' RACE inner primer DpSuc1a-5RAngsp, as shown in SEQIDNO.6,
3 ' RACE outer primer DpSuc1a-3RAgsp, as shown in SEQIDNO.7,
3 ' RACE inner primer DpSuc1a-3RAngsp, as shown in SEQIDNO.8.
According to GeneRacer Kit(Invitrogen) requirement carry out the 5 ' RACE and 3 ' of diaphania β-FFase geneRACE operation. Taking reverse transcription obtain cDNA as template, with design 5 ' RACE and 3 ' RACE outer primer carry out the first round respectivelyPcr amplification. First set reaction system is 20ul, and reaction condition is as following table:
Taking first round PCR product as template, carry out respectively second with the 5 ' RACE designing and 3 ' RACE inner primer and take turns PCR expansionIncrease, amplification reaction condition is as following table:
1% agarose gel electrophoresis detects PCR product. After cutting glue, use SanPrep pillar DNA glue to reclaim kit(Sangon) reclaim. This DNA fragmentation is connected to pMD19-T carrier (TaKaRa), is transformed into bacillus coli DH 5 alpha competence thinIn born of the same parents, picking positive colony is delivered to the order-checking of Shanghai Li Fei Bioisystech Co., Ltd. By 5 ' RACE and 3 ' RACE, obtain respectivelyThe nucleotide sequence of 5 ' RACE fragment 297bp size and 3 ' RACE fragment 995bp size, obtains total length 1762bp size after splicingCDNA sequence be diaphania β-FFase encoding gene, called after DpSuc1a, sequence is as shown in SEQIDNO.2. Diaphania β-The ORF that FFase encoding gene comprises a 1467bp, its 488 amino acid whose albumen of encoding, protein sequence is as SEQIDShown in NO.1.
2.3. diaphania β-FFase amino acid sequence analysis
Through NCBIBlastP retrieval and sequence alignment analysis, the amino acid of DpSUC1a albumen and silkworm BmSUC1 albumenThe homology the highest (66%) of sequence, belongs to glycosyl hydrolase 32 families, as shown in Figure 1.
3. 1 kinds of recombinant plasmid pET-24b/DpSuc1a and structure sides thereof of containing diaphania β-FFase gene of embodimentMethod.
Primer DpSuc1aF and DpSuc1aR that utilization contains restriction enzyme site carry out pcr amplification, amplimer DpSuc1aFRespectively as shown in SEQIDNO.9 and SEQIDNO.10, reaction condition is with DpSuc1aR: 94 DEG C of denaturation 5min, then94 DEG C of sex change 40s, 58 DEG C of annealing 40s, 72 DEG C of extension 1min30s, totally 30 circulations, last 72 DEG C are extended 10 minutes. Fig. 2 is for containingThere are primer DpSuc1aF and the DpSuc1aR of restriction enzyme site to carry out the electrophoretogram containing DpSuc1aORF that pcr amplification obtains. To expandVolume increase thing and carrier pET-24b be respectively through BamH I and Xho I double digestion after 6 hours, connects with T4 ligase (TaKaRa)Connect, be transformed in bacillus coli DH 5 alpha competent cell, picking monoclonal is in 5mlLB culture medium, and 37 DEG C, 220rpm, cultivates12h. With SanPrep pillar DNA in a small amount extraction agent box (Sangon) extract recombinant plasmid, be one contain diaphania β-The recombinant plasmid pET-24b/DpSuc1a of FFase gene.
4. 1 kinds of recombinant plasmid pET-24b/DpSuc1a that contain diaphania β-FFase gene of embodiment transform the work obtainingJourney bacterium, the diaphania β-FFase recombinant protein DpSUC1a extract, purifying obtaining.
Recombinant plasmid pET-24b/DpSuc1a is transformed into e. coli bl21 (DE3) competent cell, picking monoclonalIn 10mlLB culture medium, 37 DEG C, 220rpm, cultivates 12h.
Get bacterium liquid 5ml, extract plasmid by kit method, with BamH I and Xho I double digestion after 6 hours, then run 1%Agarose gel electrophoresis, the qualification figure of the recombinant plasmid pET-24b/DpSuc1a enzyme that contains diaphania β-FFase gene after cutting as(swimming lane 1:pET-24b empty carrier shown in Fig. 3; Swimming lane 2:pET-24b empty carrier double digestion product; Swimming lane 3: recombinant plasmid; Swimming lane4: recombinant plasmid double digestion product; M:DNA molecular weight Marker). As seen from Figure 3, a kind of weight that contains diaphania β-FFase geneGroup plasmid pET-24b/DpSuc1a successfully builds, and successfully transforms and obtained corresponding engineering bacteria.
Get in the LB fluid nutrient medium that 4ml bacterium liquid is inoculated in 250ml and expand and cultivate, 37 DEG C, 220rpm, shaken cultivation is extremelyOD600≈ 0.6-0.8, adds IPTG to final concentration 0.5mM, 30 DEG C of abduction delivering 10h. By induction after bacterium liquid in 4 DEG C,The centrifugal 5min of 5000rpm, collects thalline, 4 DEG C of centrifuging and taking supernatants after ultrasonication, and SDS-PAGE detects, and finds recombinant protein quiltObvious abduction delivering is also present in the upper albumin of solubility, the results are shown in Figure 4. Supernatant is pure through Ni-NTA affinity columnAfter change, be a master tape through Westernblot detection display, the results are shown in Figure 5. Subsequently, recombinant protein purifying being obtainedDpSUC1a is with super filter tube ultrafiltration concentration to 0.5mL, and-80 DEG C of preservations have obtained diaphania β-FFase recombinant protein DpSUC1a.
Embodiment 5. is extracted enzyme function alive and the application of the β-FFase of purifying acquisition by diaphania β-FFase genetic engineering bacterium
5.1 enzyme activity determination methods
Taking raffinose (containing the trisaccharide of fructosyl) as substrate, the β-FFase that detects the recombinant protein DpSUC1a after purifying livesProperty. Reaction system comprises the raffinose of 100mM, the phosphate buffer (pH=7.0) of 10mM, and final volume is 100 μ l. 35 DEG CAfter hatching 5min in water-bath, add 1 μ g purifying protein, 35 DEG C of reaction 15min, then boil 5min cessation reaction. InsteadAnswer in liquid and add 3 of 200 μ l, 5-edlefsen's reagent, mixes rear boiling water bath 5min, cooling rear mensuration 540nm extinctionValue. Enzyme activity unit (U) is defined as: under condition determination, the fructose of generation 1 μ mol per minute enzyme amount used is a workUnit of force.
The mensuration of 5.2 optimal reaction pH
In the time of 35 DEG C, different pH(4.0-10.0, Britton-RobinsonBufferSolution) lower point of conditionDo not measure enzyme and live, find that recombinant protein DpSUC1a demonstrates β-FFase activity, its optimal reaction pH is 7.0, with this understandingEnzyme activity is 107.95 μ mol/min/mg, as shown in Figure 6.
Data and the result of enzymatic activity show, the raffinose hydrolysis that DpSUC1a can catalysis contains fructosyl, shows its toolHave the enzymatic activity function of β-FFase, this albumen can be for relating to the Substance Transformation with β-FFase zymolyte structure.
Claims (9)
1. a diaphania saccharase, its amino acid sequence is as shown in SEQIDNO.1.
2. the gene of a coding diaphania saccharase claimed in claim 1.
3. the gene of a kind of diaphania saccharase of encoding as claimed in claim 2, is characterized by described gene orderAs shown in SEQIDNO.2.
4. the gene of a kind of diaphania saccharase of encoding described in claim 2 or 3 is being prepared recombinant beta-furans fruitApplication in glycosidase.
5. the preparation method of the gene of a kind of diaphania saccharase of encoding described in claim 2 or 3, the methodComprise: extract total RNA of intestines in diaphania, and reverse transcription becomes cDNA as template, with degenerate primer, clone obtains portion gene orderRow, then obtain by RACE method the cDNA full-length gene that length is 1762bp, and sequence, as shown in SEQIDNO.2, comprises oneThe complete ORFs ORF of individual 1467bp, the RACE primer that described degenerate primer and RACE method are used is as follows:
Degenerate primer: forward primer SUCdgpF, as shown in SEQIDNO.3,
Reverse primer SUCdgpR, as shown in SEQIDNO.4;
RACE primer: 5 ' RACE outer primer DpSuc1a-5RAgsp, as shown in SEQIDNO.5,
5 ' RACE inner primer DpSuc1a-5RAngsp, as shown in SEQIDNO.6,
3 ' RACE outer primer DpSuc1a-3RAgsp, as shown in SEQIDNO.7,
3 ' RACE inner primer DpSuc1a-3RAngsp, as shown in SEQIDNO.8.
6. a recombinant plasmid that contains a kind of diaphania saccharase gene of encoding described in claim 2 or 3PET-24b/DpSuc1a, is characterized in that, by the primer DpSuc1aF that contains double enzyme site and DpSuc1aR, ORF is carried outAfter pcr amplification, amplified production is connected to acquisition through BamH I respectively with carrier pET-24b after Xho I double digestion 6h; InstituteState two kinds of amplimer DpSuc1aF designing in PCR method and DpSuc1aR respectively as SEQIDNO.9 and SEQIDShown in NO.10.
7. by recombinant plasmid transformed BL21(DE3 claimed in claim 6) engineering strain that obtains of competent cell.
8. a recombinant protein DpSUC1a who contains diaphania saccharase as claimed in claim 1, its feature existsIn, this recombinant protein is that recombinant plasmid pET-24b/DpSuc1a claimed in claim 6 is converted into BL21 competent cell,Cultivate and abduction delivering through expanding, after ultrasonication, purifying obtains the recombinant protein of solubility.
9. the application of diaphania saccharase as claimed in claim 1, described in be applied as and utilize diaphania β-fructofuranoseThe activity of glycosides enzyme transforms the material with saccharase substrate structure.
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