CN1289367A - Plant alkaline and neutral invertases - Google Patents
Plant alkaline and neutral invertases Download PDFInfo
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Abstract
The present invention provides DNA comprising a sequence of nucleotides which can be translated into a protein with invertase activity, wherein highest activity is observed in the range of pH 6.0 to 7.5, as well as the encoded protein produced recombinantly.
Description
The protein DNA of hydrolyzable sucrose the present invention relates to encode.Specifically, the invention describes and to be translated as protein DNA with neutral sucrase active.
In most plant varieties, sucrose (α-D-glucopyranosyl beta-D-fructofuranose glycosides) is first non-phosphorylating product in the photo-assimilation, and as the mobile source of the energy and the carbon of heterotrophic plant tissue.The metabolism of sucrose is indispensable for the survival of heterotrophic plant organ, wherein must could be utilized after with the sucrose cracking by sucrase or sucrose synthase.
Sucrase is glucose and fructose with sucrose hydrolysis, thereby makes sucrose enter various bio-chemical pathways.Sucrase has several isomer, and they have different biochemical characteristics and Subcellular Localization.The feature of acid sucrase is that its acid optimal pH is 4.0 to 5.5.Found that they are to be combined on the cell walls (cell walls sucrase) or as the soluble protein in the vacuole (vacuole sucrase) with ionic species.The aminoacid sequence of described acid sucrase has conservative primitive.The sequence similarity analysis shows that they and sucrase from yeast and bacterium have the dependency in the evolution, and does not find its counterpart in zooblast.Technical, acid sucrase is used in the candy industry so that will be than easy crystalline sucrose inversion for being difficult for crystalline glucose-fructose mixture.Thereby wrapped up for example chocolate hard sucrose core and can change into the soft sweets core.According to this concrete purposes, preferably has the sucrase of neutrality or alkaline optimal pH.
Neutral or alkaline sucrase is characterised in that optimal pH is respectively 6.0 to 7.5 and 7.5 to 8.5.It is generally acknowledged that these enzymes are confined in the mature tissue, guess that usually they are accumulated in the tenuigenin, the fact that does not detect the polysaccharide of N-connection can be supported this point.Up to the present, also do not illustrate the molecular structure and the gene thereof of neutral sucrase.And this is the prerequisite of neutral or alkaline sucrase being carried out the biotechnology exploitation.Recently purifying from the corresponding enzyme (Lee and Sturm, plant physiology, 112:1513-1522,1996) of Radix Dauci Sativae (Daucus carota cvQueen Anne ＇ s Lace), and it has been carried out biochemical identification.
Cell in the Radix Dauci Sativae suspension culture contains the sucrose lytic activity of solubility, more than pH6 and following optimal pH (be respectively alkalescence and neutral sucrase) arranged respectively.Can distinguish this two kinds of activity by saturation ratio effectively at the ammonium sulfate precipitation of 20-45%.In protein precipitation, can detect neutral and alkaline sucrase active, and acid sucrase is retained in the supernatant liquor.Ammonium sulfate fraction chromatography on Q-Sepharose with 20-45% obtains two sucrase active peaks, and they have only rough separation.To contain activated fraction and merge also further chromatography purification on HA-Ultrogel, affinity chromatography on Green19 obtains effectively isolating two kinds of activity subsequently.In unconjugated protein fraction, identify sucrose lytic activity (neutral sucrase, H with neutral optimal pH
1).Optimal pH is the activity of meta-alkalescence (alkaline sucrase, H more
2) be attached on HA-Ultrogel and the Green19 dyestuff pillar, can carry out wash-out with the saliniferous damping fluid.In the purifying in this stage, neutral sucrase accounts for the about 1/3rd of sucrase active, and alkaline sucrase accounts for 2/3rds.What contain neutral and alkaline sucrase respectively respectively merges fraction separately further through gel permeation chromatography, ion exchange chromatography, the second time, gel permeation chromatography and hydrophobic interaction chromatography carried out purifying for the second time.It is the most effective step of removing contaminating protein matter in the goods that neutral sucrase is carried out the Macro-Prep anion-exchange chromatography.Although propyl group agarose chromatography can not improve the specific activity of alkaline sucrase, need carry out to obtain electrophoretically pure enzyme.When purifying finishes, be recovered to two kinds of enzymes less than 10%.This loss has been considered to adopt multistep purification step and the low result who causes of enzyme stability.
Found that the neutral sucrase that elutes from the gel-filtration pillar is the polypeptide of a kind of about 456kD, and the enzyme behind the purifying moves into the single band of about 57kD on sds polyacrylamide gel electrophoresis.Alkalescence sucrase wash-out is the polypeptide of about 504kD, moves into the single band of about 126kD behind the purifying on sds polyacrylamide gel electrophoresis.The neutral sucrase of these results suggest may form eight aggressiveness, and alkaline sucrase may form the tetramer.The optimal pH that is measured to them is respectively 6.8 and 8.0.In addition, neutral sucrase shows energy cracking raffinose and stachyose, points out this enzyme to have the saccharase activity.
Main purpose of the present invention provides DNA, and it comprises can be translated as the proteinic nucleotide sequence with sucrase active, and wherein said protein preferably has maximum activity 6.0 to 7.5 at pH6.0 to 8.5.As if although neutral and alkaline sucrase is considered to heterogeneic product, they are relevant on immunology.
Dynamic programming algorithm (dynamic programming algorithm) produces the different comparison of several classes.Usually sequence alignment there are two kinds of practices.The algorithm of Needleman and Wunsch and the algorithm of Sellers can be compared to two full length sequences, obtain the total length comparison of sequence.And the Smith-Waterman algorithm is local comparison.Zone pairing the most similar in the sequence is compared in local comparison, can select rating matrix and breach point penalty.This makes database retrieval can concentrate on the highest zone of conservative property in the sequence.Utilize it can also identify analog structure territory in the sequence.Accelerate the comparison that utilizes the Smith-Waterman algorithm to carry out, BLAST (basic local comparison gopher) and FASTA have all proposed additional restricted condition to comparison.
In literary composition of the present invention, can compare easily with BLAST (it is one group of similarity retrieval program that design is used for searching for all existing sequence libraries, and that no matter inquire about is protein or DNA).The public can obtain the BLAST 2.0 editions (Gapped BLAST) of this gopher from internet (at present at http://www.ncbi.nlm.nih.gov/BLAST/).It has utilized a kind ofly seeks the part but not the heuristic algorithm of total length comparison, therefore can detect the relation between the sequence of total separated region only.There is clear and definite statistics to explain to branch in the BLAST retrieval.What be particularly useful within the scope of the invention is the blastp program that allows to introduce breach in local sequence alignment, and PSI-BLAST program (these two kinds of programs all use amino acid search sequence and protein sequence database to compare), also have modified version blastp program, it carries out the part comparison to two sequences.Preferred said procedure moves when optional parameter is set at default value.
Total length that aminoacid sequence of the present invention and known array are carried out or local comparison show that the sequence homogeny of they and known acid sucrase or other Sucrose Metabolism enzymes is lower than 40%.Described several routine DNA in SEQ IDNO:1 and SEQ ID NO:3, they comprise can be translated as the proteinic nucleotide sequence with neutral sucrase active.In SEQ ID NO:2, provided the aminoacid sequence of coded sucrase.Can be at least separate at the related protein more than 40% and corresponding gene thereof with SEQ ID NO:2 sequence homogeny from gathering in the crops seed, fruit or storing any plant of organ.Example has the crop of protein crop, oil crops and reserve starch, beet, corn, sweet corn, soybean, Sunflower Receptacle, grass, oilseed rape, wheat, barley, Chinese sorghum, rice, melon, watermelon, pumpkin, witloof, tomato, pepper, cabbage, Cauliflower, Caulis et Folium Brassicae capitatae, cucumber, japanese radish, benas and romaine lettuce.
The protein of describing among the SEQ ID NO:2 lacks signal peptide, and wetting ability is very high.In addition, it contains 18 halfcystines and 15 methionine residues.With demonstrate it after LIM17 protein (coded) comparison the highest full length sequence homogeny (47%) arranged by the partial cDNA Cloning that derives from white trumpet lily (Lilium longiflorum).Carry out the total length comparison with other protein sequences, find that the sequence homogeny is below 40%.Coding LIM protein DNA sequence is origin to be come from the (Kobayashi etc. that identify when screen in library that the cDNA of the white trumpet lily microsporocyte of meiosis prophase obtains with the special subtracted probe of meiosis prophase at first, DNA studies 1:15-26,1994).Utilize computer program GAP, by the aminoacid sequence and Radix Dauci Sativae protein 47% identical (58% is similar) of the proteinic partial sequence deduction of white trumpet lily LIM17.Introducing several big breach so that after optimizing comparison, is 37% (47% is similar) by the relevant LIM17 albumen (ORF sII0626) of the genome encoding of single celled blue algae bacterium-collection born of the same parents cyanobacteria (Synechocystis) to the sequence homogeny of Radix Dauci Sativae enzyme.Therefore, just might in photosynthetic bacterium, find with sucrase sequence homogeny of the present invention at the related protein more than 40%.The same with Radix Dauci Sativae protein, be rich in Cys and Met from lilium and the LIM17 albumen that collection born of the same parents cyanobacteria belongs to, but as if their positions in polypeptide chain conservative.The function or the enzymic activity of LIM17 albumen (this albumen is littler than Radix Dauci Sativae sequence analogue) are not clear.
Therefore, according to the present invention, can define the neutral sucrase of gang, its member is 40% or higher at the aminoacid sequence homogeny of total length comparison back and SEQ ID NO:2.Preferred amino acid sequence homogeny is higher than 50% or even be higher than 55%.Identical sequence can be thought a subtribe more than 55%.Can also comprise to the composition sequence of 330,450 or 510 base pairs of the youthful and the elderly the composition sequence at least 60% of local comparison among these sequences and the SEQ ID NO:2,70% or even 75% above identical according to sequence of the present invention.
When carrying out multiple sequence when comparison, except the sequence homogeny, some algorithm can be considered sequence similarity, and is identical or hydrophobic/hydrophilic character is suitable such as the net charge of single amino acids.Therefore, this algorithm can be estimated an aminoacid replacement for another is possible keep to keep this protein structure and necessary physics of function and chemical property, still more may destroy this proteinic key structure and functional character.This class sequence similarity is with the amino acid whose per-cent of the positive, rather than the percentage of same amino acid recently represents, and helps protein is belonged to correct protein families under ambiguous situation.Valuable especially within the scope of the present invention protein is the sucrase that its aminoacid sequence comprises at least one following characteristic amino acid subsequence:
(a)?VGTVAA???????????????????????(SEQ?ID?NO：4)
(b)?AIGRV????????????????????????(SEQ?ID?NO：5)
(c)?DFGESAIGRVAPVDSGLWWIIL???????(SEQ?ID?NO：6)
(d)?CMIDRRMGI????????????????????????(SEQ?ID?NO：7)
(e)?PTLLVTDGSCMIDRRMGIHGHPLEIQAL?????(SEQ?ID?NO：8)
(f)?GGYLIGN??????????????????????????(SEQ?ID?NO：9)
(g)?DFRFFTLGN????????????????????????(SEQ?ID?NO：10)
Can produce the DNA that coding belongs to the sucrase of described new protein family by following general method.With SEQ ID NO:1 comprise at least 15, the single-chain fragment of preferred 20 to 30 even 100 above continuous nucleotides is as probe, filter out in the DNA library can with the clone of described fragment hybridization.At Sambrook etc., molecular cloning: the factor of having described decision hybridization in experiment guide (Cold Spring HarborLaboratory Press, 1989) 9.47-9.57 and the 11.45-11.49 chapter.To hybridize cloning and sequencing, those comprise the DNA that coding and SEQ ID NO:2 sequence homogeny are higher than the clone of 40% proteinic opening code-reading frame purifying.Can further described DNA be processed by the recombinant DNA method (such as restriction enzyme digestion, connection or polymerase chain reaction) of many routines then.
The DNA that comprises the nucleotide sequence that limits among the SEQ ID NO:1 can clone in the following manner:
Partial interior peptide sequence (the XNIYPDQIPPWLV that the neutral sucrase of Radix Dauci Sativae that purifying is crossed is decomposed to form through trypsinase, SEQ ID NO:11) compares with the est database, show by having this sequence in est t88552 (1026bp of the Arabidopis thaliana cDNA 3 ' end) amino acid sequence coded from Arabidopis thaliana.Use primer 5 '-TCTAAGGATCTAGAAAGAGCCATTA-3 ' (SEQID NO:12) and 5 '-TTCAATTGAATTCAATATAGCTTC-3 ' (SEQ ID NO:13) can be separated to the dna fragmentation (Nucleotide 100-410) of this peptide sequence of coding through PCR.With XbaI and EcoRI with the digestion of PCR product after, be connected to the corresponding site among the escherichia coli plasmid pBluescriptIIKS (Stratagene).Behind this plasmid amplification and purifying, downcut fragment, through agarose gel electrophoresis and electroelution purifying, and with [α-
32P] the ATP random labelling.With the good DNA of mark as the probe screening by wild Radix Dauci Sativae (Daucus carota cv Queen Anne ' sLace, W001C) library that suspension culture makes of growing fast.With the cloning and sequencing that obtains, may for example demonstrate a segmental clone of insertion who comprises 2447 Nucleotide, described fragment contains 5 ' non-coding sequence 29bp and 3 ' non-coding sequence 393bp, and ORF coding one has 675 amino acid whose protein, with the Arabidopis thaliana est t88552 aminoacid sequence of deriving 80% identical (86% is similar) is arranged.
Thereby those skilled in the art can improve aforesaid method and make it can be applicable to encode and belong to proteinic any gene of this sucrase new family.In addition, those skilled in the art can utilize disclosed SEQ ID NO:3 to be designed for the oligonucleotide of polymerase chain reaction, so that amplification comprises the dna fragmentation of certain nucleotide sequence from template, described nucleotide sequence is characterised in that it is among the SEQ IDNO:1 15, the continuous sequence of preferred 20 to 30 or more base pairs.Described Nucleotide comprises 20 that represent among the SEQ ID NO:1, the nucleotide sequence of preferred 20 to 30 or more base pairs.Carry out the polymerase chain reaction with at least one such oligonucleotide, its amplified production has constituted another embodiment of the invention.In addition, those skilled in the art can utilize disclosed nucleotide sequence to design conversion carrier, can adopt transformation technology known in the art (WO9627673 for example, the 17-20 page or leaf is described) to utilize described preparing carriers transgenic plant.
Another object of the present invention provides the recombinant plant sucrase with neutral optimal pH.DNA by the described sucrase of recombinant expressed coding in microorganism host (such as intestinal bacteria or yeast), preferred cDNA can realize this purpose, for example can produce the recombinant sucrose enzyme in order to following method: the cDNA of this enzyme of will encoding introduces expression vector, in pTrc99A (Pharmacia Biotech).Transform bacteria (such as intestinal bacteria), and if desired, after for example the IPTG induced protein synthesizes, with the bacterium cracking.Determine the neutral sucrase active in the solubility lysate fraction.Specifically, about 100 μ l soluble extracts are mixed with 700 μ l water, 100 μ l 0.5M potassiumphosphates (pH6.8), 100 μ l 0.5M sucrose, and in 37 ℃ of incubations 30 minutes.Measure reducing sugar with this solution of aliquot according to the method for Somogyi (Somogyi, journal of biological chemistry 195:19-23,1952).Be to determine the pH dependency of sucrose lytic activity, use the pH value at 4.5 to 8.5 0.5M potassium phosphate solution.
The sucrase of reorganization preparation has the biochemical characteristic of three keys closely similar with the enzyme of purifying from plant, i.e. the about 20mM of Km value, and the pH dependency has a sharp-pointed peak value between pH6.5 to 7.0, by the Cu of micro-molar concentration
2+Suppress.On the other hand, a recombinase sucrose hydrolysis and not cracking raffinose or stachyose unexpectedly.Therefore recombinant protein does not have the saccharase activity basically.
Embodiment
Embodiment 1: the purifying of Radix Dauci Sativae neutrality and alkaline sucrase
The preparation extract
Collect carrot cell (400g) from the suspension culture of logarithmic phase, (50mM Hepes-KOH, pH7.5 contains 0.5mMEDTA, 10mM Methionin, 0.5mM MgCl at the ice-cold buffer A of 2.5 volumes with the Polytron Syrup-homogenizing instrument
2, 0.5%2-mercaptoethanol and 100mM phenylmethylsulfonyl fluoride) at full speed homogenate 4 times, each 20 seconds.With homogenate in Sorvall GSA rotary head centrifugal 20 minutes in 6000g.Collect supernatant liquor and refrigeration.The 6000g precipitation is resuspended among the 2.5 volume ice-cold buffer A, with Polytron Syrup-homogenizing instrument full speed homogenate 3 times, each 20 seconds, and centrifugal again 20 minutes.The supernatant liquor that merges centrifugal 30 minutes in 16300g, be poured on then four layers of Miracloth (Calbiochem-Behring Corporation, La Jolla, USA) on.Filtered solution is used for further protein purification.If not point out in addition, institute all carries out at 4 ℃ in steps.
Ammonium sulfate precipitation
Solid ammonium sulfate is slowly added in the crude extract, and stir gently, by being collected in the sedimentary protein of 20% to 45% saturation ratio in centrifugal 30 minutes in 16300g.With resolution of precipitate in 100ml buffer B (25mM Hepes-KOH, pH7.5 contain 190mM NaCl, 0.5% 2 mercapto ethanol and 100mM phenylmethylsulfonyl fluoride), to the buffer B dialysed overnight.
On Q-Sepharose, carry out anion-exchange chromatography
With sample on the dialyzate to the Q-Sepharose pillar of crossing with the buffer B balance (2.5cm * 25cm, Pharmacia LKB Biotechnology, Uppsala, Sweden) in.With buffer B wash pillar until the absorbancy of 280nm less than 0.01.Be dissolved in 190-550mM NaCl linear gradient elution bonded protein among the 25mM Hepes-KOH (pH7.5) with 240ml.Merge active fraction (level partial volume 5ml), with the ammonium sulfate precipitation of 60% saturation ratio, and in 16300g centrifugal 30 minutes.Precipitation is dissolved among the 5ml damping fluid C (5mM potassium phosphate buffer, pH7.5 contain the 0.1%2-mercaptoethanol), to damping fluid C dialysed overnight.
On HA-Ultrogel, carry out chromatography
With sample on the dialyzate to the HA-Ultrogel pillar of crossing with damping fluid C balance (2.5cm * 25cm, Sigma, Buchs, Switzerland) on.C washes pillar with damping fluid, and with 5-500mM potassiumphosphate (pH7.5) the damping fluid linear gradient elution that contains 0.1% 2 mercapto ethanol.Pillar is collected the 5ml fraction with the flow velocity wash-out of 40ml/h.Merge respectively containing the fraction that contains alkaline sucrase active in the fraction of neutral sucrase active and the elutriant in the effluent liquid, with the ammonium sulfate precipitation of 60% saturation ratio, and in 16300g centrifugal 30 minutes.Two parts of protein precipitations respectively are dissolved among the 5ml damping fluid D (25mM potassiumphosphate, pH7.5 contain 0.1% 2 mercapto ethanol), to damping fluid D dialysed overnight.
On Green19 dye, carry out affinity chromatography
The protein soln (each 5ml) of dialysing is divided into the aliquot of 0.5ml, go up then sample to 10 that cross with the damping fluid D balance Green19 dye pillars that install in advance (4.5 * 0.7cm, Sigma, Buchs, Switzerland) on.D washes pillar with the 15ml damping fluid, uses NaCl (being respectively 15ml and the 25ml) stepwise elution of 0.35M and 1.5M then, and elutriant is collected in the 2ml fraction.In effluent liquid, detect neutral sucrase active, and alkaline sucrase active is by 1.5M NaCl wash-out.Merge the fraction that contains enzymic activity, precipitate in 60% saturation ratio with ammonium sulfate.Protein in centrifugal 30 minutes collecting precipitations of 16300g.
Gel permeation chromatography on Sephacryl S-300
Every part of protein precipitation is dissolved among the 7ml damping fluid E (100mM Hepes-KOH, pH7.5 contain 0.1% 2 mercapto ethanol).Protein soln is gone up sample separately to Sephacryl S-300 pillar (2.6cm * 100cm, Pharmacia LKB Biotechnology, Uppsala, Sweden) on, pillar is crossed with damping fluid E balance and was demarcated with blue dextran (Vo), thyroglobulin (669kD), apoferritin (443kD), beta-amylase (200kD), ethanol dehydrogenase (150kD), BSA (66kD) and carbonic anhydrase (29kD).With the flow velocity wash-out pillar of 110ml/h, collect the 5ml fraction.The fraction that will contain enzymic activity merges, for alkaline sucrase, to damping fluid F (25mM Hepes-KOH, pH8.0, contain 200mM NaCl and 0.1% 2 mercapto ethanol) dialysed overnight, for neutral sucrase, then to damping fluid G (25mM Hepes-KOH, pH7.2 contains 275mM NaCl and 0.1% 2 mercapto ethanol) dialysed overnight.
On Macro-Prep, carry out anion chromatography II
In order to be further purified alkaline sucrase, with sample on the dialyzate to the Macro-Prep pillar of crossing with damping fluid F balance (1.5cm * 20cm, Bio-Rad Laboratories, Richmond, CA, USA) on.F washes pillar with damping fluid, and is dissolved in the 200-450mM NaCl linear gradient elution among the 25mM Hepes-KOH (pH8) that contains 0.1% 2 mercapto ethanol with 200ml.
In order to be further purified neutral sucrase, with sample on the dialyzate to the Macro-Prep pillar of crossing with damping fluid G balance (1.2cm * 25cm, Bio-Rad Laboratories).G washes pillar with damping fluid, and is dissolved in the 275-360mM NaCl linear gradient elution among the 25mM Hepes-KOH (pH7.2) that contains 0.1% 2 mercapto ethanol with 200ml.
The fraction that will contain corresponding enzymic activity merges respectively, precipitates in 60% saturation ratio with ammonium sulfate, and in 16300g centrifugal 30 minutes.
On Propyl Agarose, carry out hydrophobic interaction chromatography
The protein precipitation that will have alkaline sucrase active is dissolved among the 5ml damping fluid H (25mMHepes-KOH, pH8.0 contain 1.5M ammonium sulfate and 0.1% 2 mercapto ethanol).With sample on the solution to the Propyl Agarose pillar of crossing with damping fluid H balance (10 * 1.5cm, Sigma, Buchs, Switzerland) on.H washes pillar with damping fluid, with 25mM Hepes-KOH (pH8.0) wash-out that contains 0.1% 2 mercapto ethanol, collects the 3ml fraction then.The fraction that will contain enzymic activity merges, and the 10mM Hepes-KOH (pH8.0) that contains 0.1% 2 mercapto ethanol is dialysed, and be kept in 50% glycerine in-20 ℃.
On Sephacryl S-300, carry out gel permeation chromatography II
The protein precipitation that will have neutral sucrase active is dissolved in 5ml damping fluid I (100mM potassium phosphate buffer, pH7.0, contain 0.1% 2 mercapto ethanol) in, and go up sample to the Sephacryl S-300 pillar of crossing with damping fluid I balance (2.6cm * 100cm, Pharmacia LKB).Collect the 5ml fraction.The fraction that will contain enzymic activity merges, and to containing 10mM potassium phosphate buffer (pH7.0) dialysis of 0.1% 2 mercapto ethanol, is kept in 50% glycerine in-20 ℃.
Embodiment 2: the cDNA clone who separates the neutral sucrase of coding Radix Dauci Sativae
The partial interior trypsinase site peptide sequence (XNIYPDQIPPWLV of the neutral sucrase of the Radix Dauci Sativae that purifying is crossed, SEQ ID NO:11) compares with the est database, show by having this sequence in est t88552 (1026bp of the Arabidopis thaliana cDNA 3 ' end) amino acid sequence coded from Arabidopis thaliana.Use primer 5 '-TCTAAGGATCTAGAAAGAGCCATTA-3 ' (SEQ IDNO:12) and 5 '-TTCAATTGAATTCAATATAGCTTC-3 ' (SEQ ID NO:13) to separate the dna fragmentation (Nucleotide 100-410) of this peptide sequence of encoding through PCR.Increase with following condition in DNA thermal cycler (PerkinElmer Cetus): 10 circulations are 95 ℃ of sex change 1 minute, 40 ℃ of annealing 0.5 minute and extended 1.5 minutes in 72 ℃, carry out 1 minute, 60 ℃ annealing of 95 ℃ of sex change of 20 round-robin then and extended 1.5 minutes for 0.5 minute, 72 ℃.With phenol/chloroform extracting PCR product, with the corresponding site that is connected to after XbaI and the EcoRI cracking among the escherichia coli plasmid pBluescriptIIKS (Stratagene).Behind plasmid amplification and purifying, downcut fragment, through agarose gel electrophoresis and electroelution purifying, with [α-
32P] the ATP random labelling.With the good DNA of mark as the cDNA library in the probe screening λ ZAP II carrier (Stratagene), this library is with wild Radix Dauci Sativae (Daucus carota cv Queen Anne ' s Lace, W001C) grow fast that the polyA+mRNA of the cell in the suspension culture makes, identify a hybridization clone.
Embodiment 3: Radix Dauci Sativae sucrase clone's sequential analysis
The described cDNA clone's of embodiment 2 insertion fragment is connected in pBluescriptII KS (+/-) carrier (Stratagene), measures double-stranded sequence automatically by the dideoxy nucleotide chain cessation method.Utilize Wisconsin Package Version9.0, Genetics ComputerGroup (GCG), Madison, Wisconsin carries out computer-aided analysis to the DNA and the protein sequence of embodiment 2 and 3.
Carry out sequence relatively with computer program GAP, described program is used Needleman and Wunsch (molecular biology magazine 48:443-453,1970) alignment algorithm is so that analyze the comparison situation of two complete sequence, allow the introducing breach to realize making coupling number maximum under the situation of best comparison, and reducing the breach number as far as possible.GAP can consider all possible comparison and gap position, and it is maximum to produce coupling base number, and the minimum comparison of breach.Can produce point penalty and breach extension point penalty with coupling base unit indication notch.In other words, for the breach of each insertion, GAP must sentence breach to the coupling number and produce point penalty.If select breach to extend point penalty greater than zero, for each breach that inserts, GAP must sentence notch length in addition and multiply by the point penalty number that breach extends point penalty.When protein sequence compares, produce and breach extension point penalty for breach, threshold value is 3.0 and 0.1 usually.
The neutral sucrase cDNA of Radix Dauci Sativae clones long 2447 Nucleotide (SEQ ID NO:3), and contains the 5 ' non-coding region of 29bp, the 3 ' non-coding region of 393bp.675 amino acid of its ORF coding, molecular weight is 75957Dalton, the iso-electric point that calculates is pI8.1.Aminoacid sequence of inferring and the speculating acid sequence 80% identical (86% is similar) of Arabidopis thaliana est t88552.
Neutral sucrase (car) aminoacid sequence that Radix Dauci Sativae cDNA clone is inferred with compare from white trumpet lily (lil) and the LIM17 protein sequence that collects born of the same parents cyanobacterias (bac), identify three conserved sequence structural domains (box 1-3).During with the sequence retrieval database of box 2, identify a function known protein matter, promptly from the cellobiose phosphorylase of Clostridium stercorarium, (β-D-Glc (1 → 4)-D-Glc) is cracked into Cori's eater Cori and glucose to this enzyme with cellobiose under the situation of tetra-sodium having.This shows that box 2 may constitute the glucosyl group binding site of this disaccharide.
Table 1: the aminoacid sequence of the neutral sucrase of the Radix Dauci Sativae of inferring by cDNA (car) with from white trumpet lily (lil) with collect the comparative result of the LIM17 protein amino acid sequence of born of the same parents cyanobacterias (bac).Aminoacid sequence uses the single-letter code, and compares homogeny is maximized by introducing breach (..).The amino-acid residue of runic is represented conserved domain (box 1-3).Asterisk under the sequence is illustrated in all identical amino-acid residue in three sequences.
Embodiment 4: the steady-state level of neutral sucrase mRNA
We have determined in three different developmental phases leaves of Radix Dauci Sativae plant and the root and the maintenance level of the neutral sucrase mRNA in the organ of multiplication.Method (molecular biology of plants report 4:219-224 by Prescott and Martin description, 1987) and a little change the total RNA of back preparation, every gram tissue had added 20mg PolyclarAT (Serva) before this method improvement part was to grind in liquid nitrogen.When carrying out RNA gel engram analysis, containing separation total RNA (10mg/ road) on 1.2% sepharose of 6% formaldehyde.What add in the northern trace is from 4-, 10-and the leaf in 16 weeks, 4-, 10-and the root in 16 weeks and petal (B), flower (F), the little seed of growing (Gs), the big seed of growing (Gl) and total RNA (10mg/ road) of mature seed (S).With
32The neutral sucrase cDNA and the blot hybridization of P-mark.
All observe the stable transcript level of the neutral sucrase of Radix Dauci Sativae at the different development stage of all organs, level is slightly high in the organ of growing.This discovery shows that this enzyme has function more general and may be relevant with growth in the Sucrose Metabolism of Radix Dauci Sativae.
Embodiment 5: the neutral sucrase of escherichia coli expression Radix Dauci Sativae
In order in coli strain JM105 (Pharmacia Biotech), to express the cDNA clone of the neutral sucrase of Radix Dauci Sativae, utilize primer 5 '-CGATTTAGCAAGGTACCATAGATATGAATAC-3 ' (SEQ ID NO:14) and 5 '-CTTATCCTTAAACTAGATCTCCATTAGACC-3 ' (SEQ ID NO:15) through pcr amplification ORF.Amplification is to carry out in following condition in DNA thermal cycler (Perkin Elmer Cetus): 1 minute, 55 ℃ annealing of 95 ℃ of sex change of 30 round-robin were extended 1.5 minutes in 0.5 minute and 72 ℃.With phenol/chloroform extraction PCR product, with the corresponding site that is connected to after Kpnl and the Xbal cracking among the expression vector pTrc99A (Pharmacia Biotech).Induce about 16 hours of protein biosynthesizing in the transform bacteria that carries expression vector with 1mM IPTG, in a small amount of 50mM potassiumphosphate (pH6.8), pass through freeze-thaw cycle cracking bacterium (Johnson and a Hecht, biotechnology 12:1357-1360,1994).According to the neutral sucrase active in the description mensuration solubility lysate fraction of Lee and Sturm (1996, the same).In brief, 100 μ l water soluble extracts are mixed with 700 μ l water, 100 μ l 0.5M potassiumphosphates (pH6.8) and 100 μ l 0.5M sucrose, and in 37 ℃ of incubations 30 minutes.Measure reducing sugar with this solution of aliquot according to the method for Somogyi.Determine the pH dependency of sucrose lytic activity, use the pH value at 4.5 to 8.5 0.5M potassium phosphate solution.
Embodiment 6: measure sucrase active
In the reaction system of the final volume 1ml that contains 50mM potassium phosphate buffer (pH6.8 or 8.0), 100mM sucrose and an amount of enzyme, measure sucrase active.With mixture in 37 ℃ of incubations 30 minutes.Measure the amount of the reducing sugar that discharges according to the method for Somogyi.Enzymic activity (unit) is expressed as the amount (μ mol) of the reducing sugar (glucose and fructose) of per minute release.The ammonium ion of high density can suppress sucrase active, and this just requires will be through the protein soln dialysis of ammonium sulfate precipitation preparation before determination of activity.
The measuring method of taking from embodiment 1 described purge process can be inferred the neutral sucrase active that about 240 units of 400g cell expressing, this means every gram cell expressing 0.6 unit.In the purge process of enzyme, lost about 95% activity.
Recombinase to expression in escherichia coli carries out activity measurement, detects the neutral sucrase active of about 0.4 unit in 100 μ l embodiment, 5 described intestinal bacteria extracts.
Sequence Table
<110> Novartis AG
<120> Plants alkaline and neutral invertase
<130> S-30364 / A
<140>
<141>
<150> GB 9802249.4
<151> 1998-02-03
<160> 15
<170> PatentIn Ver. 2.0
<210> 1
<211> 2025
<212> DNA
<213> Carrots
<220>
<221> CDS
<222> (1). . (2025)
<400> 1
atg aat act act tgt att gct gta tcg aat atg agg cct tgt tgt aga 48
Met Asn Thr Thr Cys Ile Ala Val Ser Asn Met Arg Pro Cys Cys Arg
151015
atg tta ctt agc tgt aag aat tca tcg att ttc gga tac tcg ttt cga 96
Met Leu Leu Ser Cys Lys Asn Ser Ser Ile Phe Gly Tyr Ser Phe Arg
202530
aaa tgt gat cat aga atg ggg act aat ttg tcg aaa aag caa ttt aag 144
Lys Cys Asp His Arg Met Gly Thr Asn Leu Ser Lys Lys Gln Phe Lys
35 40 45
gtg tac ggt ttg cga ggg tat gtt agt tgt agg ggt ggt aaa ggt tta 192
Val Tyr Gly Leu Arg Gly Tyr Val Ser Cys Arg Gly Gly Lys Gly Leu
50 55 60
ggt tat agg tgt ggg att gat ccg aat cgg aag ggt ttt ttt ggt tcc 240
Gly Tyr Arg Cys Gly Ile Asp Pro Asn Arg Lys Gly Phe Phe Gly Ser
65 70 75 80
ggt tct gat tgg gga cag cct agg gtt tta aca agt ggt tgt cgt cgt 288
Gly Ser Asp Trp Gly Gln Pro Arg Val Leu Thr Ser Gly Cys Arg Arg
85 90 95
gtt gat agt ggt ggt agg agt gta ctt gtt aat gtg gcg tcg gat tat 336
Val Asp Ser Gly Gly Arg Ser Val Leu Val Asn Val Ala Ser Asp Tyr
100 105 110
agg aat cat tca act tcg gtt gaa ggt cat gtt aat gat aag agt ttc 384
Arg Asn His Ser Thr Ser Val Glu Gly His Val Asn Asp Lys Ser Phe
115120125
gag agg att tat gtt cgt gga ggg ttg aat gtg aag ccg ttg gtg att 432
Glu Arg Ile Tyr Val Arg Gly Gly Leu Asn Val Lys Pro Leu Val Ile
130135140
gaa agg gtg gag aaa ggg gag aaa gta agg gaa gag gag ggt agg gta 480
Glu Arg Val Glu Lys Gly Glu Lys Vel Arg Glu Glu Glu Gly Arg Val
145150155160
gga gtt aat ggt tcg aat gta aat att ggt gat tcg aaa ggt tta aat 528
Gly Val Asn Gly Ser Asn Val Asn Ile Gly Asp Ser Lys Gly Leu Asn
165170175
ggg ggt aag gtt ttg tct ccg aag aga gag gtg tct gag gtc gaa aaa 576
Gly Gly Lys Val Leu Ser Pro Lys Arg Glu Val Ser Glu Val Glu Lys
180185190
gag gct tgg gag tta ctt cga ggt gct gtt gtt gat tat tgt gga aac 624
Glu Ala Trp Glu Leu Leu Arg Gly Ala Val Val Asp Tyr Cys Gly Asn
195200205
cct gtt ggg act gtt gca gct agt gat cca gct gat tct aca cca ctc 672
Pro Val Gly Thr Val Ala Ala Ser Asp Pro Ala Asp Ser Thr Pro Leu
210215220
aac tat gac cag gtg ttt att cgt gat ttt gtc ccc tct gct ctt gca 720
Asn Tyr Asp Gln Val Phe Ile Arg Asp Phe Val Pro Ser Ala Leu Ala
225230235240
ttc ttg ctt aat gga gaa ggg gag att gtt aag aat ttt ctg cta cat 768
Phe Leu Leu Asn Gly Glu Gly Glu Ile Val Lys Asn Phe Leu Leu His
245250255
aca ctg cag tta cag agt tgg gaa aaa act gta gac tgc cat agc cct 816
Thr Leu Gln Leu Gln Ser Trp Glu Lys Thr Val Asp Cys His Ser Pro
260265270
ggg caa ggg ttg atg ccc gca agt ttc aaa gtt aaa aac gtg gct att 864
Gly Gln Gly Leu Met Pro Ala Ser Phe Lys Val Lys Asn Val Ala Ile
275280285
gat ggg aaa att gga gaa tca gag gat att tta gat cca gat ttc ggt 912
Asp Gly Lys Ile Gly Glu Ser Glu Asp Ile Leu Asp Pro Asp Phe Gly
290295300
gaa tca gcc ata ggt cgt gtt gca cct gtt gat tct ggg tta tgg tgg 960
Glu Ser Ala Ile Gly Arg Val Ala Pro Val Asp Ser Gly Leu Trp Trp
305310315320
atc att ttg tta aga gct tat act aag ctc aca gga gat tat ggg ctg 1008
Ile Ile Leu Leu Arg Ala Tyr Thr Lys Leu Thr Gly Asp Tyr Gly Leu
325330335
caa gca cga gtg gat gtg cag aca gga ata agg ctg ata ctt aat ctg 1056
Gln Ala Arg Val Asp Val Gln Thr Gly Ile Arg Leu Ile Leu Asn Leu
340345350
tgt tta acg gat gga ttc gac atg ttt cct aca ctg tta gtc act gat 1104
Cys Leu Thr Asp Gly Phe Asp Met Phe Pro Thr Leu Leu Val Thr Asp
355360365
ggt tcc tgt atg att gac aga agg atg ggc att cat ggt cac cct ctc 1152
Gly Ser Cys Met Ile Asp Arg Arg Met Gly Ile His Gly His Pro Leu
370375380
gaa att caa gca ttg ttt tat tca gct ttg cgt tgt tct cga gag atg 1200
Glu Ile Gln Ala Leu Phe Tyr Ser Ala Leu Arg Cys Ser Arg Glu Met
385390395400
ctc att gtc aat gat tcc aca aag aat ttg gtt gct gct gtc aac aac 1248
Leu Ile Val Asn Asp Ser Thr Lys Asn Leu Val Ala Ala Val Asn Asn
405410415
cgg ctt agt gca ctg tcc ttc cac att agg gag tat tat tgg gtg gac 1296
Arg Leu Ser Ala Leu Ser Phe His Ile Arg Glu Tyr Tyr Trp Val Asp
420425430
atg aag aag atc aat gaa ata tac cga tac aaa act gaa gaa tac tca 1344
Met Lys Lys Ile Asn Glu Ile Tyr Arg Tyr Lys Thr Glu Glu Tyr Ser
435440445
act gat gcc atc aat aag ttc aac atc tat ccg gat caa ata ccc tct 1392
Thr Asp Ala Ile Asn Lys Phe Asn Ile Tyr Pro Asp Gln Ile Pro Ser
450455460
tgg ctg gta gac tgg atg cct gag acg gga ggg tat ctc att ggc aat 1440
Trp Leu Val Asp Trp Met Pro Glu Thr Gly Gly Tyr Leu Ile Gly Asn
465470475480
ctg cag cca gct cat atg gac ttt aga ttc ttt acc cta gga aat ctt 1488
Leu Gln Pro Ala His Met Asp Phe Arg Phe Phe Thr Leu Gly Asn Leu
485490495
tgg tct att gtc tca tca ctg ggt aca cct aaa caa aat gag agc att 1536
Trp Ser Ile Val Ser Ser Leu Gly Thr Pro Lys Gln Asn Glu Ser Ile
500505510
tta aat ttg ata gaa gat aaa tgg gac gat ctt gtg gca cat atg cct 1584
Leu Asn Leu Ile Glu Asp Lys Trp Asp Asp Leu Val Ala His Met Pro
515520525
tta aaa ata tgt tac cct gct ctg gag tat gag gaa tgg cga gta ata 1632
Leu Lys Ile Cys Tyr Pro Ala Leu Glu Tyr Glu Glu Trp Arg Val Ile
530535540
aca ggc agt gac ccc aag aat acg cca tgg tca tat cat aat ggg gga 1680
Thr Gly Ser Asp Pro Lys Asn Thr Pro Trp Ser Tyr His Asn Gly Gly
545550555560
tcc tgg cca aca ctt ctc tgg cag ttt aca tta gct tgc att aag atg 1728
Ser Trp Pro Thr Leu Leu Trp Gln Phe Thr Leu Ala Cys Ile Lys Met
565570575
aag aaa cca gag ctt gca aga aag gcg gtg gcg ttg gcc gag aaa aag 1776
Lys Lys Pro Glu Leu Ala Arg Lys Ala Val Ala Leu Ala Glu Lys Lys
580585590
ctt tcg gag gat cat tgg cct gaa tat tat gat aca cgg cgt gga aga 1824
Leu Ser Glu Asp His Trp Pro Glu Tyr Tyr Asp Thr Arg Arg Gly Arg
595600605
ttt att ggg aaa caa tcc aga ctt tat cag aca tgg aca att gct ggc 1872
Phe Ile Gly Lys Gln Ser Arg Leu Tyr Gln Thr Trp Thr Ile Ala Gly
610615620
ttc tta aca tct aag ttg tta ttg gaa aat cca gag atg gca tca aag 1920
Phe Leu Thr Ser Lys Leu Leu Leu Glu Asn Pro Glu Met Ala Ser Lys
625630635640
ttg ttt tgg gag gaa gac tat gaa ctg ctc gag agc tgt gtc tgt gca 1968
Leu Phe Trp Glu Glu Asp Tyr Glu Leu Leu Glu Ser Cys Val Cys Ala
645650655
att ggc aaa tct ggt aga aag aag tgc tct cgg ttt gct gcc aaa tca 2016
Ile Gly Lys Ser Gly Arg Lys Lys Cys Ser Arg Phe Ala Ala Lys Ser
660665670
caa gtg gtc 2025
Gln Val Val
675
<210> 2
<211> 675
<212> PRT
<213> Carrots
<400> 2
Met Asn Thr Thr Cys Ile Ala Val Ser Asn Met Arg Pro Cys Cys Arg
151015
Met Leu Leu Ser Cys Lys Asn Ser Ser Ile Phe Gly Tyr Ser Phe Arg
202530
Lys Cys Asp His Arg Met Gly Thr Asn Leu Ser Lys Lys Gln Phe Lys
35 40 45
Val Tyr Gly Leu Arg Gly Tyr Val Ser Cys Arg Gly Gly Lys Gly Leu
50 55 60
Gly Tyr Arg Cys Gly Ile Asp Pro Asn Arg Lys Gly Phe Phe Gly Ser
65 70 75 80
Gly Ser Asp Trp Gly Gln Pro Arg Val Leu Thr Ser Gly Cys Arg Arg
85 90 95
Val Asp Ser Gly Gly Arg Ser Val Leu Val Asn Val Ala Ser Asp Tyr
100 105 110
Arg Asn His Ser Thr Ser Val Glu Gly His Val Asn Asp Lys Ser Phe
115120125
Glu Arg Ile Tyr Val Arg Gly Gly Leu Asn Val Lys Pro Leu Val Ile
130135140
Glu Arg Val Glu Lys Gly Glu Lys Val Arg Glu Glu Glu Gly Arg Val
145150155160
Gly Val Asn Gly Ser Asn Val Asn Ile Gly Asp Ser Lys Gly Leu Asn
165170175
Gly Gly Lys Val Leu Ser Pro Lys Arg Glu Val Ser Glu Val Glu Lys
180185190
Glu Ala Trp Glu Leu Leu Arg Gly Ala Val Val Asp Tyr Cys Gly Asn
195200205
Pro Val Gly Thr Val Ala Ala Ser Asp Pro Ala Asp Ser Thr Pro Leu
210215220
Asn Tyr Asp Gln Val Phe Ile Arg Asp Phe Val Pro Ser Ala Leu Ala
225230235240
Phe Leu Leu Asn Gly Glu Gly Glu Ile Val Lys Asn Phe Leu Leu His
245250255
Thr Leu Gln Leu Gln Ser Trp Glu Lys Thr Val Asp Cys His Ser Pro
260265270
Gly Gln Gly Leu Met Pro Ala Ser Phe Lys Val Lys Asn Val Ala Ile
275280285
Asp Gly Lys Ile Gly Glu Ser Glu Asp Ile Leu Asp Pro Asp Phe Gly
290295300
Glu Ser Ala Ile Gly Arg Val Ala Pro Val Asp Ser Gly Leu Trp Trp
305310315320
Ile Ile Leu Leu Arg Ala Tyr Thr Lys Leu Thr Gly Asp Tyr Gly Leu
325330335
Gln Ala Arg Val Asp Val Gln Thr Gly Ile Arg Leu Ile Leu Asn Leu
340345350
Cys Leu Thr Asp Gly Phe Asp Met Phe Pro Thr Leu Leu Val Thr Asp
355360365
Gly Ser Cys Met Ile Asp Arg Arg Met Gly Ile His Gly His Pro Leu
370375380
Glu Ile Gln Ala Leu Phe Tyr Ser Ala Leu Arg Cys Ser Arg Glu Met
385390395400
Leu Ile Val Asn Asp Ser Thr Lys Asn Leu Val Ala Ala Val Asn Asn
405410415
Arg Leu Ser Ala Leu Ser Phe His Ile Arg Glu Tyr Tyr Trp Val Asp
420425430
Met Lys Lys Ile Asn Glu Ile Tyr Arg Tyr Lys Thr Glu Glu Tyr Ser
435440445
Thr Asp Ala Ile Asn Lys Phe Asn Ile Tyr Pro Asp Gln Ile Pro Ser
450455460
Trp Leu Val Asp Trp Met Pro Glu Thr Gly Gly Tyr Leu Ile Gly Asn
465470475480
Leu Gln Pro Ala His Met Asp Phe Arg Phe Phe Thr Leu Gly Asn Leu
485490495
Trp Ser Ile Val Ser Ser Leu Gly Thr Pro Lys Gln Asn Glu Ser Ile
500505510
Leu Asn Leu Ile Glu Asp Lys Trp Asp Asp Leu Val Ala His Met Pro
515520525
Leu Lys Ile Cys Tyr Pro Ala Leu Glu Tyr Glu Glu Trp Arg Val Ile
530535540
Thr Gly Ser Asp Pro Lys Asn Thr Pro Trp Ser Tyr His Asn Gly Gly
545550555560
Ser Trp Pro Thr Leu Leu Trp Gln Phe Thr Leu Ala Cys Ile Lys Met
565570575
Lys Lys Pro Glu Leu Ala Arg Lys Ala Val Ala Leu Ala Glu Lys Lys
580585590
Leu Ser Glu Asp His Trp Pro Glu Tyr Tyr Asp Thr Arg Arg Gly Arg
595600605
Phe Ile Gly Lys Gln Ser Arg Leu Tyr Gln Thr Trp Thr Ile Ala Gly
610615620
Phe Leu Thr Ser Lys Leu Leu Leu Glu Asn Pro Glu Met Ala Ser Lys
625630635640
Leu Phe Trp Glu Glu Asp Tyr Glu Leu Leu Glu Ser Cys Val Cys Ala
645650655
Ile Gly Lys Ser Gly Arg Lys Lys Cys Ser Arg Phe Ala Ala Lys Ser
660665670
Gln Val Val
675
<210> 3
<211> 2447
<212> DNA
<213> Carrots
<400> 3
gaattccgat ttagcaaatt gttatagata tgaatactac ttgtattgct gtatcgaata 60
tgaggccttg ttgtagaatg ttacttagct gtaagaattc atcgattttc ggatactcgt 120
ttcgaaaatg tgatcataga atggggacta atttgtcgaa aaagcaattt aaggtgtacg 180
gtttgcgagg gtatgttagt tgtaggggtg gtaaaggttt aggttatagg tgtgggattg 240
atccgaatcg gaagggtttt tttggttccg gttctgattg gggacagcct agggttttaa 300
caagtggttg tcgtcgtgtt gatagtggtg gtaggagtgt acttgttaat gtggcgtcgg 360
attataggaa tcattcaact tcggttgaag gtcatgttaa tgataagagt ttcgagagga 420
tttatgttcg tggagggttg aatgtgaagc cgttggtgat tgaaagggtg gagaaagggg 480
agaaagtaag ggaagaggag ggtagggtag gagttaatgg ttcgaatgta aatattggtg 540
attcgaaagg tttaaatggg ggtaaggttt tgtctccgaa gagagaggtg tctgaggtcg 600
aaaaagaggc ttgggagtta cttcgaggtg ctgttgttga ttattgtgga aaccctgttg 660
ggactgttgc agctagtgat ccagctgatt ctacaccact caactatgac caggtgttta 720
ttcgtgattt tgtcccctct gctcttgcat tcttgcttaa tggagaaggg gagattgtta 780
agaattttct gctacataca ctgcagttac agagttggga aaaaactgta gactgccata 840
gccctgggca agggttgatg cccgcaagtt tcaaagttaa aaacgtggct attgatggga 900
aaattggaga atcagaggat attttagatc cagatttcgg tgaatcagcc ataggtcgtg 960
ttgcacctgt tgattctggg ttatggtgga tcattttgtt aagagcttat actaagctca 1020
caggagatta tgggctgcaa gcacgagtgg atgtgcagac aggaataagg ctgatactta 1080
atctgtgttt aacggatgga ttcgacatgt ttcctacact gttagtcact gatggttcct 1140
gtatgattga cagaaggatg ggcattcatg gtcaccctct cgaaattcaa gcattgtttt 1200
attcagcttt gcgttgttct cgagagatgc tcattgtcaa tgattccaca aagaatttgg 1260
ttgctgctgt caacaaccgg cttagtgcac tgtccttcca cattagggag tattattggg 1320
tggacatgaa gaagatcaat gaaatatacc gatacaaaac tgaagaatac tcaactgatg 1380
ccatcaataa gttcaacatc tatccggatc aaataccctc ttggctggta gactggatgc 1440
ctgagacggg agggtatctc attggcaatc tgcagccagc tcatatggac tttagattct 1500
ttaccctagg aaatctttgg tctattgtct catcactggg tacacctaaa caaaatgaga 1560
gcattttaaa tttgatagaa gataaatggg acgatcttgt ggcacatatg cctttaaaaa 1620
tatgttaccc tgctctggag tatgaggaat ggcgagtaat aacaggcagt gaccccaaga 1680
atacgccatg gtcatatcat aatgggggat cctggccaac acttctctgg cagtttacat 1740
tagcttgcat taagatgaag aaaccagagc ttgcaagaaa ggcggtggcg ttggccgaga 1800
aaaagctttc ggaggatcat tggcctgaat attatgatac acggcgtgga agatttattg 1860
ggaaacaatc cagactttat cagacatgga caattgctgg cttcttaaca tctaagttgt 1920
tattggaaaa tccagagatg gcatcaaagt tgttttggga ggaagactat gaactgctcg 1980
agagctgtgt ctgtgcaatt ggcaaatctg gtagaaagaa gtgctctcgg tttgctgcca 2040
aatcacaagt ggtctaatgg aggcccagtt taaggataag tatcaataac agatgaggcg 2100
ttctttctaa tccactactc tttagataga gcttcacagt tttagactga cagtgattga 2160
ttaagaggct gttgtgaata gccacatctg gatttaaaac ttctaagaat aagtatctag 2220
ctgacttgat tagatttatg agtctgaaga caatagcaga gcagtgttga acttattata 2280
gttttcttct tgctctgttt atggttagaa atccgcattt tctttctaac cataacagca 2340
tgattctttg tttctttggt gagcaaacac tttagaacct ggtttgagga atgaagcagg 2400
gtgacatttt cttaaaaaaa aaaaaaaaaa aaaaggaatt ggaattc 2447
<210> 4
<211> 6
<212> PRT
<213> Unknown
<220>
<223> unknown creatures Description: protein fragments
<400> 4
Val Gly Thr Val Ala Ala
15
<210> 5
<211> 5
<212> PRT
<213> Unknown
<220>
<223> unknown creatures Description: protein fragments
<400> 5
Ala Ile Gly Arg Val
15
<210> 6
<211> 22
<212> PRT
<213> Unknown
<220>
<223> unknown creatures Description: protein fragments
<400> 6
Asp Phe Gly Glu Ser Ala Ile Gly Arg Val Ala Pro Val Asp Ser Gly
151015
Leu Trp Trp Ile Ile Leu
20
<210> 7
<211> 9
<212> PRT
<213> Unknown
<220>
<223> unknown creatures Description: protein fragments
<400> 7
Cys Met Ile Asp Arg Arg Met Gly Ile
15
<210> 8
<211> 28
<212> PRT
<213> Unknown
<220>
<223> unknown creatures Description: protein fragments
<400> 8
Pro Thr Leu Leu Val Thr Asp Gly Ser Cys Met Ile Asp Arg Arg Met
151015
Gly Ile His Gly His Pro Leu Glu Ile Gln Ala Leu
20 25
<210> 9
<211> 7
<212> PRT
<213> Unknown
<220>
<223> unknown creatures Description: protein fragments
<400> 9
Gly Gly Tyr Leu Ile Gly Asn
15
<210> 10
<211> 9
<212> PRT
<213> Unknown
<220>
<223> unknown creatures Description: protein fragments
<400> 10
Asp Phe Arg Phe Phe Thr Leu Gly Asn
15
<210> 11
<211> 13
<212> PRT
<213> Unknown
<220>
<223> unknown creatures Description: protein fragments
<400> 11
Xaa Asn Ile Tyr Pro Asp Gln Ile Pro Pro Trp Leu Val
1510
<210> 12
<211> 25
<212> DNA
<213> Unknown
<220>
<223> unknown creatures Description: oligonucleotide
<400> 12
tctaaggatc tagaaagagc catta 25
<210> 13
<211> 24
<212> DNA
<213> Unknown
<220>
<223> unknown creatures Description: oligonucleotides:
<400> 13
ttcaattgaa ttcaatatag cttc 24
<210> 14
<211> 31
<212> DNA
<213> Unknown
<220>
<223> unknown creatures Description: oligonucleotide
<400> 14
cgatttagca aggtaccata gatatgaata c 31
<210> 15
<211> 30
<212> DNA
<213> Unknown
<220>
<223> unknown creatures Description: oligonucleotide
<400> 15
cttatcctta aactagatct ccattagacc 30
...
Claims (10)
1. comprise the DNA that can be translated as proteinic nucleotide sequence, wherein reach maximum sucrase active at pH6.0 to 7.5 with sucrase active.
2. the coded plant protein DNA of claim 1.
3. the DNA of claim 1 wherein comprises the nucleotide sequence that coding is selected from down the aminoacid sequence of group: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.
4. the DNA of claim 1 wherein comprises the described proteinic nucleotide sequence of coding SEQ ID NO:2.
5. the DNA of claim 1 wherein comprises the described nucleotide sequence of SEQ ID NO:1.
6. one kind has sucrase active but lacks the active protein of saccharase, wherein at pH6.0 to 7.5 maximum sucrase active is arranged.
7. the plant protein of claim 6, wherein said protein comprise and are selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9 and the described aminoacid sequence of SEQ ID NO:10.
8. the plant protein of claim 6, it has the described aminoacid sequence of SEQ ID NO:2.
9. method for preparing the described DNA of claim 1, this method comprises: the clone of the fragment hybridization of the DNA that can limit with SEQ ID NO:3 in the-screening DNA library, wherein said fragment is to 15 Nucleotide of the youthful and the elderly;-measure and hybridize the sequence of cloning;-purifying clone's carrier DNA, it comprises coding and SEQ ID NO:2 sequence homogeny at the proteinic opening code-reading frame more than 40%.The DNA of-optional this purifying of further processing.
10. polymerase chain reaction, wherein at least one used oligonucleotide comprises 15 or the nucleotide sequence of representing among SEQID NO:1 or the SEQ ID NO:3 of more a plurality of base pairs.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9802249.4 | 1998-02-03 | ||
| GBGB9802249.4A GB9802249D0 (en) | 1998-02-03 | 1998-02-03 | Organic compounds |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1289367A true CN1289367A (en) | 2001-03-28 |
Family
ID=10826352
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 99802663 Pending CN1289367A (en) | 1998-02-03 | 1999-02-01 | Plant alkaline and neutral invertases |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP1053334A1 (en) |
| JP (1) | JP2002505084A (en) |
| CN (1) | CN1289367A (en) |
| AR (1) | AR014528A1 (en) |
| AU (1) | AU742048B2 (en) |
| CA (1) | CA2319316A1 (en) |
| GB (1) | GB9802249D0 (en) |
| WO (1) | WO1999040206A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101812433A (en) * | 2009-11-10 | 2010-08-25 | 中国热带农业科学院橡胶研究所 | Use of hevea brasiliensis invertase and coding gene thereof |
| CN110904079A (en) * | 2020-01-07 | 2020-03-24 | 中国科学院天津工业生物技术研究所 | β -fructofuranosidase mutant, mutant gene and application thereof in preparation of vitamin B12In (1) |
| CN116144632A (en) * | 2023-02-07 | 2023-05-23 | 青岛农业大学 | Tea tree neutral/alkaline invertase CsINV2 protein and preparation method and application thereof |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7138262B1 (en) | 2000-08-18 | 2006-11-21 | Shire Human Genetic Therapies, Inc. | High mannose proteins and methods of making high mannose proteins |
| ES2391657T3 (en) | 2006-02-07 | 2012-11-28 | Shire Human Genetic Therapies, Inc. | Stabilized protein compositions that have a free thiol moiety |
| RU2733466C2 (en) | 2009-07-28 | 2020-10-01 | Шайр Хьюман Дженетик Терапиз | Compositions and methods for treating gaucher disease |
| US9453847B2 (en) | 2010-07-19 | 2016-09-27 | Shire Human Genetic Therapies, Inc. | Mannose receptor C type 1 (MRC1) codon optimized cell line and uses thereof |
| KR20140138850A (en) | 2012-03-02 | 2014-12-04 | 샤이어 휴먼 지네틱 테라피즈 인크. | Compositions and methods for treating type iii gaucher disease |
| JP7429896B2 (en) * | 2020-12-25 | 2024-02-09 | パナソニックＩｐマネジメント株式会社 | Method for producing plant acid invertase activator, plant acid invertase activator, and method for activating plant acid invertase |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4213444A1 (en) * | 1992-04-18 | 1993-10-28 | Inst Genbiologische Forschung | Prodn. of potato plants with suppressed tuber sprouting - by genetic modification to reduce sucrose concn. |
-
1998
- 1998-02-03 GB GBGB9802249.4A patent/GB9802249D0/en not_active Ceased
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1999
- 1999-02-01 AR ARP990100411 patent/AR014528A1/en not_active Application Discontinuation
- 1999-02-01 WO PCT/EP1999/000623 patent/WO1999040206A1/en not_active Application Discontinuation
- 1999-02-01 EP EP99907465A patent/EP1053334A1/en not_active Withdrawn
- 1999-02-01 AU AU27217/99A patent/AU742048B2/en not_active Ceased
- 1999-02-01 CA CA002319316A patent/CA2319316A1/en not_active Abandoned
- 1999-02-01 CN CN 99802663 patent/CN1289367A/en active Pending
- 1999-02-01 JP JP2000530618A patent/JP2002505084A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101812433A (en) * | 2009-11-10 | 2010-08-25 | 中国热带农业科学院橡胶研究所 | Use of hevea brasiliensis invertase and coding gene thereof |
| CN110904079A (en) * | 2020-01-07 | 2020-03-24 | 中国科学院天津工业生物技术研究所 | β -fructofuranosidase mutant, mutant gene and application thereof in preparation of vitamin B12In (1) |
| CN116144632A (en) * | 2023-02-07 | 2023-05-23 | 青岛农业大学 | Tea tree neutral/alkaline invertase CsINV2 protein and preparation method and application thereof |
| CN116144632B (en) * | 2023-02-07 | 2024-04-19 | 青岛农业大学 | Tea tree neutral/alkaline invertase CsINV protein and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| AR014528A1 (en) | 2001-02-28 |
| EP1053334A1 (en) | 2000-11-22 |
| JP2002505084A (en) | 2002-02-19 |
| AU742048B2 (en) | 2001-12-13 |
| CA2319316A1 (en) | 1999-08-12 |
| AU2721799A (en) | 1999-08-23 |
| WO1999040206A1 (en) | 1999-08-12 |
| GB9802249D0 (en) | 1998-04-01 |
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