CN1264983C

CN1264983C - Heat-resistant phytase, clone and expression of gene

Info

Publication number: CN1264983C
Application number: CNB031299857A
Authority: CN
Inventors: 刘丽丽; 陈立新; 杨文博
Original assignee: TIANJIN CITY AGRICULTURAL BIO-TECH RESEARCH CENTER; Tianjin Normal University
Current assignee: TIANJIN CITY AGRICULTURAL BIO-TECH RESEARCH CENTER; Tianjin Normal University
Priority date: 2003-06-03
Filing date: 2003-06-03
Publication date: 2006-07-19
Anticipated expiration: 2023-06-03
Also published as: CN1552872A

Abstract

本发明公开了一种耐热植酸酶及其基因的克隆和表达。解决了现有植酸酶耐温性较差及营养无利用流失和必须营养的“添加浪费”等问题。技术方案：筛选耐热性植酸酶天然菌株，利用分子生物学的手段，将编码此产物的基因进行基因克隆；该基因全长1152核苷酸，编码383个氨基酸；N端的26个氨基酸为信号肽，信号肽的切割位点在+26位的丙氨酸之后；构建耐热植酸酶基因的工程菌株SDLiuTP01，表达基因产物。本发明的耐热植酸酶在较宽温度范围内具有生物活性、能促进植物生长、增加土壤肥力、不破坏农作物品质；降解食品和饲料中的不溶性磷、提高磷的利用、减少有机磷对环境的污染。The invention discloses the cloning and expression of a thermostable phytase and its gene. It solves the problems of poor temperature resistance of the existing phytase, non-utilized loss of nutrients and "waste of addition" of necessary nutrients. Technical solution: Screen the natural strains of heat-resistant phytase, and use the means of molecular biology to clone the gene encoding this product; the gene is 1152 nucleotides in full length, encoding 383 amino acids; the 26 amino acids at the N-terminal are The signal peptide, the cleavage site of the signal peptide is after the alanine at position +26; the engineering strain SDLiuTP01 of the heat-resistant phytase gene was constructed to express the gene product. The heat-resistant phytase of the present invention has biological activity in a wide temperature range, can promote plant growth, increase soil fertility, and do not damage the quality of crops; degrade insoluble phosphorus in food and feed, improve phosphorus utilization, and reduce organic phosphorus. pollution of the environment.

Description

The clone of heat-stable phytase and gene thereof and expression

Technical field

The present invention relates to a kind of phytase, the clone and the expression of particularly a kind of phytase and gene thereof.

Background technology

For a long time, the rural area is extensive use of chemical fertilizer, and chemical fertilizer can increase soil fertility, but uses chemical fertilizer continuously, and fertilizer efficiency day subtracts, and has to strengthen rate of fertilizer application.And the excessive chemical fertilizer of using then can cause soil compaction, and soil fertility weakens, and also destroys crops quality.Cause the chemical residual public hazards simultaneously, contaminate environment (as body eutrophication) causes vicious cycle, jeopardizes the descendants.At the seventies, set up " Organic farming alliance " in the world, appeal need not or to use chemical fertilizer less at world wide as far as possible, advocate energetically and use organic fertilizer.Under this common recognition, countries in the world are all making great efforts to come fertilizer for replacing chemical with bio-bacterial manure.The nothing chemistry farm without chemical fertilizer and agricultural chemicals is developed on the farm of the existing U.S. existing 5%.Germany is encouraging and is developing without chemical fertilizer, and ecological agricultural model is adopted in existing 3% farmland.USSR (Union of Soviet Socialist Republics) also has 1/2 beans farm crop to use microbial fertilizer.

The activity of existing bio-feritlizer is insoluble inorganic phosphorus of mineralising [Ca3 (PO4) 2Ca (OH) 2] and part organophosphorus mostly.Make the invalid phosphorus in the topsoil soils become the utilizable phosphorus nutrition of plant.Phosphorus 15%--95% in the topsoil soil is an organic form.Wherein a large amount of organophosphoruss exist with the form of phytic acid.Particularly promotion is returned crop stocks to the field and is used after the muck, and more phytic acid is brought into topsoil.Many decision phosphorus that studies show that are not increased organic phosphorus content in the soil to the principal element of the validity of plant, but mineralization rate.The effect substrate of phytase is phytic acid and its esters, and it can discharge available phosphorus.

Yet the coded phytase of plant is a nonsecreting type, owing to lack the outer phytic acid enzyme activity of born of the same parents, plant is very low for the utilization ratio of the main component phytic acid of organophosphorus in the soil.The phytase (3-Phytase, Myo-Inositol-hexaphosphate 3-phosphohydrolase) that is produced by certain micro-organisms under the state of nature can act on ester bond, and the hydrolysis of catalysis phosphate monoester can be inositol and inorganic phosphorus with hydrolysis of phytic acid.Concrete reaction is:

Phytase is a kind of novel monogastric animal feed additive that hydrolysis of phytic acid in the feed can be become inorganic phosphorus and inositol simultaneously, and this is one of research focus of our times.Phosphorus mainly exists with the form of phytic acid (phytinic acid) and its esters in plant.Their content in plant and oil crop seeds by using accounts for the 1%-3% of dry weight, but is storing the 60%-90% of total phosphorus in the plant, is the main source of the required phosphorus of seed germination.The required phosphorus of animal mainly obtains from plant.The growth of animal, breeding, mineralization of skeleton and metabolism all need phosphorus.Yet in the food of people and monogastric animal, the phytic acid that derives from plant has the intensive anti-oxidant action.Kentucky, United States university studies confirm that by 10 years pig can only utilize the 10%-20% of phosphorus in the corn, the 15%-35% of phosphorus in the soya-bean cake, and chicken is also lower than pig to the utilization ratio of phosphorus in corn and the dregs of beans.This shows that about 85% phosphorus fails effectively to absorb just to discharge in the food from ight soil.

Phosphorus has vital role as a kind of necessary mineral nutrient in Animal nutrition.For prevention phosphorus deficiency disease, must in food or feed, add inorganic phosphate again, to satisfy the demand of livestock and poultry, therefore, cause the serious waste of phosphoric acid to phosphorus.Human nutrition scholar studies show that this character of phytic acid has also caused the shortage and the quantity imbalance of human calcium, zinc, iron, potassium.Food utilization is low, nutrition does not have the loss of utilizing and " adding waste " phenomenon of necessary nutrition because the anti-oxidant action of phytic acid has caused, and also is a great problem that always perplexs food and fodder industry.

Phytase can be removed the anti-oxidant action of phytic acid, improves mineral element and proteinic nutrition validity and energy balance ability in food and the feed.Nayini etc. (1983) prove that after the interpolation phytase fermented through about 2 hours, phytate content can reduce 58%-70%.Phytase can get off phosphoric acid hydrolysis from the phytic acid molecule, has destroyed it to mineral substance and proteinic avidity, thereby improves the nutrition validity and the proteinic digestibility of mineral substance; Can make simultaneously some enzymes such as amylase etc. recover its original activity, improve the energy efficiency of food and feed.Simons etc. add phytase in the chicken feed to, the broiler of feeding, and test shows, adds phytase and can make the utilization ratio of phosphorus improve 60%, the phosphorus output reduces 40% in the ight soil, has significantly improved the gain in weight and the feeding effect of broiler chicken.This shows, be particular about dietary nutrition and paying attention to today of efficiency of feed utilization, study and utilize phytase to have considerable meaning.

Yet phytase is applied to agricultural and food and feed additive, need carry out industrialization processing and processing, for this reason, requires phytase after processes and processing through comparatively high temps, still can keep higher biological activity.And its temperature tolerance of existing phytase is relatively poor, and phytase is restricted in the application in agricultural and food and feed additive field.

Summary of the invention

The present invention be directed to the research that the problems referred to above are carried out, purpose be the screening a kind of bacterial strain that produces heat-stable phytase, utilize molecular biological means, with the coding this product gene carry out gene clone and expression; Provide a kind of through comparatively high temps processes and handle after the heat-stable phytase of biologically active still.

Technical scheme of the present invention is: screen a kind of natural bacterial strain that produces heat-stable phytase, and from bacterial strain, extract gene DNA, adopt PCR method (ManiatisT., et al.Molecular cloning.New York:cold spring harberlaberalory, 1982) amplify the heat-stable phytase gene.Utilize ligase enzyme that this gene is connected on the cloning vector pMD18-T, transformed into escherichia coli detects positive colony.Carry out complete sequence analysis for the entrained goal gene of positive colony.The heat-stable phytase gene DNA sequence is S1, and deriving aminoacid sequence by dna sequence dna is S2.

＜110〉Tianjin Normal University

＜120〉clone of heat-stable phytase and gene thereof and expression

<130>03152

<140>03129985.7

<141>2003-06-03

<160>2

<170>PatentIn version 3.1

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<212>DNA

＜213〉genus bacillus (Bacillus sp.)

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atgaatcatt caaaaacact tttgttaacc gcggcagccg gattgatgct cacatgcggt 60

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gcggcggaaa cggagccggt tgatacagcc ggtgatgcag ctgatgatcc tgcgatttgg 180

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gtcgtgtaca gcctagaggg aaagacgctt cattcctatc ataccgggaa gctgaacaat 300

gttgatatcc gctatgattt tccgttgaac ggaaaaaaag tcgatattgc ggcggcatcc 360

aatcggtctg aaggaaagaa taccattgag atttacgcca ttgacgggaa aaacggcaca 420

ttacaaagca ttacagatcc agaccgcccg attgcatcag caattgatga agtatacggt 480

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ggcgaatttg aacaatacga attaaatgcg gataaaaatg gatacatatc cggcaaaaag 600

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ggcagtaacg gaacggttat cgatcgtgcc gacggcaggc atttaacccc tgatattgaa 780

ggactgacga tttactacgc tgctgacggg aaaggttatc tgcttgcatc aagccagggt 840

aacagcagct acgcgattta tgaaagacag ggacagaaca aatatgttgc ggactttcag 900

ataacagacg ggcctgaaac agacggcaca agcgatacag acggaattga cgttctgggt 960

ttcgggctgg ggcctgaata tccgttcggc ctttttgtcg cacaggatgg agaaaatata 1020

gatcacggcc aaaaagtgaa tcaaaatttt aaaatggtgc cttgggaaag aatcgccgat 1080

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agcggaaaat aaacatgcaa aaagcagctt atacaagctg ctttttgcat gtgaagaacg 1200

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＜213〉genus bacillus (Bacillus sp.)

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Gly Glu Asn Ile Asp His Gly Gln Lys Val Asn Gln Asn Phe Lys Met

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Heat-stable phytase full length gene 1152 Nucleotide, 383 amino acid of encoding; 26 amino acid of N end are signal peptide, and the cleavage site of signal peptide is after+26 Ala.

The cloning process of heat-stable phytase gene comprises the steps:

(1) screening of bacterial strain:

A) soil sampling a little, put into the triangular flask of sterilized water, shake up, dilution is coated on the genetic donor bacterium screening solid medium, cultivates 24 hours for 37 ℃, selects on the single bacterium colony switching genetic donor bacterium screening culture medium;

B) collect various bacterium pure culture bacterial classifications, on the switching genetic donor bacterium screening culture medium slant medium.Jiang's strain bacterium is transferred on genetic donor bacterium screening solid medium with identical method, cultivates 7 days for 37 ℃, takes out, and relatively the situation of hydrolysis circle is selected the bacterial classification that forms obvious hydrolysis circle, obtains SD01N, SD01X, SD01B, SD01D.

(2) design synthetic primer P1 and P2:

Extract SD01N respectively according to the method that bibliography (" fine works molecular biology experiment guide " [U.S.] F. Ao Sibai etc.) provides, SD01X, SD01B, total DNA of SD01D bacterial strain, design synthetic primer P1 and P2

P1：5’CTG CAG GAT CCA TGA ATC ATT CAA AAA CAC TTT TGT 3’1

P2：5’TTT AAG CTT CGT TCT TCA CAT GCA AAA AGC 3’

(3) clone's heat-stable phytase gene

With SD01N, SD01X, SD01B, the genomic dna of SD01D bacterial strain are that template is carried out pcr amplification respectively, and reaction parameter is: 94 ℃ 1 minute, 50 ℃ 1 minute, 72 ℃ 2 minutes, 30 circulations, 72 ℃ 10 minutes.Amplify the dna fragmentation of about 1.2Kb by the genomic dna of SD01N bacterial strain, by agarose gel electrophoresis, reclaim dna fragmentation, after cutting, enzyme is connected on the carrier pMD18-T, obtain pMD18-TP, Transformed E .coli Competent CellJM109, screening positive clone carries out the analysis of DNA complete sequence determination, the sequencing result is S2,1152 Nucleotide of heat-stable phytase full length gene, 383 amino acid of encoding, its aminoacid sequence is S2; 26 amino acid of N-end are signal peptide, and the cleavage site of signal peptide is after+26 Ala.

The engineering strain preparation method of heat-stable phytase gene, it comprises the steps:

(1) the heat-stable phytase gene is downcut from pMD18-TP, the pQE30 that is connected in expression vector again goes up and forms recombinant DNA is pQE30-P;

(2) the single colony inoculation of picking E.Coli M15 is in LB (Km50 μ g/mlAp100 μ g/ml) substratum, LB substratum: 1% peptone, 0.5% yeast extract, 1%NaCl, PH7; 250rpm, 37 ℃, shaking table was cultivated 3 hours, and ice bath 10 minutes changes in the precooling centrifuge tube, frozen centrifugation, collecting precipitation is used CaCl ₂Solution is washed precipitation, and is resuspended in CaCl ₂In the solution, ice bath 30 minutes adds pQE30-P, ice bath 30 minutes, and 42 ℃ of water-baths 1 minute and 30 seconds, ice bath 2 minutes adds fresh LB nutrient solution, and 37 ℃ of 250rpm cultivated 1 hour, and culture is applied on the LB plate culture medium, puts into 37 ℃ of incubator overnight incubation.

(3) selected clone is with method screening positive clone of PCR, with positive colony switching LB slant medium; Thus, filter out heat-stable phytase engineering strain SDLiuTP01, i.e. the engineering strain of structure of the present invention, its microorganism classification is called intestinal bacteria (E.coli).

Heat-stable phytase expression of gene product of the present invention----is a biologically active in 25---95 ℃ of temperature range, and optimal reactive temperature is 65 ℃ a heat-stable phytase, sees Fig. 5.

Heat-stable phytase of the present invention, it can be used for developing fertilizer, feed, foodstuff additive such as plant growth promoter, compost additive, bio-feritlizer, bio-bacterial manure, organic fertilizer, compound manure.

Advantage of the present invention and positively effect are: the bacterial strain that is used for microbial fertilizer and plant growth promoter production at present is prokaryotic organism.Because the present invention holds the heat-stable phytase gene source in prokaryotic organism, the heat-stable phytase gene in prokaryotic organism source and these bacterial strains have the sibship on the genetics, and the difference between species is less.Therefore, the present invention holds the heat-stable phytase gene can genetic engineering means import these bacterial strains, obtain new phytase gene engineering strain, make these natural bacterial strains increase the new proterties of heat-stable phytase, can make it be applied to scientific research and production practice better with good character.

The present invention holds the heat-stable phytase gene and imports eukaryote, can more convenient expression heat-stable phytase by gene recombination.

The present invention clone's heat-stable phytase gene has unique nucleotide sequence, and the phytase of its coding is a heat-stable phytase, because have thermotolerance, biologically active in wide temperature range experimental results show that, equal biologically active is seen Fig. 5 in 25-95 ℃ of temperature range.Its product is in the industrialization operating process, for example in the operations such as composting, fermentation, granulation, pulverizing, transportation, still can keep higher biological activity, solve the high temperature resistant problem in the processes such as phytase production, manufacturing, transportation, its agricultural and industrial application value are increased.

The present invention clone's heat-stable phytase has increases soil fertility, do not destroy crops quality, improve phosphorus utilization, promote the effect of plant-growth.Can remove the anti-oxidant action of phytic acid, improve mineral element and proteinic nutrition validity and energy balance ability in food and the feed, improve the nutrition validity and the proteinic digestibility of mineral substance; Simultaneously, can make some enzymes such as amylase etc. recover its original activity, improve the energy efficiency of food and feed.Solve nutrition and do not had " adding waste " this puzzlement food of loss of utilizing and necessary nutrition and the hang-up of fodder industry.

Those of ordinary skills can obtain the heat-stable phytase gene by the present invention's method, and import the listed biology of the present invention; Also can carry out various transformations to enzyme of the present invention, as modifying with various chemically modified genes, as glycosylation, phosphorylation, N holds amination, and protein endoenzyme cuts etc., also can add signal peptide and add fusogenic peptide etc. before or after heat-stable phytase albumen for the transhipment after improving it and expressing, and do not change the basic function of enzyme of the present invention, as enzymic activity, substrate specificity.The functional deriv that these are commonly referred to as enzyme of the present invention includes within the scope of the invention.

Description of drawings:

The transparent hydrolysis circle that Fig. 1 .Bacillus sp.SD01N forms on the screening flat board.

Fig. 2. heat-stable phytase gene amplification electrophorogram

The physical map of Fig. 3 genetic engineering bacterium strain expression vector pQE30

The positive colony electrophorogram of Fig. 4 engineering strain SDLiuTP01

1, the negative clone 2 of 3PCR, 4PCR positive colony

The suitable property of the temperature of the heat-stable phytase that Fig. 5 engineering strain SD LiuTP01 produces

Embodiment

Embodiment 1:

1. material source

Biological enzyme: restriction enzyme, ligase enzyme are purchased the company in Promega, and Tag enzyme, DNA recovery kit etc. is purchased in Shanghai bio-engineering corporation;

Chemical reagent: phytic acid ca, sodium phytate are purchased the company in Sigma, and IPTG purchases in couple stars biotech firm;

Substratum: genetic donor bacterium screening culture medium: D-glucose 20%, Sodium phytate 0.01%, CaCl ₂0.2%, NH ₄NO ₃0.5%, KCl 0.05%, MgSO ₄7H ₂O 0.05%, FeSO ₄7H ₂O 0.001%, MnSO ₄H ₂O0.001%, agar 20%;

Other: the synthetic living worker biotech firm of sea base health biotech company and Shanghai of going up of primer; Precious biotech firm in determined dna sequence Dalian and associating Gene science company; E.coli JM109 and pMD18-T purchase the precious biotech firm in Dalian.

2. produce the screening of the natural bacterial strain of heat-stable phytase:

(1) soil sampling 1 restrains, and puts into the triangular flask of sterilized water, shakes up, and dilution is coated on the genetic donor bacterium screening solid medium, cultivates 24 hours for 37 ℃, selects on the single bacterium colony switching genetic donor bacterium screening culture medium.

(2) collect various bacterium pure culture bacterial classifications, on the switching genetic donor bacterium screening culture medium slant medium.Totally 203 strain bacterium are transferred on genetic donor bacterium screening solid medium with identical method, cultivate 7 days for 37 ℃, take out, relatively the situation of hydrolysis circle.Select bacterial classification totally 4 strains that form obvious hydrolysis circle.Obtain SD01N, SD01X, SD01B, SD01D sees Fig. 1.

3. synthetic primer

P1：5’CTG CAG GAT CCA TGA ATC ATT CAA AAA CAC TTT TGT 3’1

P2：5’TTT AAG CTT CGT TCT TCA CAT GCA AAA AGC 3’

4. clone the heat-stable phytase gene

With SD01N, SD01X, SD01B, the genomic dna of SD01D bacterial strain are that template is carried out pcr amplification respectively, and reaction parameter is: 94 ℃ 1 minute, 50 ℃ 1 minute, 72 ℃ 2 minutes, 30 circulations, 72 ℃ 10 minutes.Amplify the dna fragmentation of about 1.2Kb by the genomic dna of SD01N bacterial strain, see Fig. 2.By agarose gel electrophoresis, reclaim dna fragmentation, after cutting, enzyme is connected on the carrier pMD18-T, obtain pMD18-TP.Transformed E .coli CompetentCell JM109, screening positive clone carries out the analysis of DNA complete sequence determination, and sequencing the results are shown in S1.1152 Nucleotide of heat-stable phytase full length gene, 383 amino acid of encoding, 26 amino acid of N-end are signal peptide, and the cleavage site of signal peptide is after+26 Ala, and its aminoacid sequence is seen S2.

Embodiment 2

The foundation of heat-stable phytase expression system: select efficient expression system E.Coli M15 as the bio-reactor of expressing heat-stable phytase.The heat-stable phytase gene that embodiment 1 is obtained downcuts from pMD18-TP, and the pQE30 that is connected in expression vector again goes up and forms recombinant DNA is pQE30-P, sees Fig. 3.

The single colony inoculation of picking E.Coli M15 in LB (Km50 μ g/mlAp100 μ g/ml) substratum, the LB substratum: 1% peptone, 0.5% yeast extract, 1%NaCl, PH 7; 250rpm, 37 ℃, shaking table was cultivated 3 hours.Took out the horse back ice bath 10 minutes, and changed in the precooling centrifuge tube, frozen centrifugation, collecting precipitation is used cold CaCl ₂Solution is washed precipitation, and is resuspended in CaCl ₂In the solution, ice bath 30 minutes adds pQE30-P, ice bath 30 minutes, and 42 ℃ of water-baths 1 minute and 30 seconds, ice bath 2 minutes adds fresh LB nutrient solution, and 37 ℃ of 250rpm cultivated 1 hour, and culture is applied on the LB plate culture medium, puts into 37 ℃ of incubator overnight incubation.

Selected clone, with method screening positive clone of PCR, electrophoresis result is seen Fig. 4.With positive colony switching LB slant medium.Thus, filter out heat-stable phytase engineering strain SDLiuTP01, its microorganism classification is called intestinal bacteria E.coli.

Embodiment 3

The heat-stable phytase characteristic: SDLiuTP01 is incubated at LB, and (Km25ug/ml, Ap50ug/ml) in the nutrient solution, 37 ℃, 250rpm spends the night.The IPTG induced product forms.Measure the biological activity of heat-stable phytase, the temperature range of reaction is 25-95 ℃, heat-stable phytase biologically active all in this range of reaction temperature, and optimal reactive temperature is 65 ℃, sees Fig. 5.

Embodiment 4:

The cultivation of crop: the seed of surface sterilization is placed on the solid medium, cultivated 4 days.Aseptic corn seedling is transferred in the sterile test tube, and the test tube bottom adds aseptic culture fluid (nutrient solution prescription: Ca (No ₃) ₂0.88g, NaH ₂PO ₄0.006g, K ₂SO ₄0.39g, MgSO ₄0.31g, FeEDTA 0.0031g, trace element solution 1ml, sodium phytate 1.25mM, pH6.5).

Adding the crude extract 20 μ l of SDLiuTP01 bacterium in each test tube, is contrast with phytase liquid.Do not add crude extract, the test tube that adds 20 μ l deionized waters is a blank.Above-mentioned test tube was cultivated 15 days.The results plant materials is measured the plant-growth parameter.

Coerce in the nutrient solution under the 20 μ l SDLiuTP01 bacterium crude extract conditions of adding the above plant body weight of root 201mg, root weight 173mg, the long 238mm of root at the phosphorus that contains phytic acid; Coerce under the condition that adds phytase in the nutrient solution the above plant body weight of root 187mg, the heavy 181mg of root, the long 243mm of root at the phosphorus that contains phytic acid; Coerce in the nutrient solution at the phosphorus that contains phytic acid and to add under the deionized water condition the above plant body weight of root 111mg, root weight 103mg, the long 173mm of root.

Experimental result shows that the corn seedling of growing after adding the SDLiuTP01 crude extract, compares with the control group that does not add the SDLiuTP01 crude extract under the phosphorus stress conditions in containing the nutrient solution of phytic acid, the growth of plant is promoted.

Claims

1. A heat-resistant phytase gene is characterized in that the heat-resistant phytase gene is 1152 nucleotides in length, the gene encodes 383 amino acids, and 26 amino acids at the N-terminal are signal peptides, and the cleavage site of the signal peptide is at After the alanine at position +26, the DNA sequence of the gene is S1, and the amino acid sequence deduced from the DNA sequence is S2:

S1

NewSequence Sequence

ATGAATCATT CAAAAACACT TTTGTTAACC GCGGCAGCCG GATTGATGCT 50

CACATGCGGT GCGGTTTCTT CCCAGGCCAA GCATAAGCTG TCTGATCCTT 100

ATCACTTTAC CGTGAATGCG GCGGCGGAAA CGGAGCCGGT TGATACAGCC 150

GGTGATGCAG CTGATGATCC TGCGATTTGG CTGGACCCCA AGAATCCTCA 200

GAACAGCAAA TTGATCACAA CCAATAAAAA ATCAGGCTTA GTCGTGTACA 250

GCCTAGAGGG AAAGACGCTT CATTCCTATC ATACCGGGAA GCTGAACAAT 300

GTTGATATCC GCTATGATTT TCCGTTGAAC GGAAAAAAAAG TCGATATTGC 350

GGCGGCATCC AATCGGTCTG AAGGAAAGAA TACCATTGAG ATTTACGCCA 400

TTGACGGGAA AAACGGCACA TTACAAAGCA TTACAGATCC AGACCGCCCG 450

ATTGCATCAG CAATTGATGA AGTATACGGT TTCAGCTTGT ACCACAGTCA 500

AAAAACAGGA AAATATTACG CGATGGTGAC AGGGAAAGAA GGCGAATTTG 550

AACAATACGA ATTAAATGCG GATAAAAATG GATACATATC CGGCAAAAAG 600

GTAAGGGCGT TTAAAATGAA TTCTCAGACA GAAGGGATGG CAGCAGACGA 650

TGAATACGGC AGTCTTTTATA TCGCAGAAGA AGATGAGGCC ATCTGGAAGT 700

TCAGCGCTGA GCCGGACGGC GGCAGTAACG GAACGGTTAT CGATCGTGCC 750

GACGGCAGGC ATTTAACCC TGATATTGAA GGACTGACGA TTTACTACGC 800

TGCTGACGGG AAAGGTTATC TGCTTGCATC AAGCCAGGGT AACAGCAGCT 850

ACGCGATTTA TGAAAGACAG GGACAGAACA AATATGTTGC GGACTTTCAG 900

ATAACAGACG GGCCTGAAAC AGACGGCACA AGCGATACAG ACGGAATTGA 950

CGTTCTGGGT TTCGGGCTGG GGCCTGAATA TCCGTTCGGC CTTTTTGTCG 1000

CACAGGATGG AGAAAATATA GATCACGGCC AAAAAGTGAA TCAAAATTTT 1050

AAAATGGTGC CTTGGGAAAG AATCGCCGAT AAAATCGGCT TTCACCCGCA 1100

GGTCAATAAA CAGGTCGACC CGAGAAAACT GACCGACAGA AGCGGAAAAT 1150

AAACATGCAA AAAGCAGCTT ATACAAGCTG CTTTTTGCAT GTGAAGAACG 1200

S2:

NewSequence Sequence

MNHSKTLLLT AAAGLMLTCG AVSSQAKHKL SDPYHFTVNA AAETEPVDTA 50

GDAADDPAIW LDPKNPQNDK LITTNKKSGL VVYSLEGKTL HSYHTGKLNN 100

VDIRYDFPLN GKKVDIAAAS NRSEGKNTIE IYAIDGKNGT LQSITDPDRP 150

IASAIDEVYG FSLYHSQKTG KYYAMVTGKE GEFEQYELNA DKNGYISGKK 200

VRAFKMNSQT EGMAADDEYG SLYIAEEDEA IWKFSAEPDG GSNGTVIDRA 250

DGRHLTPDIE GLTIYYAADG KGYLLASSQG NSSYAIYERQ GQNKYVADFQ 300

ITDGPETDGT SDTDGIDVLG FGLGPEYPFG LFVAQDGENI DHGQKVNQNF 350

KMVPWERIAD KIGFHPQVNK QVDPRKLTDR SGK- 383

2. The heat-resistant phytase gene according to claim 1 is characterized in that it can be used to develop plant growth promoters, compost additives, biological fertilizers, biological bacterial fertilizers, organic fertilizers, compound fertilizers, feed, and food additives.