Heat-resistant beta-mannase mutant ManAK-3 and coding gene and application thereof

文档序号：502566 发布日期：2021-05-28 浏览：7次中文

阅读说明：本技术 一种耐热性β-甘露聚糖酶突变体ManAK-3及其编码基因和应用 (Heat-resistant beta-mannase mutant ManAK-3 and coding gene and application thereof ) 是由牟海津刘哲民苑明雪张芳于 2020-05-18 设计创作，主要内容包括：本发明涉及一种耐热性β-甘露聚糖酶突变体ManAK-3及其编码基因和应用,属于基因工程和酶工程领域,所述突变体是在野生β-甘露聚糖酶ManAK的氨基酸序列如SEQ ID NO.1所示基础上进行的突变,所述的突变位点选自G331C、S158Y、Q166W、S177R、S180I、K99W、N178R、N178I、D273C/V308C或N277C/T315C中的一种。本发明突变体的耐热稳定性提高,且基本保持了野生β-甘露聚糖酶ManAK的的广泛pH和最适温度。(The invention relates to a heat-resistant beta-mannase mutant ManAK-3 and a coding gene and application thereof, belonging to the field of genetic engineering and enzyme engineering, wherein the mutant is subjected to mutation on the basis that the amino acid sequence of wild beta-mannase ManAK is shown as SEQ ID NO.1, and the mutation site is selected from one of G331C, S158Y, Q166W, S177R, S180I, K99W, N178R, N178I, D273C/V308C or N277C/T315C. The mutant of the invention has improved heat-resistant stability, and basically maintains the wide pH and optimum temperature of the wild beta-mannase ManAK.)

1. A heat-resistant beta-mannase mutant ManAK-3 is characterized in that the mutant is subjected to mutation on the basis that the amino acid sequence of wild beta-mannase ManAK is shown as SEQ ID NO.1, and the mutation site is Q166W.

2. A gene encoding the mutant of claim 1.

3. A recombinant vector comprising a gene encoding the mutant of claim 1.

4. A recombinant strain comprising a gene encoding the mutant of claim 1, wherein the host of the recombinant strain is pichia pastoris X33.

5. Use of the mutant of claim 1 in feed or feed additives.

6. A feed or feed additive comprising the mutant of claim 1.

7. Use of the recombinant strain of claim 4 in feed or feed additives.

8. Use of the recombinant vector of claim 3 in feed or feed additives.

Technical Field

The invention belongs to the field of genetic engineering and enzyme engineering, and particularly relates to a beta-mannase mutant ManAK-3 with improved heat resistance, and a coding gene and application thereof.

Background

Mannan, which is a major component of plant hemicellulose, widely found in nature, is a major constituent of many plant cell walls and is considered as an anti-nutritional factor as a plant feed material. Due to the diversity and structural complexity of mannan, its complete hydrolysis requires the synergistic action of several enzymes to complete, including endo-beta-mannanase, exo-beta-mannosidase, beta-glucosidase, acetylmannan esterase and alpha-galactosidase, etc. Among them, the endo-beta-mannase can degrade beta-1, 4-glycosidic bonds of mannan backbone, and is an enzyme playing the most important role in mannan degradation process.

Beta-mannase (beta-mannase, EC 3.2.1.78) is mainly derived from microorganisms such as bacteria, fungi and actinomycetes, at present, most of strains applied to industrial production of the beta-mannase are aspergillus, bacillus subtilis, yeast and the like, and the beta-mannase is widely applied to the fields of food, medicine, feed and the like. The beta-mannase is used as a feed additive in the feed industry, can effectively degrade mannan in feed, and improves the digestion capability of animals on the feed. At present, enzymes for feed additives are required to be subjected to high-temperature granulation, so that the beta-mannase which can resist high temperature and acid and has improved digestive enzyme tolerance is prepared, and the beta-mannase has important values in feeding enzymes and industrial application. The wild beta-mannase gene can obtain better enzymological properties after heterologous expression and rational design, and plays a vital role in improving the high temperature resistance of the existing beta-mannase.

Disclosure of Invention

The invention aims to provide a beta-mannase mutant with improved heat resistance, which has better application potential in the fields of feed and the like.

Specifically, the invention aims at the heat-resistant acidic beta-mannase ManAK developed earlier to carry out necessary rational design and improve the enzymological characteristics. ManAK has an optimum reaction pH of 2 and has good stability when the pH is in the range of 2.0-6.0. The optimal action temperature is 75 ℃, and the enzyme activity can be kept at 83 percent at the high temperature of 60 ℃ for 30 min. However, the heat resistance and enzyme activity of the enzyme have yet to be further improved for use as a feed additive.

In order to improve the heat resistance of ManAK, the invention carries out molecular simulation butt joint analysis on the spatial structure of the enzyme, and carries out single-site and double-site mutation on the enzyme by analyzing intramolecular disulfide bonds to obtain the optimum temperature (T) of the mutant_opt) And t_1/2All have changes, the heat resistance is improved.

A heat-resistant beta-mannase mutant is subjected to mutation on the basis that the amino acid sequence of wild beta-mannase ManAK is shown as SEQ ID NO.1, and the mutation site is selected from one of G331C, S158Y, Q166W, S177R, S180I, K99W, N178R, N178I, D273C/V308C or N277C/T315C.

It is still another object of the present invention to provide a gene encoding the above mutant.

It is still another object of the present invention to provide a recombinant vector comprising the above mutant gene.

Still another object of the present invention is to provide a recombinant strain comprising the above mutant gene, wherein the recombinant strain is pichia pastoris X33.

The invention also provides application of the mutant in feed or feed additives.

The invention also provides a feed or feed additive containing the mutant.

The invention also provides application of the recombinant strain in feed or feed additives.

The invention also provides application of the recombinant vector in feed or feed additives.

According to the technical scheme of the invention, the amino acid sequence of the wild beta-mannase ManAK is shown in SEQ ID NO. 1.

SEQ ID NO.1：

TALPKASPAPSSSSSSSSSASTSFASTSGLQFTIDGETGYFAGTNSYWIGFLTDDSDVDLVMSHLKSSGLKILRVWGFNDVTTQPSSGTVWYQLHQDGKSTINTGADGLQRLDYVVSSAEQHDIKLIINFVNYWTDYGGMSAYVSAYGGSDETDFYTSDTMQSAYQTYIKTVVERYSNSSAVFAWELANEPRCPSCDTSVLYDWIEKTSKFIKGLDADHMVCIGDEGFGLNTDSDGSYPYQFAEGLNFTKNLGIDTIDFGTLHLYPDSWGTSDDWGNGWISAHGAACKAAGKPCLLEEYGVTSNHCSVESPWQKTALNTTGVSADLFWQYGDDLSTGKSPDDGNTIYYGTSDYECLVTDHVAAIGSA

The nucleotide sequence of the wild beta-mannase ManAK is shown in SEQ ID NO. 2.

SEQ ID NO.2：

actgctttgccaaaggcttctccagctccatcttcttcttcttcttcctcttcttctgcttctacttcttttgcttctacttctggtttgcaatttactattgatggtgaaactggttactttgctggtactaactcttactggattggttttcttactgatgattctgatgttgatttggttatgtctcatttgaagtcttctggtttgaagattttgagagtttggggttttaacgatgttactactcaaccatcttctggtactgtttggtaccaattgcatcaagatggtaagtctactattaacactggtgctgatggtttgcaaagattggattacgttgtttcttctgctgaacaacatgatattaagttgattattaactttgttaactactggactgattacggtggtatgtctgcttacgtttctgcttacggtggttctgatgaaactgatttttacacttctgatactatgcaatctgcttaccaaacttacattaagactgttgttgaaagatactctaactcttctgctgtttttgcttgggaattggctaacgaaccaagatgtccatcttgtgatacttctgttttgtacgattggattgaaaagacttctaagtttattaagggtttggatgctgatcatatggtttgtattggtgatgaaggttttggtttgaacactgattctgatggttcttacccataccaatttgctgaaggtttgaactttactaagaacttgggtattgatactattgattttggtactttgcatttgtacccagattcttggggtacttctgatgattggggtaacggttggatttctgctcatggtgctgcttgtaaggctgctggtaagccatgtttgttggaagaatacggtgttacttctaaccattgttctgttgaatctccatggcaaaagactgctttgaacactactggtgtttctgctgatttgttttggcaatacggtgatgatttgtctactggtaagtctccagatgatggtaacactatttactacggtacttctgattacgaatgtttggttactgatcatgttgctgctattggttctgcttaa

The point mutation G331C is mannanase mutant ManAK-1(G331C), which is obtained by changing 331 st amino acid glycine (G) of mannanase with an amino acid sequence of SEQ ID NO.1 into cysteine (C).

The point mutation S158Y is mannanase mutant ManAK-2(S158Y), which is obtained by changing amino acid serine (S) at position 158 of mannanase with amino acid sequence SEQ ID NO.1 into tyrosine (Y).

The point mutation Q166W is mannanase mutant ManAK-3(Q166W), which is obtained by changing 166 th amino acid asparagine (N) of mannanase with an amino acid sequence of SEQ ID NO.1 into tryptophan (W).

The point mutation S177R is mannanase mutant ManAK-4(S177R), which is obtained by changing the 177 th amino acid serine (S) of mannanase with the amino acid sequence of SEQ ID NO.1 into arginine (R).

The point mutation S180I is mannanase mutant ManAK-5(S180I), which is obtained by changing 180 th amino acid serine (S) of mannanase with an amino acid sequence of SEQ ID NO.1 into isoleucine (I).

The point mutation K99W is mannanase mutant ManAK-6(K99W) obtained by changing 99 th amino acid lysine (K) of mannanase with an amino acid sequence of SEQ ID NO.1 into tryptophan (W).

The point mutation N178R is mannanase mutant ManAK-7(N178R), which is obtained by changing amino acid asparagine (N) at position 178 of mannanase with an amino acid sequence of SEQ ID NO.1 into arginine (R). The point mutation N178I is mannanase mutant ManAK-8(N178I), which is obtained by changing amino acid asparagine (N) at position 178 of mannanase with an amino acid sequence of SEQ ID NO.1 into isoleucine (I).

The point mutation D273C/V308C is mannanase mutant ManAK-9(D273C/V308C), which is obtained by changing amino acid D at position 273 of mannanase with the amino acid sequence of SEQ ID NO.1 into cysteine (C) and changing amino acid valine (V) at position 308 into cysteine (C).

The point mutation N277C/T315C is mannanase mutant ManAK-10(N277C/T315C), which is obtained by changing 277 th amino acid asparagine (N) of mannanase with an amino acid sequence of SEQ ID NO.1 into cysteine (C) and changing 315 th amino acid threonine (T) into cysteine (C).

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention is rationally designed on the basis of disulfide bonds, and utilizes three-dimensional structure simulation combined with enzymatic property analysis to screen mutants with improved heat resistance, wherein the high-temperature resistance of single-point mutants is S177R, N178R, N178I, G331C, K99W, S180I, Q166W and S158Y, and the heat resistance of the mutants is enhanced. The stability of the enzyme under high temperature is obviously improved by introducing intramolecular disulfide bonds.

(2) Each mutant maintains the acid resistance of the wild enzyme and the stability of wide range of pH,

(3) the optimal temperature is 85 ℃ except the optimal temperature of the mutants N178R and D273-V308, the optimal temperature of all the other single-site and double-site mutants is 75 ℃, and compared with mannanase ManAK, the optimal action temperature of the mutant provided by the invention is not changed greatly.

Drawings

FIG. 1 shows the comparison of the optimum temperature of the beta-mannase mutant and the original gene ManAK

FIG. 2 shows the beta-mannase mutant and the original gene ManAK T₅₀Value comparison

FIG. 3 shows the mutant of beta-mannase and the original gene ManAK t_1/2Value comparison

Detailed Description

The technical solution of the present invention is further described in detail with reference to the accompanying drawings and specific embodiments. The present invention will be further described with reference to the following examples, which are intended to illustrate the present invention and not to limit the scope of the present invention.

Test materials and reagents

1. Strains and vectors:

expression hosts pichia X33, expression vectors pPICZ α a, Amp, zeocin were purchased from Invitrogen.

2. Enzyme and kit:

PCR enzymes and ligase were purchased from Takara, and plasmid extraction kit and gel recovery kit were purchased from Omega.

3. Culture medium:

LB culture medium: 1% tryptone, 0.5% yeast extract, 1% NaCl;

YPD medium: 1% yeast extract, 2% tryptone, 2% glucose;

MD culture medium: 1.34% YNB, 0.4mg/L biotin, 2% glucose;

BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (V/V);

BMMY medium: the components were identical to BMGY, pH4.0, except that 0.5% methanol was used instead of glycerol.

Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed by referring to the specific methods listed in molecular cloning, a manual, third edition, J. SammBruke, or according to the kit and product instructions.

Experimental methods

Example 1

Construction of beta-mannase mutant engineering bacteria

(1) Construction of expression vectors

An artificial gene was prepared by introducing a mutation at a specific site by a conventional method using a β -mannanase ManAK (nucleotide sequence disclosed in GenBank No. MN539024.1) derived from Aspergillus kawachii IFO 4308 as a template. Mutant primers are designed at corresponding sites of the genes and are shown in a table 1, PCR high-fidelity enzyme is used for amplification, and after the primers are recombined and connected with pPICZ alpha A vectors in vitro, the primers are thermally excited to transform escherichia coli competent cells DH5 alpha. Coating the strain on an LB + Zeocin flat plate, carrying out inverted culture at 37 ℃, selecting a single clone for verification after a transformant appears, selecting a positive transformant for sequencing, and using the transformant with a correct sequence for preparing a large amount of recombinant plasmids through sequencing.

TABLE 1 primer sequences for mutants

(2) Expression in Pichia

The plasmid vector DNA is expressed in a linear way by using restriction enzyme Sac1, 100uL of yeast competence and 10uL of linear vector are uniformly mixed and then transferred into a precooled electric rotating cup for electric shock transformation, and the condition of electric transformation is 1.5kV and 5 msec. After the shock, 1mL of sorbitol solution was added and transferred to a 1.5mL centrifuge tube and incubated at 30 ℃ for 1 h. Centrifuging at 5000rpm for 5min, collecting thallus, coating on YPD screening plate, inverting at 30 deg.C, culturing for 2-3 days, and selecting transformant growing on the plate for further expression experiment.

A single colony was picked from the plate with the transformant by using a sterilized toothpick, and spotted on the plate first according to the number, and the plate was cultured in an incubator at 30 ℃ for 2 days until the colony grew out. Selecting transformants from the plate according to the number, inoculating the transformants into 3ml of BMGY culture medium, carrying out shake culture at 30 ℃ for 48h, centrifugally collecting thalli, adding 1ml of BMMY induction culture medium containing 1% methanol, continuing the induction culture at 30 ℃, sampling after 48h, detecting the enzyme activity of each strain supernatant, and screening the transformants with high mannanase activity.

Example 2

Fermentation of recombinant mannanase mutants in pichia pastoris

(1) Large-scale expression of mannanase mutant in shake flask

Inoculating the screened transformant with higher enzyme activity into 300ml of BMGY liquid medium, carrying out shake culture at 30 ℃ and 200rpm for 48h, and carrying out thallus enrichment; centrifuging at 4000 Xg for 5min, gently discarding supernatant, transferring the thallus into 100ml BMMY liquid culture medium containing 1% methanol, and performing induction culture at 30 deg.C and 200rpm for 72 h. During the induction culture period, methanol solution is supplemented every 24h to keep the final concentration of methanol at about 1%, and the supernatant is collected by centrifugation at 10000 Xg for 10 min. The activity of the wild mannanase and its mutants was determined.

(2) Purification of mannanase mutants

Collecting the supernatant of the mannase mutant expressed in the shake flask, desalting and concentrating by using a 10kDa membrane package, and purifying by anion exchange column chromatography. So as to obtain the electrophoretically pure collection liquid as a sample for expressing the research of enzymology properties.

Example 3

Analysis of enzymatic Properties of recombinant mannanase enzymes

The activity of the mannanase is detected according to the national standard GB T36861-2018 of the people's republic of China; the mannanase activity is defined as the amount of enzyme required to release 1umol of reducing sugar by degradation of the sample from a mannan (Sigma G0753) solution at a concentration of 3mg/mL per minute at a pH of 5.5 and a temperature of 37 ℃ in units of enzyme activity, expressed as U.

The activity of the mannanase is analyzed by a DNS method.

Determination of β -mannanase activity: 2mL of the enzyme solution diluted appropriately was aspirated and added to a graduated tube, and 2mL of a 0.6% (w/v) mannan solution was added thereto, followed by shaking and incubation at 50 ℃ for 30 min. Adding 5mL of the LDNS reagent, shaking uniformly, heating in a boiling water bath for 10min, cooling to room temperature, adding water to a constant volume of 25mL, and measuring the absorbance at 540 nm.

(1) Optimum temperature of wild enzyme ManAK and mutant

The enzymatic reactions were carried out in a citrate-disodium hydrogen phosphate buffer system at pH 5.5 and at different temperatures (65,70,75,80,85,90 ℃) and the optimum temperatures of the wild-type enzyme and the mutant were determined. The relative enzyme activity was calculated with the highest enzyme activity as 100%.

As shown in FIG. 1, under the same pH conditions, the optimum temperature of all single-site and double-site mutants is 75 ℃ except that the optimum temperature of the mutants N178R and D273-V308 is 80 ℃, and the optimum action temperature of the mutant provided by the invention is not greatly changed compared with that of mannanase ManAK. (2) Optimum action pH and pH stability of wild enzyme ManAK and mutant

The results showed that the optimum pH of all the mutants was 2 as that of the wild enzyme, except that the optimum pH of the S180I mutant was 3. The change trend of the residual enzyme activity of each mutant enzyme is consistent with that of ManAK, so that each mutant keeps the acid resistance of the wild enzyme and the stability of wide-range pH.

(3) Wild enzyme ManAK and mutant T₅₀Value sum t_1/2Value of

T₅₀Value sum t_1/2The values are two important indicators of the heat resistance. T is₅₀The value refers to the measurement temperature when the enzyme activity is 50% of the highest enzyme activity according to the national standard measurement method of the mannase, and the higher the temperature is, the better the heat resistance is; t is t_1/2The value is the time required for processing at the same temperature for different time when the enzyme activity is half of the highest enzyme activity, and the longer the time is, the better the heat resistance is. Among them, the latter is a main index for judging heat resistance in recent years.

The results show (FIG. 2), T for the wild ManAK enzyme₅₀The value was 77.7 ℃ and T for G331C and D273-V308 in the mutant₅₀The value is over 80 ℃ and is respectively increased by 2.8 ℃ and 5.4 ℃ compared with the wild enzyme. And T of the remaining mutants₅₀The value is between 77.6 and 78.5 ℃, and the high-temperature resistance is N277-T315>S158Y>Q166W＝N178R>N178I>K99W>S177R ═ ManAK, whereas S180I was 0.1 ℃ lower than the wild ManAK enzyme. Therefore, G331C and D273-V308 are key sites for promoting the improvement of the thermal stability of the mannanase.

"Heat resistance" as used herein refers to the property of retaining enzymatic activity even after heat treatment, by t_1/2The size of the value. The samples were treated at 75 ℃ for 5, 10, 15, 20, 25, and 30min, respectively, and the relative enzyme activities were measured and calculated, and the results are shown in FIG. 3, and the time required for 50% relative enzyme activity was calculated from the fitted curve. According to the fitting result, the best heat resistance of N277-T315 in the double-site mutant is predicted, and T is measured at 75 DEG C_1/2The value reached 60.1min, followed by the double-site mutant D273-V308, which was 57.2 min. While the t 1/2 values for the remaining mutants were between 51.5-55.5 ℃ and were all higher than 32.9min for the wild ManAK enzyme. The tolerance of the single point mutant to high temperature is S177R>N178R>N178I>G331C>K99W>S180I>Q166W>S158Y, showing the enhancement of the heat resistance of the mutant. The stability of the enzyme under high temperature is obviously improved by introducing intramolecular disulfide bonds.

In conclusion, the present invention provides mannanase mutants comprising single and double site mutations based on β -mannanase ManAK. The invention confirms key heat-resistant sites in the mannase, proves the importance of the sites to the high heat resistance of the mannase, provides important clues for researching the heat stability mechanism of the mannase, and simultaneously provides reliable reference basis for improving the heat stability of other mannases.

Sequence listing

<110> China oceanic university

<120> heat-resistant beta-mannase mutant ManAK-3, and coding gene and application thereof

<160> 26

<170> SIPOSequenceListing 1.0

<210> 1

<211> 367

<212> PRT

<213> wild beta-mannase (beta-mannase)

<400> 1

Thr Ala Leu Pro Lys Ala Ser Pro Ala Pro Ser Ser Ser Ser Ser Ser

1 5 10 15

Ser Ser Ser Ala Ser Thr Ser Phe Ala Ser Thr Ser Gly Leu Gln Phe

20 25 30

Thr Ile Asp Gly Glu Thr Gly Tyr Phe Ala Gly Thr Asn Ser Tyr Trp

35 40 45

Ile Gly Phe Leu Thr Asp Asp Ser Asp Val Asp Leu Val Met Ser His

50 55 60

Leu Lys Ser Ser Gly Leu Lys Ile Leu Arg Val Trp Gly Phe Asn Asp

65 70 75 80

Val Thr Thr Gln Pro Ser Ser Gly Thr Val Trp Tyr Gln Leu His Gln

85 90 95

Asp Gly Lys Ser Thr Ile Asn Thr Gly Ala Asp Gly Leu Gln Arg Leu

100 105 110

Asp Tyr Val Val Ser Ser Ala Glu Gln His Asp Ile Lys Leu Ile Ile

115 120 125

Asn Phe Val Asn Tyr Trp Thr Asp Tyr Gly Gly Met Ser Ala Tyr Val

130 135 140

Ser Ala Tyr Gly Gly Ser Asp Glu Thr Asp Phe Tyr Thr Ser Asp Thr

145 150 155 160

Met Gln Ser Ala Tyr Gln Thr Tyr Ile Lys Thr Val Val Glu Arg Tyr

165 170 175

Ser Asn Ser Ser Ala Val Phe Ala Trp Glu Leu Ala Asn Glu Pro Arg

180 185 190

Cys Pro Ser Cys Asp Thr Ser Val Leu Tyr Asp Trp Ile Glu Lys Thr

195 200 205

Ser Lys Phe Ile Lys Gly Leu Asp Ala Asp His Met Val Cys Ile Gly

210 215 220

Asp Glu Gly Phe Gly Leu Asn Thr Asp Ser Asp Gly Ser Tyr Pro Tyr

225 230 235 240

Gln Phe Ala Glu Gly Leu Asn Phe Thr Lys Asn Leu Gly Ile Asp Thr

245 250 255

Ile Asp Phe Gly Thr Leu His Leu Tyr Pro Asp Ser Trp Gly Thr Ser

260 265 270

Asp Asp Trp Gly Asn Gly Trp Ile Ser Ala His Gly Ala Ala Cys Lys

275 280 285

Ala Ala Gly Lys Pro Cys Leu Leu Glu Glu Tyr Gly Val Thr Ser Asn

290 295 300

His Cys Ser Val Glu Ser Pro Trp Gln Lys Thr Ala Leu Asn Thr Thr

305 310 315 320

Gly Val Ser Ala Asp Leu Phe Trp Gln Tyr Gly Asp Asp Leu Ser Thr

325 330 335

Gly Lys Ser Pro Asp Asp Gly Asn Thr Ile Tyr Tyr Gly Thr Ser Asp

340 345 350

Tyr Glu Cys Leu Val Thr Asp His Val Ala Ala Ile Gly Ser Ala

355 360 365

<210> 2

<211> 1104

<212> DNA

<213> wild beta-mannase (beta-mannase)

<400> 2

actgctttgc caaaggcttc tccagctcca tcttcttctt cttcttcctc ttcttctgct 60

tctacttctt ttgcttctac ttctggtttg caatttacta ttgatggtga aactggttac 120

tttgctggta ctaactctta ctggattggt tttcttactg atgattctga tgttgatttg 180

gttatgtctc atttgaagtc ttctggtttg aagattttga gagtttgggg ttttaacgat 240

gttactactc aaccatcttc tggtactgtt tggtaccaat tgcatcaaga tggtaagtct 300

actattaaca ctggtgctga tggtttgcaa agattggatt acgttgtttc ttctgctgaa 360

caacatgata ttaagttgat tattaacttt gttaactact ggactgatta cggtggtatg 420

tctgcttacg tttctgctta cggtggttct gatgaaactg atttttacac ttctgatact 480

atgcaatctg cttaccaaac ttacattaag actgttgttg aaagatactc taactcttct 540

gctgtttttg cttgggaatt ggctaacgaa ccaagatgtc catcttgtga tacttctgtt 600

ttgtacgatt ggattgaaaa gacttctaag tttattaagg gtttggatgc tgatcatatg 660

gtttgtattg gtgatgaagg ttttggtttg aacactgatt ctgatggttc ttacccatac 720

caatttgctg aaggtttgaa ctttactaag aacttgggta ttgatactat tgattttggt 780

actttgcatt tgtacccaga ttcttggggt acttctgatg attggggtaa cggttggatt 840

tctgctcatg gtgctgcttg taaggctgct ggtaagccat gtttgttgga agaatacggt 900

gttacttcta accattgttc tgttgaatct ccatggcaaa agactgcttt gaacactact 960

ggtgtttctg ctgatttgtt ttggcaatac ggtgatgatt tgtctactgg taagtctcca 1020

gatgatggta acactattta ctacggtact tctgattacg aatgtttggt tactgatcat 1080

gttgctgcta ttggttctgc ttaa 1104

<210> 3

<211> 45

<212> DNA