阅读说明：本技术 3-酮脂酰辅酶a硫解酶基因rkacaa1-2及其应用 (3-ketoacyl-coenzyme A thiolase gene RKACAA1-2 and application thereof ) 是由 张琦 杨晓霞 陈功水 于 2021-08-05 设计创作，主要内容包括：本发明公开了一种3-酮脂酰辅酶A硫解酶基因RKACAA1-2,其核苷酸序列如SEQID NO:1所示,该基因编码的氨基酸序列如SEQ ID NO:2所示；该基因是从红冬孢酵母(Rhodosporidium kratochvilovae)YM25235中分离得到,将该基因通过转化转入到红冬孢酵母YM25235中,实验结果显示RKACAA1-2基因的过表达会引起YM25235菌株类胡萝卜素合成水平的提高；本发明通过基因工程手段对微生物进行改造,来提高微生物体内类胡萝卜素的产量,为类胡萝卜素大规模商业化生产奠定基础。(The invention discloses a 3-ketoacyl-coenzyme A thiolase gene RKACAA1 2, the nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence coded by the gene is shown as SEQ ID NO. 2; the gene is derived from Rhodosporidium toruloides ( Rhodosporidium kratochvilovae ) YM25235, transferring the gene into Rhodosporidium toruloides YM25235 by transformation, and showing the experimental results RKACAA1 Overexpression of the-2 gene leads to an increase in the carotenoid synthesis level of the YM25235 strain; the invention improves the microorganism by means of genetic engineering to improve the yield of carotenoid in the microorganism, and lays a foundation for large-scale commercial production of the carotenoid.)

1. 3-ketoacyl-coenzyme A thiolase geneRKACAA12, and the nucleotide sequence is shown as SEQ ID NO. 1.

2. The 3-ketoacyl-CoA thiolase gene of claim 1RKACAA1-2 in promoting the microbial production of carotenoids.

Technical Field

The invention belongs to the field of biotechnology and genetic engineering, and relates to a 3-ketoacyl-coenzyme A thiolase geneRKACAA1-2And applications thereof.

Background

Carotenoid (Carotenoid) is a general name of an important natural pigment, belongs to a class of terpenoids, has a plurality of conjugated double bonds, and is generally yellow, orange or red in color. The structure of carotenoids is based on isoprenoids, and most typical carotenoid chemical structures contain 40 carbon atoms and are polymerized from 8 isoprenoids. Carotenoids can be classified into two major classes depending on the type of chemical group at the end of the carbon skeleton. One class is closed-loop carotenoids containing cyclic structures, such as alpha-carotene, beta-carotene, lutein, anthocyanins, and the like. Another class is open-ring carotenoids which do not contain a cyclic structure, such as phytoene, lycopene, and the like. At present, more than 800 natural carotenoids have been found in organisms such as higher plants, animals, fungi, etc., of which about 50 can be converted into vitamin a, and alpha-carotene, beta-lutein and beta-carotene are very important as pro-vitamin a for human body.

Researches show that the carotenoid is a fat-soluble substance with various types and multiple functions, has special nutritional value, has the capacity of converting into essential vitamins, is an important natural pigment capable of providing natural colors for animals and plants, and is also an important antioxidant. Typical carotenoids include beta-carotene, lycopene, lutein and the like, wherein the beta-carotene has important physiological health care functions of oxidation resistance, cancer resistance, provitamin A, prevention of senile macular degeneration of eyes, senility delay, immunity improvement and the like, the lycopene can improve the oxidation resistance of self tissues and cells, enhance the capability of an immune system and effectively maintain the health of a human body, and the lutein is an important component of macular pigments in retinas, can filter blue light, prevent the damage of the retinas and the like. Carotenoids are in a wide variety of forms, and different carotenoids have different physiological functions, and are widely applied to various aspects such as medicine, health care, nutrition enrichment and coloring of food and feed.

Carotenoids can be obtained by biological (plant, microbial) extraction and chemical synthesis. Among them, the safety of chemically synthesized pigments is in question, and some chemically synthesized carotenoids (such as astaxanthin) have been found to be harmful and prohibited from being sold in drugstores. With the development of biotechnology and the increasing awareness of people about food safety, the demand of carotenoids derived from natural sources by bio-extraction is increasing. The production cost of natural carotenoid from plant sources is relatively high and is influenced by seasons, while the natural carotenoid from microorganism sources is favored by researchers due to the advantages of nature, high efficiency, low production cost and easy realization of industrialization.

Disclosure of Invention

The invention provides a 3-ketoacyl-coenzyme A thiolase geneRKACAA1-2, the gene is selected from Rhodosporidium toruloides (Rhodosporidium kratochvilovae) YM25235 by separation; the nucleotide sequence of the gene is shown as SEQ ID NO. 1 or the nucleotide sequence complementary to SEQ ID NO. 1, the length of the gene sequence is 1263bp (basic group), and the amino acid sequence coded by the gene is polypeptide shown as SEQ ID NO. 2.

Another object of the present invention is to provide the above-mentioned 3-ketoacyl-CoA thiolase geneRKACAA1-2 in promoting microbial production of carotenoids.

The Rhodosporidium toruloides (A), (B) and (C)Rhodosporidium kratochvilovae) YM25235 has the advantages of short production cycle, stable heredity, safe production, etc.

The above-mentioned goal is implemented by following technical scheme, extract total RNA from Rhodosporidium toruloides YM25235, then reverse transcription synthesize cDNA, use synthesized cDNA as template, obtain target fragment by PCR amplification, make carrier pRH2034 undergo the process of double digestion, recovery, connect target fragment and carrier by one-step cloning method, obtain recombinant plasmid pRHRKACAA1-2, the connected product is transferred into colibacillus, screen out positive monoclonal by PCR, recombinant plasmid pRHRKACAA1-2 usesBamHⅠ、EcoPerforming enzyme digestion verification on the two restriction endonucleases of RV, extracting plasmids after verifying that positive clones are cultured, sequencing to obtain 3-ketoacyl-coenzyme A thiolase gene with the fragment size of 1263bpRKACAA1-2; transforming the recombinant vector pRHRKACAA1-2 into Rhodosporidium toruloides YM25235 by PEG-mediated protoplast method, and screening transformants to obtainRKACAA1-2Culturing the over-expression strain, and measuring the content of the total carotenoid by using an ultraviolet-visible spectrophotometer.

The invention relates to a method for preparing a red wintergreen spore yeast (Rhodosporidium toruloides)Rhodosporidium kratochvilovae) 3-ketoacyl-CoA thiolase gene isolated from YM25235 Total RNA GeneRKACAA1-2, the full length of the gene 1263 bp; the gene is transferred into Rhodosporidium toruloides YM25235, and the experimental result shows thatRKACAA12, the overexpression of the gene can cause the transcription level of the gene in cells to be improved to a certain extent, which indicates that the exogenous gene is transcribed in thalli;RKACAA1-2 overexpression of the gene promotes the synthesis of total carotenoids; the research result provides reference for disclosing a mechanism of increasing the yield of the carotenoid by the microorganism, is beneficial to improving the carotenoid content by modifying the microorganism by a genetic engineering means, provides good application prospect and economic benefit for industrial production of the carotenoid, and lays a foundation for large-scale commercial production of the carotenoid.

Drawings

FIG. 1 shows a scheme for producing Rhodosporidium toruloides YM25235 of the present inventionRKACAA1-2A gene PCR amplification map; DNA molecular weight marker DL 2000; 2. negative control; a cDNA fragment of RKACAA 1-2;

FIG. 2 is a plasmid map of recombinant plasmid pRHRKACAA 1-2;

FIG. 3 is a PCR-verified electrophoretogram of colonies; DNA molecular weight marker DL 2000; 2. negative control; 3. RKACAA1-2a cDNA fragment of (1); 4-8 is a transformant;

FIG. 4 restriction analysis of the recombinant plasmid pRHRKACAA 1-2; DNA molecular scalar DL 10000; 2. negative control; 3. of the empty plasmid pRH2034BamHⅠ、EcoPerforming double enzyme digestion on RV; 4. of recombinant plasmid pRHRKACAA1-2BamHⅠ、EcoPerforming double enzyme digestion on RV; 5.RKACAA1-2a cDNA fragment of a gene; 6. DNA molecular scalar DL 2000;

FIG. 5 shows the verification of positive clones of YM25235 strain transformed with the recombinant plasmid pRHRKACAA 1-2; FIG. 1 DNA molecular scalar DL 2000; 2. negative control; 3. a wild type strain specific gene band; 4.RKACAA1-2; 5. verifying a transformant;

FIG. 6 shows a comparison of the total carotenoid content of the over-expressed strain YM25235/pRHRKACAA1-2 and the control strain YM 25235.

Detailed Description

The present invention is further illustrated in detail below with reference to the drawings and examples, but the scope of the present invention is not limited to the above description, and reagents and methods used in the examples are, unless otherwise specified, conventional reagents and methods are used.

Example 1: from Rhodosporidium toruloides (A)Rhodosporidium kratochvilovae) Isolation of 3-ketoacyl-CoA Thiolytic enzyme Gene from YM25235RKACAA1-2 construction of and overexpression vector pRHRKACAA1-2 thereof

Extracting total RNA of Rhodosporidium toruloides YM25235 with UNlQ-10 column Trizol total RNA extraction Kit (product number: SK 1321) from Biotechnology engineering (Shanghai) Ltd, performing reverse transcription to synthesize cDNA according to the Kit (product number: R212-02) HiScript II 1st Strand cDNA Synthesis Kit (+ gDNA wiper) from Vazyme, performing polymerase chain reaction with 1. mu.L as template, and sequencing according to the transcriptomeRKACAA12 sequence, designing specific primers RKACAA1-2-F and RKACAA1-2-R, and carrying out PCR amplification on the cDNA template obtained by the above on a PCR instrument (Beijing six Biotech Co., Ltd.) by using the following primers, components and amplification conditions:

RKACAA1-2-F: 5’-ATCACTCACCATGGCGGATCCTATGGCCAGCCTCATTTCG-3' (the single underlined part isBamH i site, double underlined homology arms);

RKACAA1-2-R：5’-CCGGTCGGCATCTACGATATCCTACTCAGCGACGAAGAC-3' (the single underlined part isEcoThe rv site, double underlined part is homology arm);

the PCR amplification system was as follows (50. mu.L):

；

amplification conditions: pre-denaturing at 95 ℃ for 5min, then denaturing at 95 ℃ for 15s, annealing at 60 ℃ for 30s, extending at 72 ℃ for 1min for 15s, performing 30 cycles, finally completely extending at 72 ℃ for 5min, taking 2 mu L of product after the reaction is finished, and then performing electrophoresis analysis in agarose gel with the concentration of 1% to obtain a fragment with the size of about 1263bp by amplification, wherein the result is shown in figure 1; pRH2034 throughBamHⅠ、EcoPerforming double enzyme digestion on the two restriction enzymes of RV; the above two fragments were recovered with a multifunctional DNA recovery Kit (Beijing Baitach Biotechnology Co., Ltd., product No.: DP 1502), and the fragments were ligated to the vector pRH2034 using a seamless Cloning Kit (Vazyme Clon express II One Step Cloning Kit, Nanjing Novozam Biotechnology Co., Ltd.) in the following recombinant vector ligation system (20. mu.L):

；

gently blowing and beating the mixture by using a pipettor, mixing the mixture evenly, centrifuging the mixture for a short time to collect reaction liquid to the bottom of the tube, and then reacting the reaction liquid for 30min at 37 ℃; cooling to 4 deg.C or immediately cooling on ice;

adding 10 muL of the ligation product obtained above to 100 muL of DH5 alpha competent cells, mixing gently, ice-bathing for 30min, immediately placing on ice to cool for 90s after heat shock at 42 ℃, adding 900 muL LB liquid culture medium into the connecting system, performing shake culture at 37 ℃ and 100rpm for 1h, centrifuging at 5000rpm for 10min, discarding 900 muL of supernatant, suspending thallus by the remaining about 100 muL of culture medium, coating an LB solid plate containing 100 mug/mL spectinomycin (Spe +), performing inverted culture at 37 ℃ overnight, randomly selecting white colonies growing on 5 plates and numbering 1-5, the result of positive cloning is verified through colony PCR, and the result is shown in figure 3, and it can be seen from the figure that the five selected monoclonal strains all amplify specific bands with the same size as the target fragment through colony PCR, which indicates that the five selected DH5 alpha strains are all successfully transferred into recombinant plasmids; inoculating positive clones into LB liquid medium (containing 100 μ g/mL spectinomycin) for overnight culture, extracting Plasmid (OMEGA Plasmid Mini Kit I, OMEGA, USA), and culturing with the obtained PlasmidBamHⅠ、EcoPerforming enzyme digestion verification on the two restriction enzymes of RV; the results of the enzyme digestion are shown in FIG. 4, from which it can be seen that the recombinant plasmid pRHRKACAA1-2 was obtainedBamHⅠ、EcoAfter double cleavage of RV, the cleavage occurs with the empty plasmid pRH2034BamHⅠ、EcoThe linear vector fragments obtained by the double restriction enzyme of RV have the same size band, andRKACAA1-2the cDNA fragments of the genes were bands of the same size, indicating recombinant plasmid formationSuccessfully transformed into Escherichia coli DH5 alpha strain; after enzyme digestion verification, plasmids were extracted and sequenced in the same manner (Kunming Optimus Biotech, Ltd.). The sequencing result shows that the amplified fragment has the size of 1263bp and the nucleotide sequence shown in SEQ ID NO. 1 and named asRKACAA1-2, withRKACAA1-2The sizes and sequences of cDNA fragments of the genes are consistent, which indicates that the plasmid map of the expression vector pRHRKACAA1-2 and the recombinant vector pRHRKACAA1-2 is shown in figure 2.

Example 2: analysis of the relationship between RKACAA1-2 Gene and Carotenoid Synthesis in Rhodosporidium toruloides

1. Transformed Rhodosporidium toruloides YM25235

Selecting a DH5 alpha strain which is successfully transferred into a correct recombinant vector pRHRKACAA1-2, inoculating the single clone into an LB liquid culture medium (containing 100 mug/mL spectinomycin) for overnight culture, extracting a Plasmid (OMEGA Plasmid Mini Kit I, OMEGA corporation, USA), measuring the concentration, and storing at-20 ℃ for later use; selecting single colony of Rhodosporidium toruloides YM25235, inoculating to 5mL YPD liquid culture medium, and shake culturing at 30 deg.C and 200rpm overnight; transferring the overnight cultured bacterial liquid into 50mL YPD liquid culture medium at 30 deg.C and 200rpm, and performing shaking culture to OD₆₀₀At 0.5, the culture was centrifuged at 4500 rpm for 5min at 4 ℃ to collect the cells; washing the thallus twice with citric acid buffer solution prepared in advance (30 mM citric acid, 83mM sodium citrate, 600mM mannitol, NaOH to adjust pH to 5.4), suspending the thallus with 1mL citric acid buffer solution, centrifuging at 4 deg.C and 4000 rpm for 5min, collecting the thallus, and placing on ice for use; preparing lyase solution (0.156 g snailase, 0.08g lywallzyme, ddH₂O to 5 mL), filtering the enzyme solution by using a sterile filter membrane with the diameter of 0.22 mu m, and placing the enzyme solution in a sterile centrifuge tube with the volume of 50mL for later use; mixing 4mL of enzyme solution with the bacterial solution, placing at 30 ℃, performing shaking culture and enzymolysis at 90rpm for 2.5h, centrifuging the culture at 4 ℃ and 1300rpm for 10min, and collecting thalli; with STC (1.2M sorbitol, 10mM Tris-HCl, 100mM CaCl)₂) Washing the collected thallus twice on ice to prepare yeast competent cells; subpackaging yeast competent cells into 5mL sterile centrifuge tubes for later use according to 100 mu L per tube; to 100. mu.L of competent cells, 2-5. mu.g of pRHRKACAA1-2 recombinant plasmid was added and gently mixed (usually in tablets)Volume of the fractions should not exceed 10. mu.L), incubated on ice for 10min, 200. mu.L of precooled PTC (50% PEG, 10mM Tris-HCl, 100mM CaCl) was added₂) Ice-bath for 10min, adding 800 μ L precooled PTC, mixing gently, ice-bath for 10min, centrifuging at 4 deg.C and 1500rpm for 10min, and collecting thallus; adding 1.6mL of 0.4M sucrose YPD liquid culture medium for suspension, and performing shaking culture at 30 ℃ and 90rpm for 12h to recover the thallus; centrifuging the recovered thallus at 1300rpm for 10min to collect thallus, discarding the supernatant to leave 100 μ L culture medium for suspending thallus, and finally spreading on 0.4M sucrose YPD solid culture medium containing 130ug/mL hygromycin B (HygB +), and performing inversion culture at 30 deg.C for 2-3 d; numbering the transformants obtained after coating, transferring the transformants to a solid culture medium containing hygromycin (Hyg B +) YPD of 150 mu g/mL, and performing inversion culture at 30 ℃ for 2 d; selecting transformants by color according to the known functions of the gene, specifically, inoculating the obtained transformants into 5mL YPD medium, carrying out shake culture at 30 ℃ and 200rpm for 120h, observing the color by using YM25235 wild strain as a control, and selecting the transformants with the color being redder than that of YM 25235; the selected transformant was selected, the genomic DNA of the yeast transformant was extracted according to the procedures of the DNA extraction kit of Shanghai Biotechnology engineering Co., Ltd, and then PCR verification was carried out, the results are shown in FIG. 5, from which it can be seen that the genomic DNA of the yeast transformant was amplified by PCR using the genome of the yeast transformant as a templateRKACAA1-2cDNA fragment of the same size, the recombinant transformant gene was verified correctly, which indicated thatRKACAAThe 1-2cDNA fragments have been successfully ligated into the genome of yeast transformants.

2、RKACAA1-2Analysis of Carotenoid content in Gene-overexpressed Rhodosporidium toruloides YM25235

Culturing overexpression strain containing pRHRKACAA1-2 at 28 deg.C for 168 hr, extracting carotenoid, and determining total carotenoid content (mg/g dry thallus) at 445nm with ultraviolet-visible spectrophotometer using wild type Rhodosporidium toruloides YM25235 strain as control, as shown in FIG. 6; as can be seen from the figure, the total carotenoid synthesis amount of the over-expressed strain YM25235/pRHRKACAA1-2 is significantly increased as compared with the wild type Rhodosporidium toruloides YM25235 strain, the carotenoid synthesis amount of the wild type Rhodosporidium toruloides YM25235 strain is 5.53 + -0.07 mg/g, and the over-expressed strainThe synthesis amount of carotenoid of the strain YM25235/pRHRKACAA1-2 is 8.38 +/-0.06 mg/g, namely the synthesis amount of carotenoid of the over-expressed strain YM25235/pRHRKACAA1-2 is 1.51 times of that of the control strain; the results showed that the 3-ketoacyl-CoA thiolase geneRKACAA1Overexpression of-2 leads to an increase in the total carotenoid content in Rhodosporidium toruloides YM25235 strain,RKACAA1-2the gene can promote the synthesis of total carotenoid.

Sequence listing

<110> university of Kunming science

<120> 3-ketoacyl-CoA thiolase gene RKACAA1-2 and use thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1263

<212> DNA

<213> Rhodosporidium toruloides YM25235(Rhodosporidium kratochvilovae YM25235)

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ctgcgcaaga gccccgacga tgtcgtcatc accatggcgg tccgtacgcc cctccagcgc 120

gcgcgcaagg gcggcttcaa ggacatgagc gtccaggagc tcctcattgc cttcttcaag 180

acctccatcc ccgagatgaa gatcgacccg gccctcattg gcgacatctg cgtcggcact 240

gtcctcccgc ccaaggcgcc gtacgacgcc cgtgctgccg ccctcgcgtc cggcattccc 300

gaaaccgtcc ctcttcagat catcaaccgc ttctgctctt cgggcctcat ggccgtcgcc 360

aacattgcaa accagatccg caacggcgag atcgagattg gtcttgcggt cggctttgag 420

agcatgagcg cgactccgga caggggcgcg gacagctttg cggaggaggt cctcgcgcac 480

ccggtcgcga aggacacgca gttccccatg ggctggacct cggagaacgt ttcgagtgac 540

tttaacatca cccgtgagcg catggacgag ttggcggcga tttcgcagca gcgcgcggcg 600

caggcgcagg cggctggcct tttcgacaag gagattatcc ccatcgaggc tctccaggcg 660

ccgtccaccc cggcggcggc gggcgcgccc gcgccgcagc gcgtcaaggt gctcgtcacc 720

aaggacgacg gcatccgccc ggggacgacc aaggagggcc tcggcaagat tcgccaggcg 780

ttcccgcagt ggggcaacgg cacgacgacg ggcggcaatg cgagccagat caccgacggc 840

gcggccggtg tcctcctcat gacccgtcgc aaggcggagg agctcggcct cgagatcctc 900

ggcaagcacg tcgcgacctc ggtcgcgggt cttccccctc gcattatggg catcggcccc 960

gccttcgcca tcccgaaggt ccttgcccgc tgcggcatct ccaaggagga cgtcgacctc 1020

tgggaggtca acgaggcttt tgcgtcgatg ctgagttact gcatcgacat cttcggcctt 1080

ccccatgaca aggtcaacgt caagggtggt gccatcgctc tcggccaccc tcttggctgc 1140

actggtgccc gccagatcgc gacgggcctg cacgagatcc gccgccgcga gggcaagatc 1200

ctcgttacct ccatgtgcat tggtctcggc atgggcgcag cgtcggtctt cgtcgctgag 1260

tag 1263

<210> 2

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<212> PRT

<213> Rhodosporidium toruloides YM25235(Rhodosporidium kratochvilovae YM25235)

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Met Ala Ser Leu Ile Ser Ser Thr Leu Asp Ser Ile Arg Gly Ser Gly

1 5 10 15

Lys Ser Lys Leu Leu Arg Lys Ser Pro Asp Asp Val Val Ile Thr Met

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Ala Val Arg Thr Pro Leu Gln Arg Ala Arg Lys Gly Gly Phe Lys Asp

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Met Ser Val Gln Glu Leu Leu Ile Ala Phe Phe Lys Thr Ser Ile Pro

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Glu Met Lys Ile Asp Pro Ala Leu Ile Gly Asp Ile Cys Val Gly Thr

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Val Leu Pro Pro Lys Ala Pro Tyr Asp Ala Arg Ala Ala Ala Leu Ala

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Ser Gly Ile Pro Glu Thr Val Pro Leu Gln Ile Ile Asn Arg Phe Cys

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Ser Ser Gly Leu Met Ala Val Ala Asn Ile Ala Asn Gln Ile Arg Asn

115 120 125

Gly Glu Ile Glu Ile Gly Leu Ala Val Gly Phe Glu Ser Met Ser Ala

130 135 140

Thr Pro Asp Arg Gly Ala Asp Ser Phe Ala Glu Glu Val Leu Ala His

145 150 155 160

Pro Val Ala Lys Asp Thr Gln Phe Pro Met Gly Trp Thr Ser Glu Asn

165 170 175

Val Ser Ser Asp Phe Asn Ile Thr Arg Glu Arg Met Asp Glu Leu Ala

180 185 190

Ala Ile Ser Gln Gln Arg Ala Ala Gln Ala Gln Ala Ala Gly Leu Phe

195 200 205

Asp Lys Glu Ile Ile Pro Ile Glu Ala Leu Gln Ala Pro Ser Thr Pro

210 215 220

Ala Ala Ala Gly Ala Pro Ala Pro Gln Arg Val Lys Val Leu Val Thr

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Lys Asp Asp Gly Ile Arg Pro Gly Thr Thr Lys Glu Gly Leu Gly Lys

245 250 255

Ile Arg Gln Ala Phe Pro Gln Trp Gly Asn Gly Thr Thr Thr Gly Gly

260 265 270

Asn Ala Ser Gln Ile Thr Asp Gly Ala Ala Gly Val Leu Leu Met Thr

275 280 285

Arg Arg Lys Ala Glu Glu Leu Gly Leu Glu Ile Leu Gly Lys His Val

290 295 300

Ala Thr Ser Val Ala Gly Leu Pro Pro Arg Ile Met Gly Ile Gly Pro

305 310 315 320

Ala Phe Ala Ile Pro Lys Val Leu Ala Arg Cys Gly Ile Ser Lys Glu

325 330 335

Asp Val Asp Leu Trp Glu Val Asn Glu Ala Phe Ala Ser Met Leu Ser

340 345 350

Tyr Cys Ile Asp Ile Phe Gly Leu Pro His Asp Lys Val Asn Val Lys

355 360 365

Gly Gly Ala Ile Ala Leu Gly His Pro Leu Gly Cys Thr Gly Ala Arg

370 375 380

Gln Ile Ala Thr Gly Leu His Glu Ile Arg Arg Arg Glu Gly Lys Ile

385 390 395 400

Leu Val Thr Ser Met Cys Ile Gly Leu Gly Met Gly Ala Ala Ser Val

405 410 415

Phe Val Ala Glu

420

<210> 3

<211> 40

<212> DNA

<213> Artificial sequence (Artificial)

<400> 3

atcactcacc atggcggatc ctatggccag cctcatttcg 40

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<213> Artificial sequence (Artificial)

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ccggtcggca tctacgatat cctactcagc gacgaagac 39