阅读说明：本技术 3-酮脂酰-CoA硫解酶基因RkACAA1-1及其应用 (3-ketoacyl-CoA thiolase gene RkACAA1-1 and application thereof ) 是由 张琦 陈功水 魏云林 季秀玲 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-CoA硫解酶基因RkACAA1-1,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；该基因是红冬孢酵母(Rhodosporidium kratochvilovae)YM25235中分离获得的,将该基因转化到红冬孢酵母YM25235中,实验结果显示,3-酮脂酰-CoA硫解酶基因RkACAA1-1的过表达能引起红冬孢酵母YM25235菌株中总类胡萝卜素增加,本发明通过基因工程手段对红冬孢酵母进行改造来提高类胡萝卜素含量,对类胡萝卜素的工业化生产提供良好的应用前景和经济效益,为大规模商业化生产类胡萝卜素奠定基础。(The invention discloses a 3-ketoacyl-CoA thiolase gene RkACAA1‑1 The nucleotide sequence 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 red wintergreen spore yeast ( Rhodosporidium kratochvilovae ) YM25235, transforming the gene into Rhodosporidium toruloides YM25235, and testing to show that 3-ketoacyl-CoA thiolase gene RkACAA1‑1 The overexpression of the gene can cause the Rhodosporidium toruloides YM25235The total carotenoid in the strain is increased, the rhodosporidium toruloides is transformed by a genetic engineering means to improve the carotenoid content, good application prospect and economic benefit are provided for the industrial production of the carotenoid, and a foundation is laid for the large-scale commercial production of the carotenoid.)

1. 3-ketoacyl-CoA thiolase geneRkACAA1-1The nucleotide sequence is shown in SEQ ID NO. 1.

2. The 3-ketoacyl-CoA thiolase gene of claim 1RkACAA1-1Application in promoting microbial production of carotenoid is provided.

Technical Field

The invention belongs to the technical field of biological and genetic engineering, and relates to a 3-ketoacyl-CoA thiolase geneRkACAA1-1And the application thereof in promoting the microbial production of carotenoids.

Background

Carotenoids (carotenoids) are an important class of natural pigments, which are isoprene polymers containing 40 carbon atoms. Carotenoids can be generally classified into two groups, depending on their molecular composition, one group being Carotenes (Carotenes) whose chemical structure contains no oxygen elements but only carbon and hydrogen elements, such as α -carotene, β -carotene and lycopene; one class is Xanthophylls (xanthylphyls) whose chemical structure contains oxygen-containing functional groups such as hydroxyl, keto, carboxyl, and methoxy, such as zeaxanthin, lutein, and astaxanthin. Currently, over 800 natural carotenoids have been found in nature, which are found in a wide range of higher plants, algae, fungi and bacteria.

Carotenoids are important components of the antioxidant system of animals and plants, and are effective antioxidants for protecting the body from oxidative damage. Research shows that human body can generate active oxygen in normal life metabolism activity, and ultraviolet radiation, environmental pollutants and the like can also induce the generation of the active oxygen. Excessive oxygen free radicals in the human body can cause normal life macromolecules to be oxidized, thereby causing damage to the body. The molecular structure of the carotenoid has isoprenoid conjugated double bonds, so that the carotenoid has stronger antioxidant activity and can protect cells and tissues from being damaged by active oxygen. Besides, the carotenoid is a precursor substance of vitamin A synthesis, and can promote the formation of the human visual system; it also has effects in inhibiting abnormal cell growth, preventing low density lipoprotein formation, and has important effects in maintaining health, preventing diseases, resisting aging and preventing cancer. To date, there is no evidence that animals are able to synthesize carotenoids themselves, relying only on ingestion from the outside.

Carotenoids are also rich in color, and exhibit different colors depending on the number of conjugated double bonds in the molecule, the chain length and the substituent groups. In the food and pharmaceutical industry, carotenoids have been widely used as a natural colorant; moreover, the carotenoid can not only provide attractive colors, but also prolong the shelf life of the food due to the fact that the carotenoid can prevent the nutritional ingredients and flavor substances in the food from being damaged due to the antioxidation of the carotenoid. Besides the field of food and medicine, carotenoids also play an important role in the breeding industry. The carotenoid is added into the feed, so that the morbidity risk of animals can be reduced, the immunity of the animals can be enhanced, the quality of meat can be improved, and the color of egg yolks of egg products can be improved.

With the increasingly wide application of the carotenoids in the fields of medical treatment, food, cultivation and the like, the demand is increasing. Although there have been considerable research by many researchers on how to increase the production of carotenoids, the production and quality of carotenoids are still not satisfactory for the market. Currently, the major carotenoid production methods include plant extraction, chemical synthesis and biological synthesis. The plant extraction method is too costly and obviously not suitable for commercial and large-scale production; the chemical synthesis method can meet the demand on yield, but has the defects of environmental unfriendliness, chemical substance residue and activity no better than that of natural carotenoid.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a strain prepared from rhodosporidium toruloides (A)Rhodosporidium kratochvilovae) Separating 3-ketoacyl-CoA thiolase gene from YM25235RkACAA1-1The 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 1275bp (basic group), and the coded amino acid sequence is polypeptide shown as SEQ ID NO. 2.

Another object of the present invention is to provide the above-mentioned 3-ketoacyl-CoA thiolase geneRkACAA1-1It is used in producing carotenoid.

The above purpose is realized by the following technical proposal, total RNA is extracted from Rhodosporidium toruloides YM25235, then cDNA is synthesized by reverse transcription, a target fragment is obtained by PCR amplification by taking the synthesized cDNA as a template,carrying out double enzyme digestion and recovery on the vector pRH2034, connecting a target fragment with the vector by a one-step cloning method to obtain a recombinant plasmid pRHRkACAA1-1, transferring the connection product into escherichia coli, screening out positive single clone by PCR, and using the recombinant plasmid pRHRkACAA1-1 as a recombinant plasmidBamHⅠ、EcoPerforming enzyme digestion verification on the two restriction endonucleases, extracting plasmids after verifying that positive clones are cultured, sequencing to obtain a 3-ketoacyl-CoA thiolase gene with the fragment size of 1275bpRkACAA1-1(ii) a Transforming the recombinant vector pRHRkACAA1-1 into Rhodosporidium toruloides YM25235 by PEG-mediated protoplast method, and screening transformants to obtainRkACAA1-1And (3) over-expressing the strain, culturing the over-expressed strain, and measuring the content of the total carotenoid in the strain by using an ultraviolet-visible spectrophotometer.

The invention relates to a method for preparing a red wintergreen spore yeast (Rhodosporidium toruloides)Rhodosporidium kratochvilovae) Separating YM25235 total RNA to obtain 3-ketoacyl-CoA thiolase geneRkACAA1-1Rhodosporidium toruloides YM25235RkACAA1-1The overexpression of the gene can cause the transcription level of the gene in the cell to be improved to a certain degree, which indicates that the exogenous gene is transcribed in thalli and then translated into corresponding protein to cause the expression quantity of enzymes related to the synthesis of the carotenoid in the cell to be improved.

Drawings

FIG. 1 shows a scheme for producing Rhodosporidium toruloides YM25235 of the present inventionRkACAA1-1A gene PCR amplification map; DNA molecular weight marker DL 2000; 2. negative control; 3.RkACAA1-1a cDNA fragment of (1);

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

FIG. 3 is a PCR-verified electrophoretogram of colonies; DNA molecular weight marker DL 2000; 2. and (3) a negative control. RkACAA1-1A cDNA fragment of (1); 4-8 is a transformant;

FIG. 4 is a restriction analysis of the recombinant plasmid pRHRkACAA 1-1; wherein: DNA molecular weight marker dl100002 negative control; 3. of the empty plasmid pRH2034BamHⅠ、EcoPerforming double enzyme digestion on RV; 4. of the recombinant plasmid pRHRkACAA1-1BamHⅠ、EcoPerforming double enzyme digestion on RV; 5.RkACAA1-1a cDNA fragment of (1); DNA molecular weight marker DL 2000;

FIG. 5 shows the verification of positive clone of recombinant plasmid pRHRkACAA1-1 transformed Rhodosporidium toruloides YM 25235; DNA molecular weight marker DL 2000; 2. negative control 3. wild type strain specific gene strip; 4.RkACAA1-1a cDNA fragment; 5. verifying a transformant;

FIG. 6 comparison of carotenoid content of the over-expressed strain YM25235/pRHRkACAA1-1 with that of 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 Gene from YM25235RkACAA1The nucleotide sequence of (a) and the construction of an overexpression vector pRHRkACAA1-1

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 cDNA as template, and sequencing according to the transcriptomeRkACAA1-1Designing specific primers RkACAA1-1-F and RkACAA1-1-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:

RkACAA1-1-F：5’-ATCACTCACCATGGCGGATCCTATGTCTCTCACGAACGCCG-3' (SEQ ID NO: 3) (double underlined is the upstream vector end homologySequence, single underlinedBamHI cleavage site)

RkACAA1-1-R：5’-CCGGTCGGCATCTACGATATCCTACTGCTCGTTGACGATGA-3' (SEQ ID NO: 4) (double underlined is the upstream vector end homologous sequence and single underlined isEcoAn rv cleavage site);

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

；

amplification conditions: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 15s, annealing at 64 deg.C for 15s, and extension at 72 deg.C for 1min15s for 30 cycles, and final extension at 72 deg.C for 5 min; after the reaction, 2. mu.L of the product was taken and subjected to electrophoresis analysis in 1% agarose gel, and the results are shown in FIG. 1; amplifying to obtain a fragment with the size of 1275 bp; pRH2034 throughBamHⅠ、EcoPerforming double enzyme digestion on the two restriction enzymes of RV; the two fragments were recovered with a multifunctional DNA recovery Kit (Beijing Baitaike Biotechnology Co., Ltd., product number: DP 1502), and the two recovered fragments were ligated to obtain a recombinant plasmid pRHRkACAA1-1 (FIG. 2) in a Clonexpress II One Step Cloning Kit (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 ℃; cooled to 4 ℃ or immediately placed on ice to cool.

Adding 10 muL of the ligation product obtained above to 100 muL DH5 alpha competent cells, mixing gently, ice-bathing for 30min, immediately placing on ice to cool for 90s after heat shock for 90s at 42 ℃, adding 900 muL LB liquid culture medium into the connecting system, carrying out shake culture at 37 ℃ and 100rpm for 1h, centrifuging at 5000rpm for 10min, then discarding 900 muL supernatant, suspending thalli by the residual culture medium of about 100 muL, coating the suspended thalli on an LB solid plate containing 100 mug/mL spectinomycin (Spe +), carrying out inversion culture at 37 ℃, and carrying out random overnight cultureSelecting white colonies growing on 5 plates and numbering as No. 1-5, verifying positive clones by colony PCR, wherein the result is shown in figure 3, and it can be seen from the figure that five selected monoclonal strains amplify specific bands with the same size as the target fragment by colony PCR, which indicates that five selected DH5 alpha strains are 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 pRHRkACAA1-1 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, andRkACAA1-1the cDNA fragments of the gene are bands with the same size, which indicates that the recombinant plasmid is successfully transferred 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 1275bp and the nucleotide sequence shown in SEQ ID NO. 1 and is named asRkACAA1-1And is andRkACAA1-1the cDNA fragments of the genes are consistent in size and sequence, which indicates that an expression vector pRHRkACAA1-1 is successfully constructed;

example 2:RkACAA1-1effect of Gene overexpression on Carotenoid Synthesis in Rhodosporidium toruloides YM25235

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), 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 OD₆₀₀When the concentration is 0.5, centrifuging the culture solution at 4 ℃ and 4500 rpm for 5min to collect thalli; using prepared in advanceWashing thallus twice with citric acid buffer solution (30 mM citric acid, 83mM sodium citrate, 600mM mannitol, NaOH to adjust pH to 5.4), centrifuging at 4 deg.C and 4000 rpm for 5min to collect thallus, suspending the thallus with 1mL citric acid buffer solution, 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 50mL centrifuge tube 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 pRHRKACAA1-1 recombinant plasmid was added and gently mixed (usually the volume of the plasmid solution should not exceed 10. mu.L) and incubated on ice for 10min, 200. mu.L of precooled PTC (50% PEG3350, 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 supernatant, and suspending thallus in 100 μ L culture medium, spreading on 0.4M sucrose YPD solid culture medium containing 130 μ g/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, and then genomic DNA of yeast transformant was extracted according to the procedure of DNA extraction kit of Shanghai Biotechnology engineering Co., Ltd, and PCR was performed, and the result is shown in FIG. 5, in which it can be seen that the genomic DNA of yeast transformant was used as a template and amplified by PCRGo out andRkACAA1-1the cDNA fragments of (1) have the same size, and the gene of the recombinant transformant is verified correctly, which indicates thatRkACAA1-1The cDNA fragments have been successfully ligated into the genome of yeast transformants.

2、RkACAA1-1Analysis of Carotenoid content in Gene-overexpressed Rhodosporidium toruloides YM25235

Culturing overexpression strain containing pRHRkACAA1-1 at 28 deg.C for 168 hr, extracting carotenoid, and determining total carotenoid content (mg/g dry thallus) at 445nm with UV-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/pRHRkACAA1-1 is obviously improved compared with that of the wild type Rhodosporidium toruloides YM25235 strain, the carotenoid synthesis amount of the wild type Rhodosporidium toruloides YM25235 strain is 4.87 +/-0.49 mg/g, and the carotenoid synthesis amount of the over-expressed strain YM25235/pRHRKACAA1-1 is 7.97 +/-0.16 mg/g, namely, the carotenoid synthesis amount of the over-expressed strain YM25235/pRHRKACAA1-1 is 1.636 times that of the control strain; the results showed that the 3-ketoacyl-CoA thiolase geneRkACAA1-1The overexpression of (a) can cause the increase of the total carotenoid content in the rhodosporidium toruloides YM25235 strain,RkACAA1-1the gene can promote the synthesis of total carotenoid.

Sequence listing

<110> university of Kunming science

<120> 3-ketoacyl-CoA thiolase gene RkACAA1-1 and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1275

<212> DNA

<213> Rhodosporidium toruloides YM25235(Rhodosporidium kratochvilovaeYM25235)

<400> 1

atgtctctca cgaacgccgc ctccgccgcc aaggaccgcc tcagtggcct cgtctcgcac 60

ctcacccccc gcggcaaggc tgccctcacg gccaagaacg ccgacgacgt tgtcatcgtc 120

gccgccgtcc gcacgcccat cacccgcgcc aagaagggcg gcctcaagga cgcgtgcccg 180

gaggacctcc tcaaggccgt ctttgagggt gtcattgccc gctcgggcgt cgacaagaac 240

ctcgtcgagg agatccaggt cggcaacgtc ctcccgcccg gcggcggtgc gaccgttgcc 300

cgcatggcgc agctcgctgc cggcttcccc accacctcgt ccgtcgcgac cgtcaaccgc 360

cagtgctcgt cgggcctcgt cgcggtcaac catatcgcgc tcatgatcgc ggccggccag 420

atcgactttg gtatcggcgc gggtgtcgag tcgatgaccc aagggtacgg cgcgggcgcg 480

atgccggaga agatgtcgga cgacatcctc tcctgccagg cggcggctga ctgcctcctc 540

cccatgggta tcacgagcga gaacgtcgcg accgagtaca acgtttcgcg cgagcagcag 600

gacgcgttcg cggccgagtc gttccagcgc gcggcggcgg cgcagaaggc gggcaagttc 660

aaggacgaga tcgttgccgt caagaccaag tgggtcgacc cgaagaccga ggaggagaag 720

gagattgtcg tcaccgagga cgacggcatc cgtgcgggcg tcaccaagga gagcctcagc 780

aagctcaagc ccgtcttctc caagactggc agcacccacg ctggcaacgc ctcgcaggtc 840

tcggacggcg ccgccgcggt cctcctcacg cgccgctcca aggcgcagga gctcggcctc 900

cccatcctcg gcaaggtctg ccataccgcc atcgcgggcg tcgagcccaa gctcatgggc 960

atcggccccg cgttcgccat ccccaaggtc ctcgagaaga cgggcctcac caaggacgac 1020

gtcgacctgt tcgagctgaa cgaggccttc gcctcgcagg cggtcatgtc gatcgagcac 1080

ctcggccttg actacaagaa ggtcaacccg aacggcggtg ccatcgcgct cggccacccg 1140

ctcggctgca ccggtgcccg ccagatcgcg accgcgctct ccgaggcgaa gcgctcgggc 1200

gccaagatca tctgcacgag catgtgcatc ggcagcggca tgggcgcggc gagcatcatc 1260

gtcaacgagc agtag 1275

<210> 2

<211> 424

<212> PRT

<213> Rhodosporidium toruloides YM25235(Rhodosporidium kratochvilovae YM25235)

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Met Ser Leu Thr Asn Ala Ala Ser Ala Ala Lys Asp Arg Leu Ser Gly

1 5 10 15

Leu Val Ser His Leu Thr Pro Arg Gly Lys Ala Ala Leu Thr Ala Lys

20 25 30

Asn Ala Asp Asp Val Val Ile Val Ala Ala Val Arg Thr Pro Ile Thr

35 40 45

Arg Ala Lys Lys Gly Gly Leu Lys Asp Ala Cys Pro Glu Asp Leu Leu

50 55 60

Lys Ala Val Phe Glu Gly Val Ile Ala Arg Ser Gly Val Asp Lys Asn

65 70 75 80

Leu Val Glu Glu Ile Gln Val Gly Asn Val Leu Pro Pro Gly Gly Gly

85 90 95

Ala Thr Val Ala Arg Met Ala Gln Leu Ala Ala Gly Phe Pro Thr Thr

100 105 110

Ser Ser Val Ala Thr Val Asn Arg Gln Cys Ser Ser Gly Leu Val Ala

115 120 125

Val Asn His Ile Ala Leu Met Ile Ala Ala Gly Gln Ile Asp Phe Gly

130 135 140

Ile Gly Ala Gly Val Glu Ser Met Thr Gln Gly Tyr Gly Ala Gly Ala

145 150 155 160

Met Pro Glu Lys Met Ser Asp Asp Ile Leu Ser Cys Gln Ala Ala Ala

165 170 175

Asp Cys Leu Leu Pro Met Gly Ile Thr Ser Glu Asn Val Ala Thr Glu

180 185 190

Tyr Asn Val Ser Arg Glu Gln Gln Asp Ala Phe Ala Ala Glu Ser Phe

195 200 205

Gln Arg Ala Ala Ala Ala Gln Lys Ala Gly Lys Phe Lys Asp Glu Ile

210 215 220

Val Ala Val Lys Thr Lys Trp Val Asp Pro Lys Thr Glu Glu Glu Lys

225 230 235 240

Glu Ile Val Val Thr Glu Asp Asp Gly Ile Arg Ala Gly Val Thr Lys

245 250 255

Glu Ser Leu Ser Lys Leu Lys Pro Val Phe Ser Lys Thr Gly Ser Thr

260 265 270

His Ala Gly Asn Ala Ser Gln Val Ser Asp Gly Ala Ala Ala Val Leu

275 280 285

Leu Thr Arg Arg Ser Lys Ala Gln Glu Leu Gly Leu Pro Ile Leu Gly

290 295 300

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

305 310 315 320

Ile Gly Pro Ala Phe Ala Ile Pro Lys Val Leu Glu Lys Thr Gly Leu

325 330 335

Thr Lys Asp Asp Val Asp Leu Phe Glu Leu Asn Glu Ala Phe Ala Ser

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Ala Lys Ile Ile Cys Thr Ser Met Cys Ile Gly Ser Gly Met Gly Ala

405 410 415

Ala Ser Ile Ile Val Asn Glu Gln

420

<210> 3

<211> 41

<212> DNA

<213> Artificial sequence (Artificial)

<400> 3

atcactcacc atggcggatc ctatgtctct cacgaacgcc g 41

<210> 3

<211> 41

<212> DNA

<213> Artificial sequence (Artificial)

<400> 3

ccggtcggca tctacgatat cctactgctc gttgacgatg a 41