阅读说明：本技术 苄基异喹啉生物碱及其衍生物合成基因Ac6OMT的应用 (Application of benzyl isoquinoline alkaloid and derivative thereof in synthesis of gene Ac6OMT ) 是由 卢善发 崔馨云 李彩丽 于 2021-09-10 设计创作，主要内容包括：本发明公开了一种Ac6OMT基因在生产苄基异喹啉生物碱及其衍生物中的应用,Ac6OMT基因的核苷酸序列如SEQ ID NO.1所示；合成的氨基酸序列如SEQ ID NO.2所示；还公开了Ac6OMT基因在选育合成/不能合成苄基异喹啉生物碱及其衍生物的转基因植物品种中的应用,并记载了调控生物体合成苄基异喹啉生物碱及其衍生物的方法。本发明对苄基异喹啉生物碱及其衍生物合成通路中的Ac6OMT编码基因进行了功能鉴定,该基因可应用于北马兜铃苄基异喹啉生物碱的生物合成和分子育种。(The invention discloses an application of Ac6OMT gene in producing benzylisoquinoline alkaloid and derivatives thereof, wherein the nucleotide sequence of the Ac6OMT gene is shown as SEQ ID NO. 1; the synthesized amino acid sequence is shown as SEQ ID NO. 2; also discloses the application of Ac6OMT gene in breeding transgenic plant varieties capable of synthesizing/not synthesizing the benzylisoquinoline alkaloid and the derivatives thereof, and records a method for regulating organism synthesis of the benzylisoquinoline alkaloid and the derivatives thereof. The invention carries out functional identification on Ac6OMT coding gene in the synthetic pathway of the benzylisoquinoline alkaloid and the derivative thereof, and the gene can be applied to biosynthesis and molecular breeding of the Aristolochia benzylisoquinoline alkaloid.)

The application of Ac6OMT gene in the production of benzylisoquinoline alkaloid and derivatives thereof, wherein the nucleotide sequence of the Ac6OMT gene is shown as SEQ ID NO. 1; the synthesized amino acid sequence is shown as SEQ ID NO. 2.

2. The use of the Ac6OMT gene in the production of benzylisoquinoline alkaloids and derivatives thereof according to claim 1, wherein the Ac6OMT gene is transformed into a microorganism engineered bacterium or plant for the synthesis of benzylisoquinoline alkaloids and derivatives thereof.

And 3, the nucleotide sequence shown in SEQ ID NO.1 is obtained by replacing, deleting or inserting one or more bases, and the nucleotide sequence is used for coding the protein with the same function as the Ac6OMT gene.

And 4, the amino acid sequence shown in SEQ ID NO.2 is obtained by replacing, deleting or inserting one or more amino acids.

The Ac6OMT gene is applied to breeding of transgenic plant varieties for synthesizing the benzylisoquinoline alkaloid and the derivatives thereof.

6. Application of the knock-out Ac6OMT gene in cultivating plant varieties incapable of synthesizing benzylisoquinoline alkaloid derivatives.

7. A method for regulating and controlling the synthesis of benzylisoquinoline alkaloids in organisms is characterized in that the yield of benzylisoquinoline alkaloids and benzylisoquinoline alkaloid derivatives is controlled or the benzylisoquinoline alkaloid derivatives in organisms are completely eliminated by regulating and controlling the expression quantity of Ac6OMT genes or knocking out the genes.

8. A gene obtained by replacing, deleting or inserting one or more bases in the nucleotide sequence shown in SEQ ID NO. 1.

Technical Field

The invention relates to the technical field of synthesis of benzylisoquinoline alkaloids and derivatives thereof, in particular to application of a benzylisoquinoline alkaloid and a derivative thereof synthetic gene Ac6 OMT.

Background

Aristolochic acid is a benzylisoquinoline alkaloid derivative, which is of great concern due to its serious nephrotoxicity and carcinogenicity, and at the same time, the use of aristolochic acid-containing Chinese medicinal materials is also limited. From 2005 onwards, the pharmacopoeia successively deleted some aristolochic acid medicinal materials such as aristolochia debilis, dutchmanspipe root, aristolochia debilis, guan tong, and 2020 edition of Chinese pharmacopoeia shows that only one aristolochic acid medicinal material is asarum. However, most of aristolochic acid medicinal materials have long use history, obvious curative effect and no suitable medicinal material substitute is found, so that the medicine is required to be safely used, and the toxicity is reduced by processing, compatibility and traditional breeding.

At present, the aristolochia medicinal materials are controversial, and the aristolochic acid is only a toxic carcinogenic substance and has no other functions through research; it is also considered that aristolochic acid is also a drug effective ingredient. Therefore, pharmacological toxicological experiments are carried out on the aristolochic acid-free aristolochic acid, so that the aristolochic acid can be further verified to have no medicinal efficacy, and guidance is provided for the medication safety in the future.

The biosynthesis pathway of aristolochic acid is less researched at present, only possible intermediate compounds in the pathway are preliminarily researched by an isotope tracing method, and only one gene TyrDC is considered to be possibly involved in the synthesis of aristolochic acid at present. Because aristolochic acid belongs to derivatives of benzylisoquinoline alkaloids, and the biosynthesis pathway of benzylisoquinoline alkaloids is relatively comprehensive at present, the inference of aristolochic acid biosynthesis pathway can refer to the benzylisoquinoline alkaloid synthesis pathway. However, the biosynthesis pathway of aristolochic acid, a benzylisoquinoline alkaloid of aristolochia debilis, has been studied in a blank space, and thus, the genes for synthesizing aristolochic acid, a benzylisoquinoline alkaloid of aristolochia debilis, and a derivative thereof have been also studied less.

Therefore, how to apply the synthetic genes of the aristolochia benzyl isoquinoline alkaloid and the derivatives thereof is a problem which needs to be solved by the technical personnel in the field.

Disclosure of Invention

In view of the above, the present invention provides a method for screening and function identification of Ac6OMT coding gene in biosynthetic pathway of benzylisoquinoline alkaloid and its derivative aristolochic acid by using plant aristolochia debilis as research object. The method lays a foundation for the analysis of biosynthesis routes of the benzylisoquinoline alkaloid and the aristolochic acid derivative thereof and the molecular breeding of plants without the aristolochic acid.

In order to achieve the purpose, the invention adopts the following technical scheme:

the application of Ac6OMT gene in producing benzylisoquinoline alkaloid and derivatives thereof, wherein the nucleotide sequence of the Ac6OMT gene is shown as SEQ ID NO. 1; the synthesized amino acid sequence is shown as SEQ ID NO. 2.

As a preferable technical scheme of the technical scheme, the Ac6OMT gene is transformed into microbial engineering bacteria or plants and is used for synthesizing the benzylisoquinoline alkaloid and the derivatives thereof.

The nucleotide sequence shown in SEQ ID NO.1 is obtained by replacing, deleting or inserting one or more bases, and the nucleotide sequence is used for coding the protein with the same function as the Ac6OMT gene.

The amino acid sequence shown as SEQ ID NO.2 is obtained by replacing, deleting or inserting one or more amino acids.

As the same inventive concept, the invention also claims the application of the Ac6OMT gene in breeding transgenic plant varieties for synthesizing the benzylisoquinoline alkaloid and the derivatives thereof.

The invention also claims the application of the knock-out Ac6OMT gene in the cultivation of plant varieties incapable of synthesizing benzylisoquinoline alkaloid derivatives as the same inventive concept.

As the same inventive concept, the invention also claims a method for regulating and controlling the synthesis of benzylisoquinoline alkaloids in organisms, which controls the yield of benzylisoquinoline alkaloids and derivatives thereof or completely eliminates benzylisoquinoline alkaloid derivatives in organisms by regulating and controlling the expression amount of Ac6OMT genes or knocking out the genes.

A gene obtained by replacing, deleting or inserting one or more bases in the nucleotide sequence shown in SEQ ID NO. 1.

According to the technical scheme, compared with the prior art, the application of the norcoclaurine 6-oxymethyltransferase Ac6OMT gene in the synthetic pathway of the benzylisoquinoline alkaloid and the derivative aristolochic acid thereof is disclosed, the Ac6OMT gene has the function of catalyzing the norcoclaurine to generate the coclaurine, a key gene in the biosynthesis of the benzylisoquinoline alkaloid and the derivative aristolochic acid thereof is disclosed, and a foundation is laid for the analysis of the biosynthesis pathway of the benzylisoquinoline alkaloid and the derivative aristolochic acid thereof and the molecular breeding.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a graph showing the content of aristolochic acid in various tissues of Aristolochia according to the present invention;

FIG. 2 is a diagram of the identification and evolutionary analysis of the Aristolochia genome-based Ac6OMT gene provided by the present invention;

FIG. 3 is a graph showing the differential expression of the Ac6OMT encoding gene in different tissues of Aristolochia debilis;

FIG. 4 is a schematic diagram showing the catalytic mechanism of Ac6OMT protein;

FIG. 5 is a schematic representation of the catalytic pathway for the Ac6OMT protein.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention discloses screening and application of Aristolochia debilis benzylisoquinoline alkaloid and derivative synthetic genes.

EXAMPLE 1 determination of Aristolochia acid content in Aristolochia root, Stem, leaf, and flower

1) Sample pretreatment

Taking out the four tissues of the root, stem, leaf and flower from a refrigerator at the temperature of-80 ℃, and grinding the four tissues into fine powder by using liquid nitrogen. 0.1g of the powder is weighed into a centrifuge tube, 2ml of 70% methanol is added, the mixture is shaken and soaked for 15min at room temperature, and then ultrasonic treatment is carried out for 30 min. Centrifuging, collecting supernatant, adding 2ml 70% methanol into the rest precipitate, shaking, and performing ultrasonic treatment for 30 min. After centrifugation, the supernatant was collected and combined with the previously collected supernatant. Filtering with 0.22 μm filter membrane, and loading;

2) UPLC for detecting content of aristolochic acid A, B, C and D

Using an ACQUITYUPLC BEH C18 column (2.1X100nm, 1.7 μm; Waters); detection wavelength: 254 nm; column temperature: 30 ℃; flow rate: 0.4mL min^-1Sample size; 10 mu L of the solution; mobile phase: acetonitrile-0.2% acetic acid water; isocratic elution conditions were: 40% acetonitrile. And recording peak areas of the aristolochic acid A, B, C and D components, and calculating to obtain the content of the aristolochic acid A, B, C and D in each tissue by combining the peak areas of quantitative standard products. The results showed that total aristolochic acid was highest in roots, higher in flowers, and lower in stems and leaves (fig. 1).

Example 2 screening and phylogenetic analysis of Ac6OMT genes based on Aristolochia debilis genome and transcriptome

Extracting an OMT protein sequence of arabidopsis thaliana from a Tair database, comparing Aristolochia genome data through BLASTP, and identifying to obtain Aristolochia AcOMTs. The 6OMT protein sequences (Ps6OMT, Tf6OMT, Cj6OMT, Ps4 ' OMT1, Ps4 ' OMT2, Cj4 ' OMT) of other species related to the synthesis of benzylisoquinoline alkaloids, which are obtained by searching in a Unit database, and AcOMTs are compared together through MUSCLE to construct an NJ phylogenetic tree, and the selection of bootstrap is repeated for 1000 times. As in FIG. 2, the focus was on 7 AcOMTs, AcOMT1-7, grouped together with 6 OMTs of known function. Based on the transcriptome data, the FPKM values of the expression levels of the 7 AcOMTs in different tissues were extracted and plotted in a heat map (FIG. 3) for comparison. It was found that the expression level of Ac6OMT was significant in different tissues and positively correlated with aristolochic acid accumulation. Presumably it is involved in the synthesis of aristolochic acid.

Example 3 cloning of Aristolochia debilis Ac6OMT Gene

The full-length gene amplification primer is designed according to the open reading frame of the Ac6OMT gene sequence, cDNA of the Aristolochia debilis is taken as a template, the nucleotide sequence of the Ac6OMT gene is obtained by PCR amplification, is shown as SEQ ID No.1, and the full length of the gene is 1056 bp. The nucleotide sequence was translated to derive the amino acid sequence of the Ac6OMT, which included 351 amino acid residues, as shown in SEQ ID No. 2. Full-length amplification primers for the Ac6OMT gene are shown in Table 1:

TABLE 1

Example 4 inducible expression and purification of Ac6OMT protein

Cloning Ac6OMT gene into pET30a vector to form pET30a-AcOMT2 recombinant expression vector, and separately transforming the vector and pET30a into BL21(DE3) Escherichia coli expression strain. 1mL of the overnight activated bacterial suspension was added to 100mL of LB liquid medium (containing 50. mu.g/mL kanamycin), cultured at 37 ℃ for 3.5 hours at 200rpm, and when the OD reached 0.6 to 0.8, IPTG was added to a final concentration of 0.5mM, and induction was started at 16 ℃ at 200 rpm. After inducing for 20h, the thalli were collected by centrifugation, and after ultrasonication, the obtained supernatant was purified further. The solution obtained above was filtered through a 0.45 μm filter, loaded onto an equilibrated Ni column, and washed three times with 10mL of a washing buffer (500mM NaCl, 20mM Tris-HCl pH 8.0, 100mM imidazole). 6mL of elution buffer (500mM NaCl, 20mM Tris-HCl pH 8.0, 500mM imidazole) was washed three times and collected. Concentrating the eluent to 500 μ L by using an ultrafiltration tube, and measuring the protein concentration to obtain the purified protease liquid.

Example 5 functional verification of the Key enzyme Gene Ac6OMT involved in biosynthesis of Aristolochia benzylisoquinoline alkaloid and its derivative Aristolochic acid

The purified protease solution was functionally tested in vitro in accordance with the following reaction system (50. mu.L): 25mM Tris-HCl (pH 8.0),25mM sodium ascorbate, 0.1mM S-adenosylmethionine, 100mM higenamine, 10. mu.g of purified protein. Incubate 1 hour at 37 ℃, mix with 50 μ L methanol to stop the reaction, centrifuge and filter 0.22 μm, detect the chemical components using UPLC. Using an ACQUITY UPLC BEH C18 column (2.1X100nm, 1.7 μm; Waters); detection wavelength: 280 nm; column temperature: 35 ℃; flow rate: 0.3mL min^-1Sample size; 10 mu L of the solution; mobile phase: methanol (a), 0.1% formic acid water (B); the gradient elution conditions were: maintaining for 0-0.5 min at 5% A; after 0.5-4 min, 5% A is increased to 95% A; maintaining 95% A for 4-6 min; 6-6.1 min, reducing 95% A to 5% A; keeping the concentration of A at 5% for 6.1-8 min. Referring to FIGS. 4 and 5, in vitro catalysis results show that AcOMT2 can catalyze higenamine to generate a chromatogram with the same chromatographic behavior (such as retention time, spectrogram, etc.) as higenamine; whereas the unloaded protein is not catalytic. AcOMT2 was known to have the function of catalyzing higenamine to form higenamine, and was thus named Ac6 OMT.

The Ac6OMT gene nucleotide sequence is shown in SEQ ID NO. 1;

ATGGAGACACCGAAAGACGATCAATTGGCTCAGGCGAAGCTGTGGAAGTCCGTCTACGCTTTGGCCGACTCCCTCGTCCTCCGTGCCGCCGTAGAGCTCGGCCTCGTCGACATCGTCCACGCCCACGGAGGACCCATCTCAGTTTCCGACATCTCCTCCCGCCTCCGGCTTCCCTCCGTCGACTCCGGCCGCCTTCAACGCCTCTTGCGCTACCTCGTCCGAATGGAGATCTTCTCTACGGAACCGAGCACCCTCGACGACGATGGCGAGTATCGGTACGGCCTCGCCCCCGCCGCCGCCTACCTCGTCTCCGGCAAGGACAAGAACATCATTCCCGCCCTGCTCTTTGTCACCGACAAGGACATGATGACGCCGTACTACTTCATCAAGGACGCACTCCGCCGGAGCGGGCCCACCGCATTCGAGATGGCCCTGGGGAAGAGCATCTGGGATTTCATGGCGGACCAGCCTGAGAAGAACAAGCTCTTCAACGCCGCCATGGCGTCCGATTCTCGGCTCCTCACCTCAGCCCTGGTCGGCGAATGCGAGGACGTCTTCGCCGGAATTGGGTCCCTCGTCGACGTCGGCGGCGGCACTGGAACGGCGGTGAGGGCCATCGCCGAGGCCTACCCGGGAATCAAATGTACCGTCTTCGACCTGCCGCATGTCATCGCCGACTCGCCCCACTATCCGGAGGTGACCCGCGTCGAAGGGGACATGTTTAAGTCCATTCCCGCCGCAGATGCCATTCTAATGAAGTGCATCCTCCACGATTGGGGCGACGAGGATTGCGTGAAGATCCTAAAGAAGTGCAAGGAAGCTGTGCCCACAAAAGGGGGGAAGGTGATTATTGTGGACATCGTGTTGGATACTGATACCCACCGTCCCCAAACCCACCTGAAGCAGTGTATGGATTTGGACATGCTTCTCACCACGGGAGGAAAAGAGAGAACGGAAGCAGAATGGAAGAAACTGATTCACGACGCAGGGTTTAAAGGGTACAAGATCAAACACACTTCCGCGGTACAATCTGTGATTGAGGCATTTCCTTACTAA, as set forth in SEQ ID No. 1;

the amino acid sequence of the Ac6OMT protein is shown as SEQ ID NO. 2;

METPKDDQLAQAKLWKSVYALADSLVLRAAVELGLVDIVHAHGGPISVSDISSRLRLPSVDSGRLQRLLRYLVRMEIFSTEPSTLDDDGEYRYGLAPAAAYLVSGKDKNIIPALLFVTDKDMMTPYYFIKDALRRSGPTAFEMALGKSIWDFMADQPEKNKLFNAAMASDSRLLTSALVGECEDVFAGIGSLVDVGGGTGTAVRAIAEAYPGIKCTVFDLPHVIADSPHYPEVTRVEGDMFKSIPAADAILMKCILHDWGDEDCVKILKKCKEAVPTKGGKVIIVDIVLDTDTHRPQTHLKQCMDLDMLLTTGGKERTEAEWKKLIHDAGFKGYKIKHTSAVQSVIEAFPY, as shown in SEQ ID NO. 2.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> institute of medicinal plants of academy of Chinese medical science

Application of <120> benzylisoquinoline alkaloid and derivative thereof in synthesis of gene Ac6OMT

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<170> SIPOSequenceListing 1.0

<210> 1

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ttggccgact ccctcgtcct ccgtgccgcc gtagagctcg gcctcgtcga catcgtccac 120

gcccacggag gacccatctc agtttccgac atctcctccc gcctccggct tccctccgtc 180

gactccggcc gccttcaacg cctcttgcgc tacctcgtcc gaatggagat cttctctacg 240

gaaccgagca ccctcgacga cgatggcgag tatcggtacg gcctcgcccc cgccgccgcc 300

tacctcgtct ccggcaagga caagaacatc attcccgccc tgctctttgt caccgacaag 360

gacatgatga cgccgtacta cttcatcaag gacgcactcc gccggagcgg gcccaccgca 420

ttcgagatgg ccctggggaa gagcatctgg gatttcatgg cggaccagcc tgagaagaac 480

aagctcttca acgccgccat ggcgtccgat tctcggctcc tcacctcagc cctggtcggc 540

gaatgcgagg acgtcttcgc cggaattggg tccctcgtcg acgtcggcgg cggcactgga 600

acggcggtga gggccatcgc cgaggcctac ccgggaatca aatgtaccgt cttcgacctg 660

ccgcatgtca tcgccgactc gccccactat ccggaggtga cccgcgtcga aggggacatg 720

tttaagtcca ttcccgccgc agatgccatt ctaatgaagt gcatcctcca cgattggggc 780

gacgaggatt gcgtgaagat cctaaagaag tgcaaggaag ctgtgcccac aaaagggggg 840

aaggtgatta ttgtggacat cgtgttggat actgataccc accgtcccca aacccacctg 900

aagcagtgta tggatttgga catgcttctc accacgggag gaaaagagag aacggaagca 960

gaatggaaga aactgattca cgacgcaggg tttaaagggt acaagatcaa acacacttcc 1020

gcggtacaat ctgtgattga ggcatttcct tactaa 1056

<210> 2

<211> 351

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Glu Thr Pro Lys Asp Asp Gln Leu Ala Gln Ala Lys Leu Trp Lys

1 5 10 15

Ser Val Tyr Ala Leu Ala Asp Ser Leu Val Leu Arg Ala Ala Val Glu

20 25 30

Leu Gly Leu Val Asp Ile Val His Ala His Gly Gly Pro Ile Ser Val

35 40 45

Ser Asp Ile Ser Ser Arg Leu Arg Leu Pro Ser Val Asp Ser Gly Arg

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Leu Gln Arg Leu Leu Arg Tyr Leu Val Arg Met Glu Ile Phe Ser Thr

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Glu Pro Ser Thr Leu Asp Asp Asp Gly Glu Tyr Arg Tyr Gly Leu Ala

85 90 95

Pro Ala Ala Ala Tyr Leu Val Ser Gly Lys Asp Lys Asn Ile Ile Pro

100 105 110

Ala Leu Leu Phe Val Thr Asp Lys Asp Met Met Thr Pro Tyr Tyr Phe

115 120 125

Ile Lys Asp Ala Leu Arg Arg Ser Gly Pro Thr Ala Phe Glu Met Ala

130 135 140

Leu Gly Lys Ser Ile Trp Asp Phe Met Ala Asp Gln Pro Glu Lys Asn

145 150 155 160

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

165 170 175

Ala Leu Val Gly Glu Cys Glu Asp Val Phe Ala Gly Ile Gly Ser Leu

180 185 190

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

195 200 205

Ala Tyr Pro Gly Ile Lys Cys Thr Val Phe Asp Leu Pro His Val Ile

210 215 220

Ala Asp Ser Pro His Tyr Pro Glu Val Thr Arg Val Glu Gly Asp Met

225 230 235 240

Phe Lys Ser Ile Pro Ala Ala Asp Ala Ile Leu Met Lys Cys Ile Leu

245 250 255

His Asp Trp Gly Asp Glu Asp Cys Val Lys Ile Leu Lys Lys Cys Lys

260 265 270

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

275 280 285

Leu Asp Thr Asp Thr His Arg Pro Gln Thr His Leu Lys Gln Cys Met

290 295 300

Asp Leu Asp Met Leu Leu Thr Thr Gly Gly Lys Glu Arg Thr Glu Ala

305 310 315 320

Glu Trp Lys Lys Leu Ile His Asp Ala Gly Phe Lys Gly Tyr Lys Ile

325 330 335

Lys His Thr Ser Ala Val Gln Ser Val Ile Glu Ala Phe Pro Tyr

340 345 350

<210> 3

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggatccatgg agacaccgaa agacgatca 29

<210> 4

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gcggccgctt agtaaggaaa tgcctcaatc acaga 35