阅读说明：本技术 GhMYB44基因在棉花愈伤组织分化发育中的应用 (Application of GhMYB44 gene in differentiation and development of cotton callus ) 是由 王晔 陈全家 葛晓阳 刘理森 于 2021-07-08 设计创作，主要内容包括：本发明公开了GhMYB44基因在棉花愈伤组织分化发育中的应用,属于转基因技术领域。本申请通过超表达GhMYB44基因,使中棉所24植株外植体7d形成层中细胞结构发生了重组、30d愈伤组织的细胞呈现小而圆的特征、45d愈伤组织的有类似胚性愈伤组织细胞团出现,这些表型说明该基因能够促进受体材料更快从愈伤组织分化为胚性愈伤组织,加快再生苗进程,从外植体得到再生苗时间仅需182d,比对照缩短了24.7％,为棉花基因转化和快速育种做出贡献。(The invention discloses an application of GhMYB44 gene in differentiation and development of cotton callus, belonging to the technical field of transgenosis. According to the application, the GhMYB44 gene is overexpressed, so that the cell structure in the cambium of the 7d of the explant of the plant 24 of the Zhongmiao institute is recombined, the cells of the 30d callus present small and round characteristics, and the cells of the 45d callus have embryogenic-like callus groups, and the phenotypes show that the gene can promote the receptor material to be quickly differentiated into the embryogenic callus from the callus, so that the process of the regenerated seedling is accelerated, the time for obtaining the regenerated seedling from the explant is only 182d, is shortened by 24.7% compared with the reference, and contributes to the gene transformation and quick breeding of cotton.)

The application of GhMYB44 gene in accelerating the differentiation and development of cotton callus; the GhMYB44 gene sequence is shown in SEQ ID NO. 1.

The application of GhMYB44 protein in accelerating the differentiation and development of cotton callus; the sequence of the GhMYB44 protein is shown in SEQ ID NO. 2.

3. A callus induction culture medium of a GhMYB44 gene over-expression cotton plant is characterized by comprising: MSB medium containing 0.5 mol/L2,4-D and 0.5mol/L KT.

4. An embryogenic callus induction culture medium of a GhMYB44 gene over-expression cotton plant, which is characterized by comprising: MSB medium containing 0.5mol/L KT.

5. A tissue culture method of a GhMYB44 gene over-expression cotton plant is characterized by comprising the following steps:

(1) placing the lower culture shaft of cotton seedling in the callus induction culture medium of claim 3, and culturing for 30-40 days under proper conditions;

(2) after callus growth, transferring the callus to the embryogenic callus induction medium according to claim 4, and culturing under suitable conditions to obtain embryogenic callus.

6. The tissue culture method of the GhMYB44 gene over-expression cotton plant as claimed in claim 5, wherein the suitable conditions in step (1) and step (2) are: the illumination intensity is 2000Lx at 28 +/-2 ℃, and the illumination period is 16h/8 h.

7. The tissue culture method of a GhMYB44 gene overexpressed cotton plant according to claim 5, wherein the culture period in the step (1) is 30 days.

8. The tissue culture method of a GhMYB44 gene over-expression cotton plant as recited in claim 5, further comprising the following steps:

(3) and (4) subculturing the embryonic callus into an embryoid induction culture medium for embryoid induction to obtain an embryoid.

9. The tissue culture method of a GhMYB44 gene over-expression cotton plant as recited in claim 5, further comprising the following steps:

(4) and subculturing the embryoid into a seedling culture medium to obtain the transgenic seedling.

10. The tissue culture method of a GhMYB44 gene over-expression cotton plant as recited in claim 5, further comprising the following steps:

(5) subculturing the transgenic seedling to a rooting culture medium, hardening the seedling after growing white roots, and transplanting the seedling to a greenhouse.

Technical Field

The invention belongs to the technical field of transgenosis, and particularly relates to application of a GhMYB44 gene in cotton genetic transformation.

Background

Cotton is one of the leading economic crops in the world, the fiber of the cotton is an important raw material for the textile industry, the seed is an important oil source, and the cotton textile processing industry and the planting industry are important components of national economy. At present, the comprehensive competitiveness of cotton varieties in China is weak, the improvement on the aspects of fiber quality, yield, disease resistance, drought tolerance, salt and alkali tolerance and the like is urgently needed, and the cultivation of competitive new varieties is urgent, and the improvement of the cotton varieties by using genetic engineering is a quick and effective way. However, the existing transgenic system has the problems of long transformation period, high somatic embryo aberration rate, small number of normal regenerated seedlings and the like, and the application of genetic engineering in cotton variety improvement is severely limited.

In order to solve the bottleneck problem, a regulation network for regulating cotton somatic embryogenesis needs to be analyzed, key genes for improving a cotton genetic transformation system are screened, the cotton somatic embryogenesis system is optimized, and a foundation is laid for efficiently creating new cotton varieties.

Therefore, how to provide a gene capable of accelerating the differentiation and development of cotton callus is a problem to be solved in the field.

Disclosure of Invention

The invention discloses application of a GhMYB44 gene in differentiation and development of cotton embryogenic callus.

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

the application of GhMYB44 gene in accelerating the differentiation and development of cotton callus; the GhMYB44 gene sequence is shown in SEQ ID NO. 1;

the application of GhMYB44 protein in accelerating the differentiation and development of cotton callus; the GhMYB44 protein sequence is shown in SEQ ID NO. 2;

a callus induction culture medium of a GhMYB44 gene over-expression cotton plant comprises: MSB medium containing 0.5 mol/L2,4-D and 0.5mol/L KT;

an embryogenic callus induction culture medium of a GhMYB44 gene over-expression cotton plant, comprising: MSB medium containing 0.5mol/L KT;

a tissue culture method of a GhMYB44 gene over-expression cotton plant comprises the following steps:

(1) placing cotton seedling hypocotyls in the callus induction culture medium of claim 3, and culturing for 30-40 days under appropriate conditions;

(2) after callus growth, transferring the callus to the embryogenic callus induction medium according to claim 4, and culturing under suitable conditions to obtain embryogenic callus.

Preferably, in step (1) and step (2), the suitable conditions are: the temperature is 28 +/-2 ℃, the illumination intensity is 2000Lx, and the illumination period is 16h/8 h;

preferably, in the step (1), the culture period is 30 days;

further comprising the steps of:

(3) subculturing the embryonic callus into an embryoid induction culture medium for embryoid induction to obtain an embryoid;

further comprising the steps of:

(4) subculturing the embryoid into a seedling culture medium to obtain a transgenic seedling;

further comprising the steps of:

(5) subculturing the transgenic seedlings to a rooting culture medium, hardening the seedlings after white roots grow out, and transplanting the seedlings to a greenhouse;

the cotton variety is a Chinese cotton institute 24;

in conclusion, the invention discloses the application of the GhMYB44 gene in the differentiation and development of cotton callus; by over-expressing GhMYB44 gene, the cell structure in the cambium of the explant 7d of the plant 24 of the Zhongmiao cotton plant is recombined, the cells of the callus of 30d present small and round characteristics, the cell mass of the callus of 45d appears, the tissue co-culture time is only 182d, the tissue co-culture time is shortened by 24.7 percent compared with the control, and the tissue co-culture method contributes to the tissue culture and rapid breeding of cotton.

Drawings

FIG. 1: for histological observation pictures after explant induction culture for 7D, A and B are overexpression GhMYB44 gene materials MYB44-559 and MYB44-572, C is control ZM24, and D and E are suppressor materials MY B44-SRDX-9 and MYB 44-SRDX-55. .

FIG. 2: line graphs of embryogenic callus differentiation rates of the transgenic material and the control material; MYB44-559 and MYB44-572 are overexpression materials, ZM24 is a conventional control, and MYB44-SRDX-9 and MYB44-SRDX-55 are MYB44 gene repressor materials.

FIG. 3: culturing the transgenic material and the control material on a callus induction culture medium for 30 days, and then subculturing to a shape 20 days after the embryogenic callus induction culture medium; a and B are transgenic lines, C and D are control ZM 24.

FIG. 4: optical and scanning electron microscope pictures of the transgenic material and the reference material; a is a dissecting mirror picture which is cultured for 30 days on a callus culture medium by MYB44-559, MY B44-572, ZM24, MYB44-SRDX-9 and MYB44-SRDX-55 in sequence; b is scanning electron microscope picture of MYB44-559, MYB44-572, ZM24, MYB44-S RDX-9 and MYB44-SRDX-55 cultured on callus culture medium for 30 days in sequence; c is a dissecting mirror picture of culturing MYB44-559, MYB44-572, ZM24, MYB44-SRDX-9 and MYB44-SRDX-55 on a callus culture medium for 30 days in sequence, and then subculturing the callus culture medium for 15 days; d is MYB44-559, MYB44-572, ZM24, MYB44-SRD X-9 and MYB44-SRDX-55 in sequence, and the obtained product is cultured on a callus culture medium for 30 days and then is subcultured on an embryogenic callus induction culture medium to obtain a scanning electron microscope picture of 15 days.

FIG. 5: transgenic material shortens the cotton transformation period.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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 cotton institute 24 in the cotton variety upland cotton (Gossypium hirsutumL.) in the following examples is derived from the germplasm bank of cotton institute of chinese academy of agricultural sciences, hereinafter referred to as the "cotton institute 24". The hormone sigma company product designed in the following examples; media and other products from reagent companies;

MSB culture medium: the solute and the concentration thereof are as follows: NH (NH)₄NO₃ 1650mg/L，KNO₃ 1900mg/L，K H₂PO₄ 170mg/L，MgSO₄·7H₂O 370mg/L，CaCl₂·2H₂O 440mg/L，FeSO₄·7H₂O 27.85mg/L，Na₂EDTA 37.25mg/L，MnSO₄·4H₂O 22.3mg/L，ZnSO₄·7H₂O 8.6mg /L，H₃BO₃ 6.2mg/L，KI 0.83mg/L，Na₂MOO₄·2H₂O 0.25mg/L，CuSO₄·5H₂O 0. 025mg/L，COCl₂·6H₂O0.025 mg/L, inositol 100mg/L, VB 110 mg/L, VB 61.0 mg/L, nicotinic acid 1.0mg/L, sucrose 28g/L, agar 5.6 g/L; the solvent is distilled water; pH5.8;

callus induction medium: adding 2,4-D and KT into the MSB culture medium to ensure that the contents of the 2,4-D and KT are both 0.5mol/L, thus obtaining a callus induction culture medium;

embryogenic callus induction medium: adding KT and IAA into MSB culture medium to the content of 0.5mol/L to obtain embryogenic callus induction culture medium

Embryoid induction medium: KT and 6-BA were added to the MSB medium to a content of 0.01 mol/L to obtain an embryoid induction medium.

Seedling culture medium: adding IAA and 6-BA into MSB culture medium to the content of 0.05mol/L to obtain seedling culture medium.

Rooting culture medium: and 6-BA is added into the MSB culture medium until the content is 0.1mol/L, so as to obtain the seedling culture medium.

Sterile seedling culture medium: the solute and the concentration thereof are as follows: NH (NH)₄NO₃ 1650mg/L，KNO₃ 1900mg/L， KH₂PO₄170mg/L，MgSO₄·7H₂O 370mg/L，CaCl₂·2H₂O440 mg/L, 25g/L sucrose, 2g/L plant gel; the solvent is distilled water; pH 5.8.

Example 1 acquisition of plants of GhMYB44 that were over-expressed in Cotton plant 24

Construction of GhMYB44 overexpression vector:

DNA between a recognition sequence of a restriction endonuclease XbaI and a recognition sequence of a restriction endonuclease KpnII of a plant expression vector pCambia2300 (the plant expression vector pCambia2300 is cut into a large fragment and a small fragment by the restriction endonucleases XbaI and KpnII, the DNA is the small fragment) is replaced by a DNA molecule with a nucleotide sequence of SEQ ID No.1(GhMYB44), and other sequences of the plant expression vector pCambia2300 are kept unchanged to obtain a GhMYB44 gene expression vector which is named pCambia2300-35S: GhMYB 44.

Obtaining of Positive Agrobacterium

The recombinant vector pCambia2300-35S: GhMYB44 is introduced into the competence of Agrobacterium tumefaciens LBA4404, and the recombinant Agrobacterium containing the recombinant vector pCambia2300-35S: GhMYB44 is obtained after PCR identification.

And (3) transgenic process:

inoculating positive agrobacterium to LB liquid culture medium containing 50 ug/mL kanamycin, 25 ug/mL rifampicin and 50 ug/mL streptomycin for activation; on the day of transformation of cotton, activated bacteria were transformed as 1: 100 of the ratio was inoculated into 50mL of a new LB liquid medium containing 50. mu.g/mL kanamycin, 25. mu.g/mL rifampicin, 50. mu.g/mL streptomycin, and cultured at 28 ℃ in a shaker at 190rpm to a value of OD600 of 0.3 to 0.4.

Cutting cotton hypocotyls into sections with the length of 0.5-0.8cm in a sterilized culture dish, pouring cultured agrobacterium into the culture dish to dip-stain for about 3 minutes, pouring out bacterial liquid, absorbing residual bacterial liquid on the hypocotyl sections by using sterile filter paper, transferring the hypocotyls onto an MSB basic culture medium, and co-culturing for 48 hours in an incubator at 23 ℃ under a dark condition.

After two days, transferring the hypocotyl to a callus induction culture medium containing kanamycin (50mg/L) and cephalosporin (200mg/L), and culturing for about 30 days at 28 +/-2 ℃, with the illumination intensity of 2000Lx and the illumination cycle of 16h/8h to obtain a callus;

transferring the formed callus to an embryogenic callus induction culture medium containing kanamycin and cephalosporin, and culturing for about 60 days at 28 +/-2 ℃ under the conditions of illumination intensity of 2000Lx and illumination cycle of 16h/8h to obtain embryogenic callus;

subculturing the differentiated embryogenic callus on an embryoid induction culture medium for 15-20 days at 28 +/-2 ℃ under the conditions of illumination intensity of 2000Lx and illumination period of 16h/8h until sprouts grow out;

subculturing the bud tissue on a seedling culture medium, further transferring the bud tissue to a regenerated seedling rooting culture medium for rooting culture after the seedlings grow out, taking the regenerated seedlings out of the culture medium, soaking the regenerated seedlings in tap water at 28 +/-2 ℃, the illumination intensity of 2000Lx and the illumination period of 16h/8h for 15-20d for rooting for two weeks, hardening the seedlings under the conditions of 28 +/-2 ℃, the illumination intensity of 2000Lx and the illumination period of 16h/8h, transplanting the seedlings into a flowerpot after one week, and obtaining 24 over-expression 35S of the medium cotton, namely a GhMYB44 plant.

Example 2

Removing the cotton linters of seeds GhMYB44-559 and GhMYB44-574 of different strains of cotton over-expressing GhMYB44-1 obtained by transgenosis, peeling shells of the cotton seeds, soaking and sterilizing the cotton seeds for 5min by using 0.1% mercuric chloride, then washing the cotton seeds for 3-5 times by using sterile water, and sowing the cotton seeds on a sterile seedling culture medium to germinate and grow into sterile seedlings. Cutting hypocotyls of 7-day aseptic seedlings into 0.5 cm small segments on an aseptic workbench by using an alcohol lamp flame sterilized surgical blade, placing the small segments into a cotton callus induction culture medium, and performing tissue culture for 30 days under the conditions of 28 +/-2 ℃, artificial illumination intensity of 2000Lx and illumination period of 16h/8h to obtain calluses.

Subculturing the explant segments with the callus to a cotton embryogenic callus induction culture medium, culturing for 30 days at 28 +/-2 ℃ under the conditions of artificial illumination intensity of 2000Lx and illumination period of 16h/8h to obtain the embryogenic callus.

Subculturing the embryogenic callus into an embryoid induction culture medium, and performing embryoid induction culture for 30d under the conditions of the artificial illumination intensity of 2000Lx and the illumination period of 16h/8h at the temperature of 28 +/-2 ℃ to obtain an embryoid.

Subculturing the embryoid on a seedling culture medium, culturing for 60 days under the conditions of 28 +/-2 ℃, artificial illumination intensity of 2000Lx and illumination period of 16h/8h, further transferring the embryoid to a regenerated seedling rooting culture medium for rooting culture under the conditions of 28 +/-2 ℃, illumination intensity of 2000Lx and illumination period of 16h/8h, rooting for 30 days, then taking out the regenerated seedling from the culture medium, soaking the regenerated seedling in tap water at 28 +/-2 ℃, illumination intensity of 2000Lx and illumination period of 16h/8h, hardening the seedling, transplanting the regenerated seedling into a flowerpot after one week, obtaining plant seedlings, and counting the culture time and obtaining the result shown in figure 5.

Observing the differentiation condition of the callus from 40d of tissue culture, and counting the differentiation efficiency at 40d, 50d, 60d and 80d respectively, wherein the result is shown in figure 2; further observation by scanning electron microscopy revealed that the callus of explant 45d was overexpressed, and the results are shown in FIG. 4.

Through transcriptome data analysis of a somatic embryogenesis process, the GhMYB44 gene is found to be significantly up-regulated in a somatic embryo development process, and therefore, the GhMYB44 gene is presumed to play an important role in the cotton somatic embryo development process. To verify the function of GhMYB44 in cotton somatic embryogenesis, we constructed over-expression and transformation cotton. The result shows that the overexpression of GhMYB44 inhibits the initiation and proliferation of non-embryogenic callus, and the overexpression of GhMYB44 promotes the generation of embryogenic callus and the generation of somatic embryos. Histological observation and electron microscope scanning of the overexpressed material from GhMYB44 revealed that after induction on callus induction medium for 7 days, the cell structure reorganized in the cambium of 35S:: MYB44, whereas in control ZM24, vascular cells proliferated excessively and the cambium region did not start significantly (FIG. 1). To further observe that the cells of the 30d callus of the over-expressed explant exhibited small and round characteristics, the control ZM24 explant callus cells were large and long. Further observation through a scanning electron microscope shows that the 45d callus of the over-expression explant has cell clusters which appear and is the prophase morphology of the embryogenic callus. Whereas control ZM24 explant calli cells were loose and not clumped (fig. 2). Transgenic material and control cotton plant 24 were cultured on callus induction and differentiation medium. Differentiation efficiency was counted at 40d, 50d, 60d, 80d, respectively, and it was found that the differentiation rate of the over-expressed material was much higher than that of the control cotton field 24 (FIG. 3).

Comparative example 1

The procedure of example 2 was repeated at 182d in Zhongmiao station 24, and seedlings could not be obtained, and the results are shown in FIGS. 1-5.

Comparative example 2

Synthesizing a KpnI-SRDX-SacI sequence in Beijing Jinzhi Biotechnology company to obtain a recombinant plasmid which is formed by connecting SRDX with KpnI and SacI double restriction enzyme sites and pUC19-T, constructing the recombinant plasmid on a pAMBIA2300 vector, and transforming cotton to obtain two strains which are chimeric inhibitor expression vector materials; GhMYB44-SRDX-9 and GhMYB44-SRDX-55 the procedure of example 2 was repeated, and the results are shown in FIGS. 1-5.

Comparative example 3

The Zhongmiao plant 24 was cultured according to the normal tissue culture method (as shown in FIG. 5), i.e., the culture time of the embryogenic callus induction medium and that of the embryoid body induction medium were both prolonged by 30d, and the rest of the procedure was the same as in example 2, and plantlets were obtained after 242 d.

Analysis of results

Through transcriptome data analysis of a somatic embryogenesis process, the GhMYB44 gene is found to be significantly up-regulated in a somatic embryo development process, so that the GhMYB44 gene is presumed to play an important role in the cotton somatic embryo development process. To verify the function of GhMYB44 in cotton somatic embryogenesis, we constructed over-expression and transformation cotton. The result shows that the overexpression of GhMYB44 inhibits the initiation and proliferation of non-embryogenic callus, and the overexpression of GhMYB44 promotes the generation of embryogenic callus and the generation of somatic embryos. Histological observations and scanning by electron microscopy of the overexpressed material from GhMYB44 revealed that after induction on MSB medium for 7 days, the cell structure reorganized in the cambium of 35S:: MYB44, whereas in control ZM24 the vascular cells were hyperproliferative and the cambium region was not evident initially (FIG. 3). To further observe that the cells of the 30d callus of the over-expressed explant exhibited small and round characteristics, the control ZM24 explant callus cells were large and long. Further observation through a scanning electron microscope shows that the 45d callus of the over-expression explant has cell clusters which appear and is the prophase morphology of the embryogenic callus. Whereas control Z M24 explant calli cells were loose and not clumped (FIG. 4). Transgenic material and control cotton plant 24 were cultured on callus induction and differentiation medium. Differentiation efficiency was counted at 40d, 50d, 60d, 80d, respectively, and it was found that the differentiation rate of the over-expressed material was much higher than that of the control cotton field 24 (FIG. 2). Co-cultivation time control ZM24 was 242d, while ZM24-35S:: MYB44 transgenic plants were 182 days, with the transgenic plants being 24.7% shorter than the control (as shown in FIG. 5).

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 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 the above-described 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> Sinkiang university of agriculture

Cotton Research Institute Chinese Academy of Agricultural Sciences

Application of <120> GhMYB44 gene in differentiation and development of cotton callus

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 855

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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atggcttgta caaggaaaga tattgatcgg attaaaggtc catggagtcc agaagaagat 60

gaggctttaa agcgtctggt tcaaacctac ggttccagga actggtcttt ggtaagtaaa 120

tcgataccgg gtcgatctgg aaagtcttgt aggctacggt ggtgcaacca gctttcccct 180

gatgttgaac accgaccctt cactcccgag gaagacgata ccatagcccg agcccatact 240

cgattcggta acaaatgggc taccattgct cgattcctca atggtcgaac cgataacgcg 300

attaagaacc actggaactc tactctcaag cgaaaatgct cttcgatgac cgatgatatg 360

aacgacgatt cacctcagcc gcttaaaaag tcagctagtc tcaatactgg taatggtgga 420

ctgggtctct acttgaactc gagaagtcct tcaggatccg atttgagtga tttgagtttg 480

cccattgcct caccggtaac aataacgggg tctttggtgc cttcaactca aacagcatcc 540

tcggctactg atcctcctac tttactcagc ctctcgttac ccggatcaga tactagtgaa 600

atcactgact taggacccgt atccaactct ttcccaagtt ctaccttggt ggcagaacca 660

gcaactgttc cggctctgaa gttgcagatg gagaagcagt ttttgaacgc tgagttattg 720

gcagtgatgc aagagatgat aagaaaagaa gtacggaagt acatgagtgg gagtgaatcc 780

aatgggcttt gttttcgaac ggaggccatt agaaaagccg tcgttaagcg tatcgggatt 840

agtaagatcg agtga 855

<210> 2

<211> 302

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Met Asp Arg Ile Lys Gly Pro Trp Ser Pro Glu Glu Asp Asp Leu Leu

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Gln Lys Leu Val Gln Lys Tyr Gly Pro Arg Asn Trp Ser Leu Val Ser

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Lys Ser Ile Pro Gly Arg Ser Gly Lys Ser Cys Arg Leu Arg Trp Cys

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Asp Glu Thr Ile Ile Arg Ala His Ala Arg Phe Gly Asn Lys Trp Ala

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

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His Trp Asn Ser Thr Leu Lys Arg Lys Cys Leu Ser Val Gly Glu Glu

100 105 110

Ser Tyr Phe Ile Thr His Gly Gly Tyr Asp Gly Asn Leu Gly Gly Glu

115 120 125

Gly Glu Gln Gln Pro Leu Lys Arg Ser Val Ser Ala Gly Leu Tyr Met

130 135 140

Ser Pro Gly Ser Pro Ser Gly Ser Asp Leu Ser Asp Ser Ser Val Pro

145 150 155 160

Val Leu Ser Ser Ser His Val Tyr Lys Pro Ile Pro Arg Thr Gly Gly

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Val Asn Val Asp Val Asn Val Met Pro Leu Pro Gly Ala Glu Ala Ala

180 185 190

Ala Ser Ser Ser Asn Asp Pro Pro Thr Ser Leu Ser Leu Ser Leu Pro

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Gly Ala Glu Ser Tyr Glu Leu Ser Val Ser Thr Leu Pro Val Thr Glu

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Ser Thr Gln Thr Arg Asn Glu Ala Lys Asn Asp Gly Lys Gly Gly Gly

225 230 235 240

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

245 250 255

Arg Met Glu Val Arg Asp Tyr Met Val Gln Met Gln Gln Gln Asn Gly

260 265 270

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

275 280 285

Arg Asn Val Leu Ser Met Ser Arg Val Gly Val Asn Lys Ile

290 295 300