Quorum sensing signal molecule DSF degradation gene fadT, encoded degradation enzyme FadT and application thereof
阅读说明：本技术 群体感应信号分子DSF降解基因fadT及和编码的降解酶FadT及其应用 (Quorum sensing signal molecule DSF degradation gene fadT, encoded degradation enzyme FadT and application thereof ) 是由 陈少华 许旭丹 叶田 张文平 周田 张炼辉 于 2021-07-23 设计创作，主要内容包括：本发明公开了一种群体感应信号分子DSF降解基因fadT,其核苷酸序列如SEQ ID NO.1所示。本发明从贪铜菌(Cupriavidus pinatubonensis)菌株HN-2中克隆得到了一种新的负责脂肪酸降解的基因-DSF降解酶编码基因fadT。该编码基因fadT在野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv.campestris,Xcc)中表达后,能够显著降低Xcc的致病力,对Xcc引起的黑腐病有显著的防治效果。(The invention discloses a quorum sensing signal molecule DSF degradation gene fadT, the nucleotide sequence of which is shown in SEQ ID NO. 1. The invention clones a new gene responsible for fatty acid degradation, namely DSF degrading enzyme coding gene fadT from a cuprianus cupriaefolius (cupriavidius pinatubonensis) strain HN-2. The coding gene fadT can obviously reduce the pathogenicity of Xcc after being expressed in Xanthomonas campestris wild rape pathogenic variants (Xcc), and has obvious prevention and treatment effect on black rot caused by Xcc.)
1. A quorum sensing signal molecule DSF degradation gene fadT is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. The degrading enzyme FadT encoded by the degrading gene fadT of claim 1, wherein the amino acid sequence is shown in SEQ ID NO. 2.
3. A recombinant plasmid comprising the gene fadT according to claim 1 or a fragment thereof, or capable of expressing the degrading enzyme FadT according to claim 2.
4. A recombinant microorganism capable of expressing the gene fadT or a fragment thereof according to claim 1 or the degrading enzyme FadT according to claim 2.
5. Use of the gene fadT according to claim 1, the enzyme FadT according to claim 2, the recombinant plasmid according to claim 3 or the recombinant microorganism according to claim 4 for controlling plant black rot or for producing a product for controlling plant black rot.
6. Use of the gene fadT according to claim 1, the enzyme FadT according to claim 2, the recombinant plasmid according to claim 3 or the recombinant microorganism according to claim 4 for degrading quorum sensing signal molecules DSF or for controlling quorum sensing signal molecules DSF-mediated pathogenic bacteria.
7. A medicine for preventing and treating plant black rot, which is characterized in that the medicine contains a recombinant plasmid of the fadT sequence of the gene or the fragment thereof in the claim 1, or contains a recombinant bacterium obtained by transforming the recombinant plasmid.
8. A medicament for controlling black rot in plants, comprising the enzyme FadT according to claim 2, or a recombinant plasmid comprising an open reading frame encoding the enzyme FadT protein, or a recombinant bacterium expressing the enzyme FadT.
9. The medicament of claim 7 or 8, wherein the recombinant plasmid is pGEX-fadT and the recombinant bacterium is Escherichia coil BL 21/pGEX-fadT.
10. A method for preventing and controlling plant black rot is characterized by comprising the following steps:
(1) constructing a recombinant bacterium containing a fadT sequence of the gene of claim 1 or a fragment thereof, or a recombinant bacterium capable of expressing the enzyme FadT of claim 2;
(2) inoculating the recombinant strain in the step (1) into plants.
Technical Field
The present invention belongs to the field of gene engineering technology. More particularly, relates to the application of the gene fadT in the aspect of preventing and controlling plant black rot.
Background
Xanthomonas campestris wild rape pathogenic variants (Xanthomonas campestris pv. campestris, Xcc) cause black rot in plants. Black rot is a worldwide plant disease that affects mainly the aerial parts of plants at any growth stage, causing severe yield losses. All cruciferous vegetables, including pakchoi, broccoli, chinese cabbage, radish, cauliflower, kale, asparagus, mustard and turnip, are susceptible to infection by Xcc to black rot.
Xcc expresses pathogenic genes by accumulation of a Diffusible Signal Factor (DSF), which is also called quorum sensing Signal molecule DSF, causing the occurrence of black rot in plants. The signals of DSF family members are closely related to extracellular enzyme formation, extracellular polysaccharide formation and antibiotic resistance of pathogens of bacteria. DSF is a fatty acid molecule with the chemical structure cis 11-methyl-2-dodecenoic acid, which is representative of a family of widely conserved quorum sensing signals involved in regulating the production of virulence factors by various gram-negative bacteria. DSF is found not only in all Xanthomonas (Xanthomonas sp.), but also in a wide range of Pseudomonas aeruginosa (Pseudomonas aeruginosa), Burkholderia sp, and various types of marine bacteria. However, the current prevention and treatment means for black rot are mainly chemical pesticides and antibiotics, and the use of the chemical pesticides and the antibiotics is easy to cause pathogenic bacteria to generate drug resistance, even multiple drug resistance. In addition, improper use or abuse of pesticides can cause a series of safety problems such as pesticide residue and environmental pollution, ecological balance destruction, food safety and human health threats. Therefore, the development of a method for preventing and treating the black rot of the plant, which is efficient, environment-friendly and free of drug resistance, has important significance and ecological benefit.
The invention patent ZL 201810997336.8 discloses a cuppriavium (cuprianidus pinatubonensis) strain HN-2 which can degrade DSF and prevent and control plant black rot. However, the mechanism of control is not clear. In addition, the biocontrol bacteria have the degradation problem, a bacteria bank needs to be continuously filled, a large amount of work is needed, and the biocontrol bacteria can possibly have negative effects on plants, the environment and the like while preventing and controlling the bacteria. Therefore, for the means for quenching the signal molecules and preventing and treating the related pathogenic bacteria, the degrading bacteria and the degrading enzyme are adopted, and compared with the degrading bacteria, the degrading enzyme has the advantages that negative effects or uncontrollable factors possibly existing on plants or environment caused by the degrading bacteria cannot be generated, and the method can be realized by using modern technical means such as simpler genetic engineering and the like. For example, the prior patent (application No. 201910731498.1) discloses DSF quorum sensing signal degradation genes dig1, dig2, dig3 and dig4, which can be widely used in the degradation of DSF family signals. However, the patent shows that the screened degradation genes only partially inhibit the pathogenicity of the strain after being expressed in pathogenic bacteria, and are not genes which can effectively degrade the DSF. Therefore, it is important to find genes and degrading enzymes which have better inhibitory effect on pathogenic bacteria and can efficiently degrade DSF.
Disclosure of Invention
The invention aims to solve the technical problem of realizing biological control of plant black rot by using a quorum sensing signal molecule DSF degradation gene fadT/enzyme. In the invention, the recombinant bacterium introduced with the fadT target gene coexists with plants, and the occurrence of plant black rot caused by the Xcc bacterium can be effectively prevented.
The invention aims to provide a quorum sensing signal molecule DSF degradation gene fadT.
Another object of the present invention is to provide a degrading enzyme FadT encoded by the quorum sensing signal molecule DSF degrading gene fadT.
Another object of the present invention is to provide a recombinant plasmid.
Another object of the present invention is to provide a recombinant microorganism.
The invention also aims to provide application of a quorum sensing signal molecule DSF degradation gene fadT, or a degradation enzyme FadT coded by the quorum sensing signal molecule DSF degradation gene, or a recombinant plasmid containing a gene fadT sequence or a fragment of the gene fadT sequence, or application of a recombinant microorganism obtained by transforming the recombinant plasmid in the aspect of preventing and treating plant black rot or in the aspect of preparing a product for preventing and treating plant black rot.
The invention also aims to provide application of the quorum sensing signal molecule DSF degradation gene fadT, or the encoded degradation enzyme FadT, or the recombinant plasmid containing the gene fadT sequence or the fragment thereof, or the recombinant microorganism obtained by transforming the recombinant plasmid in degrading the quorum sensing signal molecule DSF or preventing and treating quorum sensing signal molecule DSF-mediated pathogenic bacteria.
The invention also aims to provide a medicament for preventing and treating the black rot of plants.
Another object of the present invention is to provide a method for controlling black rot in plants.
The above purpose of the invention is realized by the following technical scheme:
the invention uses a whole genome sequencing method, searches genes which can possibly exist in the bacterial strain HN-2 and can degrade DSF and corresponding proteins in the fatty acid metabolic pathway related genes by carrying out genome feature analysis and gene function gene annotation on the bacterial strain HN-2 of cupriasis (Cupriavidus pinatubonensis); a gene knockout method is utilized to construct related gene mutants for verification, and a gene fadT for degrading DSF and an enzyme FadT for degrading DSF with high efficiency are found. Therefore, the following technical solutions are to be protected:
a quorum sensing signal molecule DSF degradation gene fadT has a nucleotide sequence shown in SEQ ID NO. 1.
The amino acid sequence of the degrading enzyme FadT coded by the degrading gene fadT is shown in SEQ ID NO. 2.
A recombinant plasmid comprising the above-mentioned gene fadT or a fragment thereof, or capable of expressing the above-mentioned degrading enzyme FadT.
A recombinant microorganism capable of expressing the above-mentioned gene fadT or a fragment thereof or capable of expressing the above-mentioned degrading enzyme FadT.
The gene fadT, the enzyme FadT, the recombinant plasmid or the recombinant microorganism are applied to the prevention and treatment of plant black rot or the preparation of products for preventing and treating plant black rot.
The gene fadT, the enzyme FadT, the recombinant plasmid or the recombinant microorganism are applied to degrading quorum sensing signal molecules DSF or preventing and treating quorum sensing signal molecules DSF-mediated pathogenic bacteria.
A medicine for preventing and treating plant black rot contains the recombinant plasmid of the fadT sequence of the gene or the fragment thereof, or contains the recombinant bacterium obtained by transforming the recombinant plasmid.
A medicine for preventing and treating plant black rot contains the said enzyme FadT, or contains the recombinant plasmid of open reading frame for coding the enzyme FadT protein, or contains the recombinant bacteria for expressing the enzyme FadT.
Wherein, preferably, the recombinant plasmid is pGEX-fadT, and the recombinant bacterium is Escherichia coil BL 21/pGEX-fadT.
A method of controlling black rot in a plant, comprising the steps of:
(1) constructing a recombinant bacterium containing the gene fadT sequence or a fragment thereof, or a recombinant bacterium capable of expressing the enzyme FadT;
(2) inoculating the recombinant strain in the step (1) into plants.
Wherein, preferably, the recombinant bacterium is Escherichia coil BL 21/pGEX-fadT;
wherein, preferably, the recombinant bacterium is in a bacterium liquid state, and the concentration is 5-7 multiplied by 108CFU/mL。
The invention has the following beneficial effects:
(1) after the coding gene fadT is expressed in pathogenic bacteria Xcc, the pathogenicity of the pathogenic bacteria Xcc can be obviously reduced, and the coding gene fadT has obvious control effect on black rot caused by the Xcc.
(2) Chemical drugs (reducing the use of pesticides) are not needed for preventing and treating the black rot of the plants, the plants cannot generate drug resistance, and the method is environment-friendly.
Drawings
FIG. 1 shows the results of the biological control effect of gene fadT on radish black rot; wherein, the picture (A) is that radish is inoculated by xanthomonas campestris Xcc; (B) the figure shows that radish is inoculated with xanthomonas campestris Xcc + wild type HN-2; (C) FIG. shows radish inoculated with Xanthomonas campestris Xcc + mutant Δ fadT; (D) the figure shows radish inoculated with sterile water.
FIG. 2 shows the expression and purification of the enzyme FadT; wherein M represents Marker, 1 represents the recombinant protein FadT obtained without adding IPTG inducer, and 7 represents the supernatant obtained with adding IPTG inducer.
FIG. 3 shows the results of the bio-control effect of DSF-degrading enzyme FadT on radish black rot; wherein, the picture (A) is that radish is inoculated by xanthomonas campestris Xcc; (B) the figure shows that radish is inoculated with xanthomonas campestris Xcc + wild type HN-2; (C) FIG. shows radish inoculated with Xanthomonas campestris Xcc + mutant Δ fadT; (D) FIG. shows the radish inoculated with Xanthomonas campestris Xcc + alexin Δ fadT (fadT); (E) FIG. 2 shows radish inoculated with Xanthomonas campestris Xcc (fadT) transformed with fadT gene; (F) the figure shows radish inoculated with sterile water.
FIG. 4 shows the results of the bio-control effect of DSF-degrading enzyme FadT on cabbage black rot; wherein, the picture (A) is that Chinese cabbage is inoculated by xanthomonas campestris Xcc; (B) FIG. shows that Chinese cabbage is inoculated with Xanthomonas campestris Xcc + wild type HN-2; (C) FIG. shows the Chinese cabbage inoculated with Xanthomonas campestris Xcc + mutant Δ fadT; (D) FIG. shows the dot complement Δ fadT (fadT) of Xanthomonas campestris Xcc + fadT gene for Chinese cabbage; (E) FIG. shows that Chinese cabbage is inoculated with Xanthomonas campestris Xcc (fadT) into which fadT gene has been transferred; (F) the figure shows Chinese cabbage inoculated with sterile water.
FIG. 5 is a result of measurement of the biocontrol effect of DSF-degrading enzyme FadT on Shanghai green black rot; wherein, the picture (A) is that the Shanghai green is inoculated with xanthomonas campestris Xcc; (B) the figure shows that the Shanghai green is inoculated with xanthomonas campestris Xcc + wild type HN-2; (C) the graph shows that the Shanghai green is inoculated with xanthomonas campestris Xcc + mutant delta fadT; (D) FIG. shows the inoculation of the Haematococcus Shanghai with Xanthomonas campestris Xcc + complement Δ fadT (fadT); (E) FIG. 2 shows that the Haicha japonica was inoculated with Xanthomonas campestris Xcc (fadT) into which fadT gene was transferred; (F) the figure shows the inoculation of the Shanghai green with sterile water.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The cuppriavium (cupriaviridus pinatubonensis) strain HN-2 used in the following examples was deposited with the Guangdong province collection of microorganisms under accession number: GDMCC 60432(ZL 201810997336.8).
EXAMPLE 1 Whole genome sequencing analysis of the cuprum greedy (Cupriavidus pinatubonensis) Strain HN-2
In this example, a whole genome sequencing method was used to analyze DSF-degrading genes that may be present in strain HN-2 by performing genome characterization and functional gene annotation on strain HN-2. The specific experimental methods and results are as follows:
(1) genomic characterization of Strain HN-2
In order to understand the gene function and structure of the cuprum bulimia (Cupriavidus pinatubonensis) strain HN-2, whole genome sequencing was performed on the strain HN-2. According to the whole genome sequencing result, the total length of the genome of the strain HN-2 is 7,548,664bp, wherein N50 is 10,533 bp. There are 7,101 coding genes, the total length of the coding gene is 6,556,812bp, and the total length of the coding gene accounts for 86.86% of the total length of the genome. The total length of the interspersed repetitive sequences is 6,886bp, and accounts for 0.0912% of the total length of the genome. The number of gene islands and prophages was 14 and 4, respectively.
(2) KEGG database annotation
KEGG is a comprehensive database that is broadly divided into three major categories, systematic, genomic and chemical information. Each database contains a large amount of useful information. The GENES database stores genomic information, including complete and partially sequenced genomic sequences; the PATHWAY database stores higher level functional information including information about cellular biochemical processes such as metabolic PATHWAYs, signaling, membrane transport, cell cycle, and homeologously conserved sub-PATHWAYs.
Analysis and annotation are carried out on the whole genome sequencing result of the strain HN-2 by KEGG, and according to a metabolic pathway classification diagram, 1150 overview genes, 161 lipid metabolism genes, 48 polysaccharide biosynthesis and metabolism genes, 65 terpene and polyketide metabolism genes, 356 carbohydrate metabolism genes, 408 amino acid metabolism genes and 223 heterotypic biomass degradation and metabolism genes are known.
Example 2 obtaining of DSF-degrading Gene fadT
In this example, based on the results of genome feature analysis and functional gene annotation of the strain HN-2 of example 1, possible gene mutants were constructed by gene knockout to perform phenotypic validation, so as to accurately find efficient degradative enzymes. The specific experimental methods and results are as follows:
(1) experimental methods
Based on the functional annotation of the KEGG database in example 1, we selected 8 genes for further study; four of these genes encode long-chain acyl-CoA synthetase (HN.2_ GM000231, HN.2_ GM003041, HN.2_ GM005970, HN.2_ GM000743 are annotated with K01897 for the KO system, which is also a homologue of rpfB), three 3-hydroxyacyl-CoA dehydrogenases (HN.2_ GM000404, HN.2_ GM003236, HN.2_ GM004234, which is annotated with K07516 for the KO system), and one butyryl-CoA dehydrogenase (HN.2_ GM 004237).
Then, wild-type HN-2 was used as a parent to construct a wild-type HN-2 deletion mutant, and finally 4 of the 8 genes were selected to construct 4 mutants, i.e., Δ 0404, Δ 4237, Δ 3041 and Δ 0743, respectively.
These four mutants were validated and the experiment was divided into four treatments: uniformly inoculating bacterial liquids with the same concentration on the white radish slices, and inoculating the bacterial liquids with sterile water as blank controls, wherein the bacterial liquids are Xcc respectively; xcc + wild type HN-2; the Xcc + mutant Δ 0404/Δ 4237/Δ 3041/Δ 0743. Finally, the mixture is placed in an artificial incubator with the temperature of 28 ℃, the humidity of 60 percent and the illumination of 0 for 2 days, and then photographed and recorded. Meanwhile, a DSF degradation experiment is carried out, a wild type HN-2 is taken as a parent, wild type HN-2 deletion mutants delta 0404/delta 4237/delta 3041/delta 0743 are respectively constructed, and a complement is used for carrying out a complementary experiment to further verify whether the 4 genes influence the degradation of the DSF, and the experiment is divided into 4 treatment groups: blank control group CK, wild type HN-2 group, mutant group delta 0404/delta 4237/delta 3041/delta 0743 and complement group; 2mM of DSF is added into MSM basal medium as a sole carbon source, after 24h of culture, the DSF degradation conditions of a blank control group CK, a wild type HN-2 group, a mutant group delta 0404/delta 4237/delta 3041/delta 0743 and a complement group are respectively measured by using high performance liquid chromatography.
(3) Results of the experiment
The nucleotide sequence of the DSF degradation gene fadT is shown in SEQ ID NO. 1.
Finally, among these four mutants, GM004237 was identified as a gene essential for HN-2 to have DSF-degrading ability and black rot control ability, and was designated fadT. Degradation experiment results show that the wild HN-2 group completely degrades DSF after 24 hours; neither the mutant delta fadT group nor the blank control group CK can grow in an MSM culture medium which takes DSF as a sole carbon source, and the ability of degrading the DSF is lost, but the complementary delta fadT (fadT) group recovers the phenotype after complementing gene fadT, and can degrade more than 90% of DSF after being cultured for 24 h; the results show that: the gene fadT has important influence on the degradation of DSF.
The biological activity determination effect is shown in figure 1: when the sliced radish is singly inoculated with the Xcc bacterial solution, the typical black rot symptom appears; when the carrot slices are inoculated with Xcc + wild type HN-2, no black rot symptom appears; when the carrot slices are inoculated with the Xcc + mutant delta fadT, black rot symptoms also appear; the radish in the blank control group had no black rot symptoms. The results show that the strain HN-2 can well inhibit the Xcc pathogenicity mediated by the DSF, while the fadT deletion mutant delta fadT has no inhibition effect on the Xcc pathogenicity mediated by the DSF, and the anaplerotic delta fadT (fadT) can restore the Xcc pathogenicity mediated by the DSF. Therefore, the gene fadT is a gene necessary for HN-2 to degrade DSF.
Example 3 expression and purification of DSF-degrading enzyme FadT
(1) Experimental methods
According to the results of whole genome sequencing analysis of the H.greedy (Cupriavidus pinatubonensis) strain HN-2 of example 1, the gene fadT is presumed to encode a butyryl-CoA dehydrogenase.
In order to obtain purer and higher yields of degradative enzyme, the gene fadT codon was first optimized. The DNA fragment which is subjected to codon optimization and codes the DSF degrading enzyme FadT is amplified through PCR, and is subcloned to an expression vector pGEX-6p-1 to obtain a recombinant plasmid pGEX-fadT, and the recombinant plasmid pGEX-fadT is transferred into Escherichia coli BL21 to obtain a recombinant bacterium Escherichia coil BL 21/pGEX-fadT. The cells were inoculated into LB medium (containing ampicillin at a final concentration of 100. mu.g/mL) and cultured at 37 ℃ and OD600When about 0.6 was reached, IPTG inducer (final concentration 1mM) was added and the cells were incubated at 18 ℃ for 24 hours with no IPTG inducer added as a control. The suspension was centrifuged and the supernatant discarded, and the pellet was resuspended in Phosphate Buffered Saline (PBS). After ultrasonication, the solution is filtered by a 0.45 mu m microporous filter (micropore), then the filtrate is purified by a protein purifier, the purified and recovered recombinant protein FadT-GST is subjected to SDS-PAGE electrophoresis, and finally the size and the purity of the recombinant protein FadT-GST are analyzed by an electrophoresis result.
(2) Results of the experiment
The amino acid sequence of the enzyme FadT is shown in SEQ ID NO. 2.
The expression and purification results of the FadT enzyme are shown in FIG. 2, and the degradation enzyme FadT is successfully expressed in lane 7, which shows that the degradation enzyme FadT can be successfully prepared by using E.coil BL21/pGEX-FadT under the condition of IPTG inducer. And a single, clear, high-purity and 93kDa recombinant protein FadT-GST band is amplified.
Example 4 biocontrol effect of DSF-degrading enzyme FadT on Black rot of radish, cabbage and Shanghai green
(1) Experimental methods
Planting healthy radish, cabbage and Shanghai green seed in a flowerpot, arranging an insect-proof net cover, watering regularly, and planting healthily for 30 days without applying pesticide. With a concentration of 6X 108And (3) respectively treating the radish, the Chinese cabbage and the Shanghai green seedling leaves by using CFU/mL bacterial solution or sterile water. The experiment comprises 6 treatments, wherein sterile water inoculation is used as a blank control, and the treated bacteria liquid is Xcc respectively; xcc + wild type HN-2; xcc + mutant Δ fadT; xcc + complement Δ fadt (fadt); xcc + Xanthomonas campestris Xcc (F. campestris) into which fadT gene has been transferred: (fadT). The plant leaves are continuously planted in the pot plants after being inoculated, symptoms are recorded every day, radishes and cabbages are respectively harvested and photographed after 8 days of inoculation, and Shanghai green is harvested and photographed after 10 days of inoculation. During the experiment, the day and night temperature, light period and humidity were the same as the ambient natural environment.
(2) Results of the experiment
FIG. 3 shows the results of the enzyme FadT for the biocontrol effect of radish black rot; the result of the measurement of the biocontrol effect of the enzyme FadT on the black rot of Chinese cabbage is shown in FIG. 4; the results of the measurement of the biocontrol effect of the enzyme FadT on Shanghai black rot are shown in FIG. 5. The leaf symptoms of three plants can be seen, the leaves inoculated with Xanthomonas campestris Xcc and the Xcc + mutant delta fadT mixed bacterial liquid alone are diseased, yellow spots are developed inwards in a V shape, and the radish black rot symptom is obvious, which indicates that the pathogenicity of the Xcc cannot be inhibited; the leaves inoculated with the Xcc + wild type HN-2 mixed bacterial liquid, the Xcc + alexin delta fadT (fadT) mixed bacterial liquid and the Xanthomonas campestris Xcc (fadT) with the fadT gene do not have black rot symptoms, and the pathogenicity of the Xcc is obviously inhibited.
The above results show that: the enzyme FadT has obvious inhibition effect on the pathogenicity of Xanthomonas campestris Xcc and has obvious biological control effect on black rot caused by Xcc.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> southern China university of agriculture
<120> quorum sensing signal molecule DSF degradation gene fadT, encoded degradation enzyme FadT and application thereof
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Asp Asn Ile Val His Leu Val Leu Ala Lys Ile Pro Gly Pro Asp Gly
210 215 220
Lys Leu Val Pro Gly Val Lys Gly Ile Ser Leu Phe Ile Val Pro Lys
225 230 235 240
Tyr Leu Val Asn Ala Asp Gly Ser Leu Gly Glu His Asn Asp Val Val
245 250 255
Leu Ala Gly Leu Asn His Lys Met Gly Tyr Arg Gly Thr Thr Asn Cys
260 265 270
Leu Leu Asn Phe Gly Glu Gly Met Lys Tyr Lys Pro Ala Gly Lys Ala
275 280 285
Gly Ala Ile Gly Tyr Leu Val Gly Glu Pro Gly Lys Gly Leu Ala Cys
290 295 300
Met Phe His Met Met Asn Glu Ala Arg Ile Gly Val Gly Leu Gly Ala
305 310 315 320
Val Met Leu Gly Tyr Thr Gly Tyr Leu His Ala Val Asp Tyr Ala Arg
325 330 335
Asn Arg Pro Gln Gly Arg Pro Val Gly Pro Gly Gly Lys Asp Pro Ala
340 345 350
Ser Pro Gln Val Lys Leu Val Glu His Ala Asp Ile Arg Arg Met Leu
355 360 365
Leu Ala Gln Lys Ser Tyr Val Glu Gly Gly Leu Ala Leu Asn Leu Tyr
370 375 380
Cys Ala Lys Leu Val Asp Glu Glu Arg Ala Ala Ser Ala Asp Ala Val
385 390 395 400
Lys His Glu Gln Leu Ser Leu Leu Leu Asp Ile Leu Thr Pro Ile Ala
405 410 415
Lys Ser Trp Pro Ser Gln Trp Cys Leu Glu Ala Asn Asn Leu Ala Ile
420 425 430
Gln Val His Gly Gly Tyr Gly Tyr Thr Arg Glu Tyr Asn Val Glu Gln
435 440 445
Phe Tyr Arg Asp Asn Arg Leu Asn Pro Ile His Glu Gly Thr His Gly
450 455 460
Ile Gln Gly Leu Asp Leu Leu Gly Arg Lys Val Val Met Lys Asp Gly
465 470 475 480
Ala Ala Phe Arg Leu Leu Gly Glu Arg Val Arg Glu Thr Cys Glu Cys
485 490 495
Ala Leu Ser Ser Gly Asp Lys Glu Leu Gly Ser Gln Ala Arg Ala Leu
500 505 510
Gly Ala Ala Ala Thr Arg Leu Ala Glu Val Thr Lys Val Leu Trp Ser
515 520 525
Ala Gly Asp Ala Asn Val Thr Leu Ala Asn Ala Ser Ile Tyr Leu Glu
530 535 540
Ala Phe Gly His Val Val Val Ala Trp Ile Trp Leu Glu Gln Ala Leu
545 550 555 560
Val Ala Gln Ala Ala Leu Ala Gly Gly Ala Ser Gly Glu Asp Glu Thr
565 570 575
Phe Tyr Arg Gly Lys Leu Ala Ala Ala Thr Tyr Phe Ser Arg Trp Glu
580 585 590
Leu Pro Lys Val Gly Pro Gln Leu Glu Leu Leu Ala Thr Leu Asp Arg
595 600 605
Thr Thr Leu Asp Met Gln Asp Ala Trp Phe
610 615