阅读说明：本技术 一种终极腐霉菌中诱导HR的Elicitin类基因及其表达载体的应用 (Elicitin gene for inducing HR in pythium ultimum and application of expression vector thereof ) 是由 窦道龙 杨坤 景茂峰 李佳露 董小华 于 2020-12-25 设计创作，主要内容包括：本发明属于基因工程技术领域,公开了一种终极腐霉菌中诱导HR的Elicitin类基因及其表达载体的应用。本发明鉴定到来自终极腐霉菌(Pythium ultimum)的三个Elicitin类基因PYOD6、PYOD7和PYOD20,所述PYOD6、PYOD7和PYOD20基因的核苷酸序列依次分别如SEQ ID NO.1—3所示。本发明从终极腐霉菌中鉴定到三个Elicitin类基因能够产生显著的HR反应,激发植物免疫。(The invention belongs to the technical field of genetic engineering, and discloses an Elicitin gene for inducing HR in pythium ultimum and application of an expression vector thereof. The invention identifies three Elicitin genes PYOD6, PYOD7 and PYOD20 from Pythium ultimum, wherein the nucleotide sequences of the PYOD6, the PYOD7 and the PYOD20 are respectively shown in SEQ ID NO. 1-3 in sequence. According to the invention, three elicidin genes identified from pythium ultimum can generate obvious HR reaction to stimulate plant immunity.)

1. An Elicitin gene for inducing HR in pythium ultimum is at least one of PYOD6, PYOD7 and PYOD20, and nucleotide sequences of PYOD6, PYOD7 and PYOD20 are sequentially and respectively shown in SEQ ID No. 1-3.

2. The Pythium ultimum immunity inducing protein coded by the elicidin gene of claim 1, wherein the amino acid sequences of the protein are respectively shown as SEQ ID NO 4-6 in sequence.

3. An expression cassette, a recombinant expression vector, a transgenic cell line or a transgenic recombinant bacterium containing the elicidin gene of claim 1.

4. The recombinant expression vector according to claim 3, wherein the starting vector of the recombinant expression vector is the expression vector PBIN-PLUS.

5. A plant immunity inducer, which comprises the Pythium ultimum immunity inducer protein of claim 2 or a fermentation broth of the Pythium ultimum immunity inducer protein of claim 2.

6. Use of the elicidin-like gene according to claim 1, the protein according to claim 2 or the recombinant expression vector according to claim 3 or 4 for inducing necrosis in a plant.

7. Use of the elicidin gene of claim 1, the protein of claim 2 or the recombinant expression vector of claim 3 or 4 for the development of a plant immune response-inducing agent.

8. Use of the elicidin gene of claim 1 in breeding disease resistant crop varieties.

9. A method for stimulating plant immunity, which is characterized in that the Elicitin gene of claim 1 or the recombinant expression vector of claim 3 or 4 is introduced into a plant to stimulate plant immune response and improve plant resistance.

10. A method for cultivating disease-resistant crop varieties, which is characterized in that the Elicitin gene of claim 1 or the recombinant expression vector of claim 3 or 4 is introduced into plants, positive transformation plants are obtained through resistance screening, and disease-resistant crop varieties are obtained.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to an Elicitin gene for inducing HR in pythium ultimum and application of an expression vector thereof.

Background

During the long-term co-evolution of plants and pathogens, pathogens have evolved a number of host-attacking weapons, such as effector proteins (effectors), in order to better infect the plant. Pathogenic bacteria secrete these effector proteins into plants in order to promote their better infestation of the host plant, however, accompanying the co-evolution of plants, some effector proteins are recognized by the genes involved in the regulation of disease resistance by the plants themselves. Recognition of pathogens often triggers a local resistance response, called Hypersensitivity (HR), characterized by rapid cell death at the site of infection. This apparent HR response is also an important signal for the development of plant immunity for the study of plant interactions with pathogenic bacteria.

Pythium ultimum is a pathogenic bacterium, and can widely infect more than 150 economic plants such as soybean, kidney bean, pea, sweet potato, pine seedling, coffee, apple, orange, peach, cotton, chrysanthemum, dahlia, pumpkin, watermelon, sugarcane, alfalfa, tomato and the like to cause various diseases such as seedling withering, damping-off, root rot, foot rot, withering and the like. We obtained three genes derived from Pythium ultimum which are capable of triggering plant allergic reactions (HR) by mass screening. All three genes contain an elicidin domain and are typical immune elicitors. The HR is stimulated, the plant immunity is triggered, the plant disease resistance response is induced, and the research and development of the plant resistance inducer are facilitated. The powerful functions of the three genes can be used as candidate genes for developing plant resistance inducers.

Disclosure of Invention

The invention aims to provide an Elicitin gene for inducing HR in pythium ultimum and application of an expression vector thereof. The three immune elicitor genes identified by the invention can be used as theoretical knowledge supplement for developing immune elicitors, and provide theoretical guidance for preventing and controlling crop diseases and insect pests.

The purpose of the invention can be realized by the following technical scheme:

the invention identifies three Elicitin genes PYOD6, PYOD7 and PYOD20 from Pythium ultimum, wherein the nucleotide sequences of the PYOD6, the PYOD7 and the PYOD20 are respectively shown in SEQ ID NO. 1-3 in sequence. The proteins coded by the three Elicitin genes are PYOD6, PYOD7 and PYOD20, and the amino acid sequences are respectively shown in SEQ ID NO. 4-6 in sequence.

An expression cassette, a recombinant expression vector, a transgenic cell line or a transgenic recombinant bacterium containing the Elicitin gene.

The recombinant expression vectors containing the Elicitin genes comprise PBIN-PLUS (PYOD 6), PBIN-PLUS (PYOD 7) and PBIN-PLUS (PYOD 20).

Constructing recombinant expression vectors PBIN-PLUS of the Elicitin genes PYOD6, PYOD7 and PYOD20, namely PYOD6, PBIN-PLUS, PYOD7 and PBIN-PLUS, PYOD 20. The plant expression vector PBIN-PLUS is used as a starting vector, and the genes PYOD6, PYOD7 and PYOD20 are respectively inserted into the Sma1 enzyme cutting site of the PBIN-PLUS to obtain the gene.

The Elicitin gene and the expression vector thereof can stimulate plant immune response, provide a knowledge base for developing immune elicitors and provide theoretical guidance for preventing and controlling crop diseases and insect pests.

A plant immunity inducer, which contains the pythium ultimum immunity inducer protein or the fermentation liquor of the pythium ultimum immunity inducer protein.

The use of the elicidin gene, the protein or the recombinant expression vector in inducing necrosis in plants.

The Elicitin gene, the protein or the recombinant expression vector are applied to the development of plant immunity inducer.

The application of the Elicitin gene in breeding disease-resistant crop varieties.

A method for stimulating plant immunity is characterized in that the Elicitin gene or the recombinant expression vector is introduced into a plant, so that the plant immune response can be stimulated, and the plant resistance can be improved. The gene or the recombinant expression vector is introduced into crop plants, and positive transformation plants are obtained through resistance screening to obtain disease-resistant crop varieties.

The invention has the beneficial effects that:

the invention identifies three Elicitin genes from pythium ultimum, and inserts the three Elicitin genes into an expression vector PBIN-PLUS. The vector is introduced into a plant, so that a remarkable HR reaction can be generated, and the immunity of the plant is stimulated. The three PYOD6, PYOD7 and PYOD20 genes can be used as potential candidates for developing plant immune inducer and are a theoretical support for preventing and controlling plant diseases.

Drawings

FIG. 1 is a table chart and a quantitative calibration bar chart of HR degree of PYOD6, PYOD7 and PYOD20 genes inducing HR effect in plants.

Wherein A is a phenotype graph of significant effect of inducing plant HR, the first left is treated by a negative control GFP gene, the second left is treated by a positive control INF1 gene, and the right three are respectively treated by PYOD6, PYOD7 and PYOD20 genes; it can be clearly seen that three genes PYOD6, PYOD7 and PYOD20 stimulate HR reaction. B is a quantitative calibration bar chart of HR levels, and the numbers corresponding to FIG. 1A clearly show that the level of HR elicited by the three genes was similar to that of the positive control, significantly higher than that of the negative control (Student's t test:. about. P < 0.0001).

Detailed Description

The present invention will be described in detail with reference to specific examples. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Example 13 amplification and sequencing of Elicitin-like genes

1. Experimental strains

The test strain Pythium ultimum is preserved in 10% V8 solid culture medium in the interacting laboratory of plant disease system and phytophthora of Nanjing university of agriculture at 10 deg.c.

2. Preparation of test Nicotiana benthamiana seedlings

Nicotiana benthamiana (Nicotiana benthamiana) is sown in a plastic flowerpot (d is 10cm) filled with vermiculite (2-4mm), and is placed in a greenhouse with the light intensity of 14 h/10 h dark. The plant is grown for 7 days, after two true leaves grow, the plant is transplanted to vermiculite black soil with the volume ratio of 5: 1 for 30 days, taking the same leaf blade with 3, 4 and 5 leaf positions as an expression experiment gene, and continuously observing whether the leaf blade phenotype has obvious HR or not for 7 days.

3. Extracting Pythium ultimum (Pythium ultimum) RNA and obtaining cDNA of target gene

Collecting pythium ultimum hyphae, quickly freezing with liquid nitrogen, putting into a mortar, cooling and grinding. And extracting and purifying the pythium ultimum total RNA according to a method recommended by a total RNA extraction and purification kit of Tiangen company. Referring to the method provided by the RNA reverse transcription kit of Novozam, oligo (dT) is used as a primer for reverse transcription synthesis of the cD NA. PCR was performed using the above-described cD N A as a template and the corresponding upstream and downstream primers (see Table 1 for primer sequences).

TABLE 1 primer sequences

name	sequence
		PBIN-PLUS-Sma1-PYOD6-F	CGATAGGGTACCCCCATGTACACCAAGTTCGCC
PBIN-PLUS-Sma1-PYOD6-R	GGATCCGTCGACCCCGCAAGCTGGCTTGACGGT
		PBIN-PLUS-Sma1-PYOD7-F	CGATAGGGTACCCCCATGTACACCAAGTTCGCC
PBIN-PLUS-Sma1-PYOD7-R	GGATCCGTCGACCCCGCAAGCTGGCTTGACGGT
		PBIN-PLUS-Sma1-PYOD20-F	CGATAGGGTACCCCCATGAAGTTCCAAGCCGTC
PBIN-PLUS-Sma1-PYOD20-R	GGATCCGTCGACCCCGAAGCAGCTGCCCTCGAA

The PCR reaction system is as follows: 2.5. mu.L of 10 XPCR reaction buffer; 1.5 μ L1.5mM MgCl 2; 0.5. mu.L of 2.5 mM dNTPs; 0.25. mu.L Taq DNA polymerase (5.0U/. mu.L); 0.5. mu.L of primer; 0.5 μ L template; make up to 25. mu.L with sterile water.

The reaction procedure is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 1:30min, and 35 cycles; extending for 10min at 72 ℃; storing at 4 ℃.

And (3) carrying out agarose gel electrophoresis and recovery on the PCR amplification product, and obtaining a sequence which is the nucleotide sequence of the 3 Elicitin genes after sequencing and no error. The Elicitin genes comprise three Elicitin genes PYOD6, PYOD7 and PYOD20 from Pythium ultimum respectively, and the nucleotide sequences of the PYOD6, PYOD7 and PYOD20 genes are sequentially and respectively shown in SEQ ID NO. 1-3. The amino acid sequences of the proteins coded by the three Elicitin genes are respectively shown in SEQ ID NO. 4-6 in sequence. Subsequent research finds that the plant growth regulator has strong functions of inducing plants to generate necrosis and active oxygen accumulation.

Example 2 construction of expression vectors for PBIN-PLUS GFP, PBIN-PLUS INF1, PBIN-PLUS PYOD6, PBIN-PLUS PYOD7 and PBIN-PLUS PYOD20

The empty vector plasmid of PBIN-PLUS (BIOVECTOR China plasmid vector strain cell line gene collection center) was digested with SmaI. The cDNA and the vector were ligated using a homologous recombinase from Novowed.

The reaction system is as follows: 2 μ L of 10 × CE II reaction buffer; mu.L of the PCR product; 2 mu L of the empty vector after enzyme digestion; 1 μ L of homologous recombinase; make up to 10. mu.L sterile water.

The reaction procedure is as follows: the reaction was carried out at 37 ℃ for 30 min. And transforming the ligation product into escherichia coli DH5 alpha, screening transformants on a Kan resistance culture medium, and selecting positive clones to extract plasmids for colony PCR identification. The identified positive clones were sequenced and correct expression vectors of PBIN-PLUS GFP, PBIN-PLUS INF1, PBIN-PLUS PYOD6, PBIN-PLUS PYOD7 and PBIN-PLUS PYOD20 were obtained.

EXAMPLE 3 transformation of Agrobacterium with expression vector and functional verification

1. Obtaining agrobacterium-infected cells

Carrying out streak culture on agrobacterium GV3101 strain on an LB solid culture medium plate, and carrying out inverted culture for 18-20h at the temperature of 28 ℃; placing the single colony in LB liquid culture medium (containing 100mg/L Rif), and performing shaking culture at 28 ℃ for 18-20 h; adding 100 times of the volume of the antibiotic-free bacterial liquid into the bacterial liquid, and performing shaking culture at 28 ℃ to obtain OD600 of 0.3-0.5; cooling on ice, centrifuging at 4000r/min at 4 deg.C for 5min, and removing supernatant; adding 20mmol/L CaCl₂Suspending the precipitate with the solution, centrifuging at 4000r/min at 4 deg.C for 5min, and removing the supernatant; adding 20mmol/L CaCl₂The solution again suspended the pellet for further use.

2. GFP in PBIN-PLUS, INF1 in PBIN-PLUS, PYOD6 in PBIN-PLUS, PYOD7 in PBIN-PLUS, PYOD20 in PBIN-PLUS

Transforming Agrobacterium cells with expression vectors of PBIN-PLUS GFP, PBIN-PLUS INF1, PBIN-PLUS PYOD6, PBIN-PLUS PYOD7 and PBIN-PLUS PYOD20 by electrotransformation; the cuvette was rinsed three times with 70% alcohol and then completely air dried. 100ng of the expression vector was added to the Agrobacterium-infected state and left on ice for 30 min. The agrobacterium-infected cells are transferred to an electric shock cup, and electric shock transformation is carried out by using the voltage of 2.5 kV. After the electric shock is finished, the agrobacterium competent cells are added into a 400 mu LLB liquid culture medium and are cultured for 2h under shaking at the temperature of 28 ℃. 20 mu L of the bacterial liquid is sucked and evenly smeared on a solid LB culture medium plate containing 50mm kanamycin and 25mm rifampicin, and inverted culture is carried out for 48h at the temperature of 28 ℃. And selecting a single colony for colony PCR to obtain a positive clone.

3. Transient expression of the proteins PBIN-PLUS GFP, PBIN-PLUS INF1, PBIN-PLUS PYOD6, PBIN-PLUS PYOD7 and PBIN-PLUS PYOD20 in Nicotiana benthamiana

The positive clones are placed in an LB liquid culture medium and shake-cultured for 30h at 28 ℃. The bacterial suspension was collected, centrifuged at 8000 rpm for 2min, and washed three times with 10mM MgCl 2. Finally, the inoculum was diluted with 10mM MgCl2 to an OD600 of 0.3. Bacterial suspension and bacterial suspension 1 containing suppressor for silencing P19: 1 and mixing. All genes were injected onto the leaf tobacco pieces with 1mL syringes, each at least 5 replicates, and the experiment was repeated three times, with the results shown in fig. 1.

PYOD6 gene

ATGTACACCAAGTTCGCCATCCTCGCCCTTGCCGCCTTCGCCGCTACGGCCGCCAACGC CGCGTCCACTGCTCCTTGCCCAAGCAGTGAACTCGCCAAGCTTGCTGGCTTGGCGTCGT CGCAGAACGTGTTCCCGTGCCAGGCCGTGTCGGGTGGTTTCAACATGATCCCACCATCG GGTCTGCCAACCACTGAGCAGCGCGCCAAGATGTGCGCGGCGCCAGTGTGCCACGCCC TGATCAAGGAAATCATCGCCCTCAACCCAACCGACTGCGTGCTCTCGCTCGGCAACTTG AACGTGAACGAACTCGCGAACGGCTTCGAGGCTTCATGCACGGCTTCGTCGCCAGCTC CAGCTGTGACCCCAGCGCCAGCGACTTCGGCTCCATCGACCCCATCCACGACGGCCCCT GGCACCCCATCCACGACGGCTCCATCGACCCCATCCACGACGGCGCCAGCCACCAACG GTACCCCAGCCCCAGCTGCCACCACCGTCAAGCCAGCTTGCTAA

PYOD7 gene

ATGTACACCAAGTTCGCCATCCTCGCCCTTGCCGCCTTCGCCGCTACGGCCGCCAACGC CGCGTCCACTGCTCCTTGCCCAAGCAGTGAACTCGCCAAGCTTGCTGGCTTGGCGTCGT CGCAGAACGTGTTCCCGTGCCAGGCCGTGTCGGGTGGTTTCAACATGATCCCACCATCG GGTCTGCCAACCACTGAGCAGCGCGCCAAGATGTGCGCGGCGCCAGTGTGCCACGCCC TGATCAAGGAAGTTGTCGCCCTCAACCCAACCGACTGCGTGCTCTCGATCGGCAACTTG AACGTGTACGAACTCGCGAACGGCTTCGAGGCTTCATGCACGGCTTCGTCGCCAGCTCC AGCTGTGACCCCAGCGCCAGCGACTTCGGCTCCATCGACCCCATCCACGACGGCCCCTG GCACCCCATCCACGACGGCGCCAGCCACCAACGGTACTCCAGCCCCAGCTGCCACCAC CGTCAAGCCAGCTTGCTAA

PYOD20 gene

ATGAAGTTCCAAGCCGTCCTCTTCGCCGCCGCTGCCGTCTTCGGCCTTGCCGCCGCCTAC GATGAAGTCACCGAGTGCCCAGCCACTGAATTCGTCAAGCTCGCGCCACTTGCGGCCA ACCCGAACTTGAACACCTGCCAAGCGGCATCGGAGGGCTGGCAGATGCTCCCACCAGT GGGTTACCCAACGGACACCCAGCGCGCTGCGATGTGCCTTGAGCCAACGTGCTTCAACT TGATCGACGCCATCAAGGCCCTGAACCCAAGCGACTGCATGTTGGTGTTTGGCGACGTC AAGTTGAACGTAAAGAAGCTCGCTGAAGAGTTCGAGGGCAGCTGCTTCTAA

PYOD6 protein

MYTKFAILALAAFAATAANAASTAPCPSSELAKLAGLASSQNVFPCQAVSGGFNMIPPSGLP TTEQRAKMCAAPVCHALIKEIIALNPTDCVLSLGNLNVNELANGFEASCTASSPAPAVTPAPA TSAPSTPSTTAPGTPSTTAPSTPSTTAPATNGTPAPAATTVKPAC*

PYOD7 protein

MYTKFAILALAAFAATAANAASTAPCPSSELAKLAGLASSQNVFPCQAVSGGFNMIPPSGLP TTEQRAKMCAAPVCHALIKEVVALNPTDCVLSIGNLNVYELANGFEASCTASSPAPAVTPAP ATSAPSTPSTTAPGTPSTTAPATNGTPAPAATTVKPAC*

PYOD20 protein

MKFQAVLFAAAAVFGLAAAYDEVTECPATEFVKLAPLAANPNLNTCQAASEGWQMLPPVG YPTDTQRAAMCLEPTCFNLIDAIKALNPSDCMLVFGDVKLNVKKLAEEFEGSCF*。

Sequence listing

<110> Nanjing university of agriculture

<120> application of Elicitin gene for inducing HR in pythium ultimum and expression vector thereof

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<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Pythium ultimum (Pythium ultimum)

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atgtacacca agttcgccat cctcgccctt gccgccttcg ccgctacggc cgccaacgcc 60

gcgtccactg ctccttgccc aagcagtgaa ctcgccaagc ttgctggctt ggcgtcgtcg 120

cagaacgtgt tcccgtgcca ggccgtgtcg ggtggtttca acatgatccc accatcgggt 180

ctgccaacca ctgagcagcg cgccaagatg tgcgcggcgc cagtgtgcca cgccctgatc 240

aaggaaatca tcgccctcaa cccaaccgac tgcgtgctct cgctcggcaa cttgaacgtg 300

aacgaactcg cgaacggctt cgaggcttca tgcacggctt cgtcgccagc tccagctgtg 360

accccagcgc cagcgacttc ggctccatcg accccatcca cgacggcccc tggcacccca 420

tccacgacgg ctccatcgac cccatccacg acggcgccag ccaccaacgg taccccagcc 480

ccagctgcca ccaccgtcaa gccagcttgc taa 513

<210> 2

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<212> DNA

<213> Pythium ultimum (Pythium ultimum)

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atgtacacca agttcgccat cctcgccctt gccgccttcg ccgctacggc cgccaacgcc 60

gcgtccactg ctccttgccc aagcagtgaa ctcgccaagc ttgctggctt ggcgtcgtcg 120

cagaacgtgt tcccgtgcca ggccgtgtcg ggtggtttca acatgatccc accatcgggt 180

ctgccaacca ctgagcagcg cgccaagatg tgcgcggcgc cagtgtgcca cgccctgatc 240

aaggaagttg tcgccctcaa cccaaccgac tgcgtgctct cgatcggcaa cttgaacgtg 300

tacgaactcg cgaacggctt cgaggcttca tgcacggctt cgtcgccagc tccagctgtg 360

accccagcgc cagcgacttc ggctccatcg accccatcca cgacggcccc tggcacccca 420

tccacgacgg cgccagccac caacggtact ccagccccag ctgccaccac cgtcaagcca 480

gcttgctaa 489

<210> 3

<211> 345

<212> DNA

<213> Pythium ultimum (Pythium ultimum)

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atgaagttcc aagccgtcct cttcgccgcc gctgccgtct tcggccttgc cgccgcctac 60

gatgaagtca ccgagtgccc agccactgaa ttcgtcaagc tcgcgccact tgcggccaac 120

ccgaacttga acacctgcca agcggcatcg gagggctggc agatgctccc accagtgggt 180

tacccaacgg acacccagcg cgctgcgatg tgccttgagc caacgtgctt caacttgatc 240

gacgccatca aggccctgaa cccaagcgac tgcatgttgg tgtttggcga cgtcaagttg 300

aacgtaaaga agctcgctga agagttcgag ggcagctgct tctaa 345

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Met Tyr Thr Lys Phe Ala Ile Leu Ala Leu Ala Ala Phe Ala Ala Thr

1 5 10 15

Ala Ala Asn Ala Ala Ser Thr Ala Pro Cys Pro Ser Ser Glu Leu Ala

20 25 30

Lys Leu Ala Gly Leu Ala Ser Ser Gln Asn Val Phe Pro Cys Gln Ala

35 40 45

Val Ser Gly Gly Phe Asn Met Ile Pro Pro Ser Gly Leu Pro Thr Thr

50 55 60

Glu Gln Arg Ala Lys Met Cys Ala Ala Pro Val Cys His Ala Leu Ile

65 70 75 80

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

85 90 95

Asn Leu Asn Val Asn Glu Leu Ala Asn Gly Phe Glu Ala Ser Cys Thr

100 105 110

Ala Ser Ser Pro Ala Pro Ala Val Thr Pro Ala Pro Ala Thr Ser Ala

115 120 125

Pro Ser Thr Pro Ser Thr Thr Ala Pro Gly Thr Pro Ser Thr Thr Ala

130 135 140

Pro Ser Thr Pro Ser Thr Thr Ala Pro Ala Thr Asn Gly Thr Pro Ala

145 150 155 160

Pro Ala Ala Thr Thr Val Lys Pro Ala Cys

165 170

<210> 5

<211> 162

<212> PRT

<213> Pythium ultimum (Pythium ultimum)

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Met Tyr Thr Lys Phe Ala Ile Leu Ala Leu Ala Ala Phe Ala Ala Thr

1 5 10 15

Ala Ala Asn Ala Ala Ser Thr Ala Pro Cys Pro Ser Ser Glu Leu Ala

20 25 30

Lys Leu Ala Gly Leu Ala Ser Ser Gln Asn Val Phe Pro Cys Gln Ala

35 40 45

Val Ser Gly Gly Phe Asn Met Ile Pro Pro Ser Gly Leu Pro Thr Thr

50 55 60

Glu Gln Arg Ala Lys Met Cys Ala Ala Pro Val Cys His Ala Leu Ile

65 70 75 80

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

85 90 95

Asn Leu Asn Val Tyr Glu Leu Ala Asn Gly Phe Glu Ala Ser Cys Thr

100 105 110

Ala Ser Ser Pro Ala Pro Ala Val Thr Pro Ala Pro Ala Thr Ser Ala

115 120 125

Pro Ser Thr Pro Ser Thr Thr Ala Pro Gly Thr Pro Ser Thr Thr Ala

130 135 140

Pro Ala Thr Asn Gly Thr Pro Ala Pro Ala Ala Thr Thr Val Lys Pro

145 150 155 160

Ala Cys

<210> 6

<211> 114

<212> PRT

<213> Pythium ultimum (Pythium ultimum)

<400> 6

Met Lys Phe Gln Ala Val Leu Phe Ala Ala Ala Ala Val Phe Gly Leu

1 5 10 15

Ala Ala Ala Tyr Asp Glu Val Thr Glu Cys Pro Ala Thr Glu Phe Val

20 25 30

Lys Leu Ala Pro Leu Ala Ala Asn Pro Asn Leu Asn Thr Cys Gln Ala

35 40 45

Ala Ser Glu Gly Trp Gln Met Leu Pro Pro Val Gly Tyr Pro Thr Asp

50 55 60

Thr Gln Arg Ala Ala Met Cys Leu Glu Pro Thr Cys Phe Asn Leu Ile

65 70 75 80

Asp Ala Ile Lys Ala Leu Asn Pro Ser Asp Cys Met Leu Val Phe Gly

85 90 95

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

100 105 110

Cys Phe