CN112442494B - Two TK1 receptor kinase genes in arabidopsis thaliana and soybean and application thereof - Google Patents

Two TK1 receptor kinase genes in arabidopsis thaliana and soybean and application thereof Download PDF

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CN112442494B
CN112442494B CN202011157754.XA CN202011157754A CN112442494B CN 112442494 B CN112442494 B CN 112442494B CN 202011157754 A CN202011157754 A CN 202011157754A CN 112442494 B CN112442494 B CN 112442494B
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景茂峰
窦道龙
艾干
朱海
李田丽
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Nanjing Agricultural University
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Abstract

The invention belongs to the field of genetic engineering, and discloses two TK1 receptor kinase genes in arabidopsis thaliana and soybean and application thereof. The application effect of the two TK1 receptor kinases screened from arabidopsis thaliana and soybean in resisting phytophthora infection of plants is remarkable. Taking the Nicotiana benthamiana as an example, the TK1 gene in Arabidopsis and soybean is transiently expressed in the Nicotiana benthamiana, so that the resistance of the Nicotiana benthamiana to phytophthora can be obviously improved. Under the condition that no particularly good method for preventing and controlling phytophthora exists, cloning of TK1 gene is of great significance for cultivating crop varieties with phytophthora resistance by using genetic engineering means.

Description

Two TK1 receptor kinase genes in arabidopsis thaliana and soybean and application thereof
Technical Field
The invention belongs to the field of genetic engineering, and discloses application of two TK1 receptor kinase genes in arabidopsis thaliana and soybean and an expression vector thereof in resisting phytophthora infection of plants.
Background
Phytophthora capsici is a broad-spectrum pathogenic bacterium and can cause serious harm to various economic crops such as capsicum, tobacco and the like. Phytophthora capsici belongs to oomycetes, although the pathogenic bacteria are similar to fungi in form, the pathogenic bacteria are closely related to diatoms and blue-green algae in evolution and belong to the kingdom of pilos-penis organisms, so that common fungus bactericides are often ineffective to Phytophthora sojae and other oomycetes. The application of the plant disease-resistant variety overcomes the problems of phytotoxicity, environmental pollution and the like, and becomes an important means for realizing disease resistance in agricultural production.
During the long-term co-evolution of plants and pathogenic bacteria, a large number of disease-resistant related genes are evolved by the plants in order to resist the pathogenic bacteria, including LRR-RLK and LRR-RLP genes which play a role in the disease-resistant process. Wherein the LRR-RLK comprises an extracellular domain, a transmembrane domain, and an intracellular kinase domain, and the LRR-RLP lacks the intracellular kinase domain. Some RLKs and RLPs act as PRRs recognizing danger signals activating plant immunity, others modulate plant growth development, reproduction, symbiosis and tolerance to abiotic stress. TK1 is a newly identified LRR-RLK, has a typical RLK structure, and plays an important role in endoplasmic reticulum stress and plant immunity. TK1 is introduced into crops to improve the resistance of crops to phytophthora capsici. Taking Nicotiana benthamiana as an example, the TK1 gene of arabidopsis thaliana and soybean is transiently expressed in Nicotiana benthamiana, so that the resistance of Nicotiana benthamiana to phytophthora capsici can be obviously improved. Under the condition that no particularly good method for preventing and controlling phytophthora capsici exists, the cloning of the TK1 gene has important significance for cultivating crop varieties with phytophthora capsici resistance by using a genetic engineering means.
Disclosure of Invention
The invention aims to make up the deficiency of preventing and treating crop epidemic diseases at present, and provides application of two TK1 receptor kinase genes in arabidopsis and soybean and expression vectors thereof in plant resistance to phytophthora infection.
The purpose of the invention can be realized by the following technical scheme:
two TK1 receptor kinase genes AtTK1 and GmTK1 from two plant materials, namely Arabidopsis thaliana (Arabidopsis thaliana) and Glycine max (Glycine max) Williams 82, wherein the nucleotide sequences of the AtTK1 and GmTK1 genes are respectively shown as SEQ ID No.1 and SEQ ID No.2 in sequence.
The proteins coded by the two LRR-RLK TK1 genes are AtTK1 and GmTK1, and the amino acid sequences are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.
An expression cassette, a recombinant expression vector, a transgenic cell line or a transgenic recombinant strain containing the TK1 receptor kinase gene.
The recombinant expression vector containing the TK1 receptor kinase gene is psupper-flag: AtTK1 and psuper-flag: GmTK 1. Constructing recombinant expression vectors psuper-flag-AtTK1 and psuper-flag-GmTK1 of the receptor-like kinase AtTK1 and GmTK1 genes, taking a plant expression vector psuper-flag as a starting vector, and respectively inserting the AtTK1 and GmTK1 genes into Xba1 and Sam1 enzyme cutting sites of the psuper-flag vector to obtain the receptor-like kinase AtTK1 and GmTK1 genes.
The TK1 receptor kinase gene and its expression vector are used in constructing phytophthora resisting crop variety.
A method for constructing phytophthora-resistant crop varieties is characterized in that the genes or the recombinant expression vectors are introduced into crop plants, and positive transformation plants are obtained through resistance screening to obtain the phytophthora-resistant crop varieties.
The advantages and positive effects of the invention are shown in:
the invention clones two TK1 receptor kinase genes from arabidopsis thaliana and soybean, and inserts the genes into an expression vector psupper-flag. The vector is introduced into crops, so that the resistance of the crops to phytophthora can be improved. The TK1 gene can be used for genetic engineering to construct epidemic disease-resistant crops, and provides powerful guidance for formulating disease-resistant strategies and controlling diseases in agricultural production.
Drawings
FIG. 1 transient expression of TK1 gene in Nicotiana benthamiana improves resistance of Nicotiana benthamiana to Phytophthora capsici.
FIG. 2 shows statistics of lesion diameter 48h after phytophthora capsici is inoculated after the left and right leaves of Nicotiana benthamiana respectively express psuper-flag and TK1 proteins.
Detailed Description
The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.
Example 1 transient expression of AtTK1 and GmTK1 in Nicotiana benthamiana improves resistance of Nicotiana benthamiana to Phytophthora capsici
1. Test strains
The test strain phytophthora capsici LT263 is preserved in a plant disease line interaction laboratory of Nanjing university of agriculture and phytophthora (published). The strain is preserved in a V8 solid culture medium, and the preservation temperature is 10 ℃.
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, and leaves at the same 3, 4, 5 leaf positions were used to express GFP and NAC089 proteins, followed by inoculation with phytophthora capsici.
3. Preparation of test Soybean seedlings
The differential host soybean is sowed in a plastic flowerpot (d is 10cm) filled with vermiculite (2-4mm), 15 seeds are planted in each pot, the pot is placed in a greenhouse with 14h of light (strong light irradiation)/10 h of darkness for growth, after 7 days, two true leaves grow out, and 10 seedlings are left for inoculation. The variety used was Williams.
4. Extracting Arabidopsis thaliana and soybean RNA, and obtaining cDNA of TK1 gene
Collecting leaves of Arabidopsis thaliana and soybean, picking, quickly freezing with liquid nitrogen, placing into a mortar, cooling and grinding. And (4) extracting and purifying the arabidopsis thaliana and soybean total RNA by referring to a recommended method of the Tiangen total RNA extraction and purification kit. According to the method provided by the RNA reverse transcription kit of Novodka, oligo (oligo) (dT) is used as a primer for reverse transcription synthesis of cDNA. The cDNA is taken as a template, PCR amplification is carried out by using corresponding upstream and downstream primers, and the sequences of the primers are sequentially shown in SEQ ID NO. 5-8 (namely, the cDNA of arabidopsis thaliana is amplified by using the primers shown in SEQ ID NO. 5-6, and the cDNA of soybean is amplified by using the primers shown in SEQ ID NO. 7-8).
The PCR reaction system is as follows: 2.5. mu.L of 10 XPCR reaction buffer; 1.5 μ L1.5 mM MgCl2(ii) a 0.5. mu.L of 2.5mM 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 ℃.
Electrophoresis: the PCR product was detected by 1% agarose gel electrophoresis, and the desired fragment (size: about 1023bp) was excised using an ultraviolet gel imager. The PCR product was recovered by TaKaRa gel recovery kit and sent to Kingsry for sequencing. Two LRR-RLK TK1 genes, namely AtTK1 and GmTK1, are obtained through amplification, and sequencing results show that the AtTK1 is shown as SEQ ID No.1 and the GmTK1 gene is shown as SEQ ID No. 2. The amino acid sequences of the proteins coded by the two TK1 genes are sequentially shown as SEQ ID NO. 3-4.
5. Construction of expression vectors of psuper-flag AtTK1 and psuper-flag GmTK1
The psuper-flag empty vector plasmid was digested with Xba1 and SamI. The cDNA and the vector were ligated using 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. Sequencing the identified positive clones, and obtaining the expression vectors of psuper-flag AtTK1 and psuper-flag GmTK 1.
6. Obtaining agrobacterium-infected cells
Carrying out streak culture on agrobacterium GV3101 strains 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 volume of bacterial liquid without antibiotics 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 ℃ 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 suspends the bacterial pellet for later use.
7. psuper-flag AtTK1 and psuper-flag GmTK1 expression vector and psuper-flag empty vector transformation Agrobacterium tumefaciens
Transforming agrobacterium cells by utilizing a PSUPer-flag, namely AtTK1, a PSUPer-flag, namely GmTK1 expression vector and a PSUPer-flag empty vector by utilizing an electric transformation method; the electric shock cup was rinsed three times with 70% alcohol and then the alcohol was completely air dried. 100ng of the expression vector was added to the Agrobacterium-infected state and left on ice for 30 min. And transferring the agrobacterium competent cells into an electric shock cup, and performing electric shock transformation by using a voltage of 2.5 kV. After the electric shock is finished, the agrobacterium competent cells are added into 400 mu L of LB 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.
8. Transient expression of the psuper-flag and TK1 proteins in Nicotiana benthamiana
The positive clones are placed in an LB liquid culture medium and shake-cultured for 30h at 28 ℃. The bacterial solution was collected, centrifuged at 8000rpm for 2min using 10mM MgCl2And washing three times. Finally, 10mM MgCl was used2Diluting the bacterial liquid to OD600The value is 0.3. Bacterial suspension and bacterial suspension 1 containing suppressor for silencing P19: 1 and mixing. The suppressor of silencing P19 has the function of promoting gene expression. The left and right sides of the same Benz tobacco leaf were each filled with an injection using a 1mL syringe.
9. Detection of resistance to disease of Bunsen tobacco by transient expression of psuper-flag and TK1 protein
The leaf sections for which the expression of the protein was confirmed were cut off and placed in a filter paper-moist tray. Punching holes on the growing edge of a flat plate of phytophthora capsici LT263 by using a 7mm puncher, and placing the punched bacterium and dish bacterium surfaces downwards on the left side and the right side of the Nicotiana benthamiana leaves in bilateral symmetry. The trays were placed in an incubator at 25 ℃ in the dark for 36 h. The leaf was removed and then irradiated with a hand-held uv-luminometer. The darkened area is the infected area of phytophthora capsici LT 263. The diameter of the infected area was measured with a measuring ruler, and the leaf surface expressing the psuper-flag protein was used as a control to compare the effect of the two proteins on the resistance to Nicotiana benthamiana. Two TK1 proteins from Arabidopsis thaliana and soybean were found to significantly improve the resistance of Nicotiana benthamiana to Phytophthora capsici LT 263.
Sequence listing
<110> Nanjing university of agriculture
<120> two TK1 receptor kinase genes in arabidopsis thaliana and soybean and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3051
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 1
atgggcaaac agagaagaac catgatctca tttactctgt ttctcacatt gacaatgatg 60
tcatcactca tcaatggcga taccgactca attcaactaa acgacgacgt tttaggactc 120
atcgtcttca aatcagacct caacgaccca ttttcacacc ttgagtcatg gaccgaagac 180
gacaacactc cttgttcatg gagttacgtc aaatgcaacc ccaaaacatc tcgagtcatc 240
gagctttctc tcgacggttt agccttaacc ggaaaaatca accgcggaat ccaaaagctt 300
caacgtttaa aagtactctc actttccaac aacaacttca ccggaaacat caacgctctc 360
tcaaacaaca accatctcca aaagctagat cttagccaca acaatctctc cggtcaaatc 420
ccttcttctc tcggttcaat aacctccttg caacaccttg acttaaccgg aaactccttc 480
tccggtacac tctctgacga tttgttcaac aactgttcct ctcttagata tctttctctc 540
tcccacaacc acctcgaagg tcaaatccca agtactctgt ttcgatgctc tgttttaaac 600
agtctcaatc tttcaagaaa ccgtttctcc ggtaacccta gcttcgtctc aggaatctgg 660
agacttgaga ggttaagagc tttagatcta tcttctaact cactttctgg ttcaatacct 720
ttggggatac tctctctaca taacttgaaa gagttgcaac tacagaggaa tcagttctct 780
ggagcattgc cttcagatat tggactctgt cctcatttaa acagagttga tctaagttcc 840
aatcatttct ctggtgaact tccaagaact cttcagaagc tgaaatcttt aaatcacttc 900
gatgtatcga acaatttgct ctccggtgat ttcccgccat ggatcggtga catgaccggt 960
ttagtacact tggatttctc cagcaatgag ttaaccggaa agcttccttc ttctattagt 1020
aacttgaggt ctctaaagga tctaaacttg tctgagaaca aactctccgg cgaggtacct 1080
gagtctttgg aatcatgcaa agagcttatg attgttcagc ttaaaggcaa tgacttctcc 1140
ggtaacattc ctgatggttt ttttgatctt ggtcttcaag aaatggattt ttcgggtaac 1200
ggtttaaccg gttcgatccc aagaggctca agcaggcttt tcgagtcact cataaggctc 1260
gatctttcgc ataacagtct cactggaagt atacctggtg aagtaggact cttcatccac 1320
atgagatacc ttaatttatc atggaaccat ttcaacacaa gagttcctcc tgaaattgag 1380
tttctacaga acttaacggt attggatctt aggaacagtg cactgattgg ttcggttcct 1440
gctgatatat gtgagtctca gagtcttcag atacttcaac tggatggtaa ctcactaacc 1500
ggttctatac cggaaggaat cggaaactgc tcttctctta aattgttgag tttgtctcat 1560
aacaatctta ccggtcctat tcctaaatct ctttcaaact tacaagagct caagatttta 1620
aagctagagg ctaataagct tagtggagaa ataccgaaag agcttgggga tttgcagaat 1680
ctgttattgg ttaacgtttc gtttaaccga cttatcggaa ggttaccatt gggagatgtg 1740
ttccaaagct tagaccagag tgctatacaa ggaaacctag gtatttgttc accgttgttg 1800
agaggtcctt gtacactgaa tgttccaaag cctcttgtca tcaatccaaa ctcctacggg 1860
aatgggaaca atatgcctgg aaaccgagca agcggtggtt ctggaacatt ccaccgcaga 1920
atgttcctga gtgtttcagt gattgtagca atatcagctg cgattctcat cttctctgga 1980
gtcataatta taacgctgct taacgcgtct gttagaagac ggcttgcatt tgtagacaat 2040
gcgttggaaa gcattttctc ggggtcttcg aaatcaggaa gaagcttaat gatgggtaaa 2100
cttgttctgt taaactcaag aacttcacgt tcctcgtctt cgtctcaaga gttcgaaaga 2160
aacccagagt cacttctcaa caaagcttca agaatcggtg aaggggtttt tggaacagtc 2220
tacaaagcac ctttaggaga gcaagggaga aacttggctg ttaagaaact tgtcccgtct 2280
ccgattcttc aaaacctaga agattttgat cgcgaagttc ggatattggc gaaagcgaag 2340
caccctaatc tagtatcgat caaagggtat ttctggacac cggacttgca tcttctggta 2400
tcagaataca tccccaatgg aaacttgcag tccaagttac acgaacgaga accctcaaca 2460
ccgcctcttt cttgggacgt aagatacaaa atcatcctcg gtacagctaa aggactcgct 2520
tatctccatc acacattccg tccaacaacc atccacttca acctgaaacc gacaaacatc 2580
ctcctcgacg agaaaaacaa cccgaaaatc tctgatttcg ggctatctcg tctcctaaca 2640
acacaagacg ggaacacaat gaacaacaac aggtttcaaa acgctttagg ctacgtagcg 2700
cctgaactag agtgtcagaa cttaagggtc aacgagaaat gcgatgttta cgggttcggg 2760
gttttgatac tcgaactggt gactggtcgg agaccggtgg agtatggtga agacagcttt 2820
gtgatactta gtgaccatgt tcgagttatg ttagaacaag ggaatgtgtt ggagtgtatt 2880
gatcctgtga tggaggaaca atactctgaa gatgaggttt tgcctgtctt gaaacttgct 2940
ctggtctgta cttctcagat accttcgaat cggccaacaa tggccgagat tgttcagatc 3000
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<210> 2
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<212> DNA
<213> Soybean (Glycine max)
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attgtgttca aatcagacct tgatgaccct tcttcttatc ttgcttcatg gaatgaagat 180
gatgccaatc catgttcatg gcagttcgtg cagtgcaatc ctgagagtgg aagagtttct 240
gaggtctcat tggatggctt aggactatct gggaagattg gaagaggtct tgagaagtta 300
cagcatctaa cggtattatc cctttctcac aacagcttga gtgggagtat tagcccatca 360
cttactcttt ccaatagtct tgagaggctg aaccttagtc acaatgctct ttctggttcc 420
attcctactt cttttgtgaa tatgaattca ataagatttc ttgatctttc agagaattca 480
ttctcaggac cagtccctga gagttttttt gagagctgtt cttcccttca ccacatttct 540
ctagctagga acatatttga tggaccaatt cctggctcac tgtctagatg ttcctcattg 600
aatagcatta atctttccaa taaccgtttc tccggtaatg tagacttcag tgggatttgg 660
tcattgaaca ggcttaggac tttggatcta tccaacaatg ccttatcagg gtctttacct 720
aatggaattt catccataca taactttaaa gagatcttat tacaaggtaa ccagttttca 780
ggtccattat ccactgatat tggattctgc ctgcatctga gtaggttgga tttcagtgat 840
aatcagttga gtggagaact acctgagtca ctagggatgc taagttcttt gagctatttc 900
aaggcatcaa acaatcattt caacagtgaa ttccctcaat ggattggtaa catgaccaat 960
ctggagtacc tagagctctc aaacaatcag ttcactggaa gcatcccaca gtcaataggg 1020
gaactgagat cactgactca cctgagcatt tcaaataaca agcttgttgg aactattcca 1080
tcatcattga gttcctgcac aaagttatct gtggtccagc tcagaggtaa tggtttcaat 1140
ggcaccattc cagaggcttt gtttggccta ggattggagg acatagactt gtcccataat 1200
ggattgagtg gctcaattcc accaggatca agcaggctct tggaaactct caccaacttg 1260
gatctttcag ataaccatct ccaagggaat atccctgcag aaactggtct tctttcaaaa 1320
ctaagatatt tgaatctgtc ttggaatgat cttcattctc agatgcctcc agaattcggc 1380
ctccttcaga acctgacggt tttggatctt cgcaacagtg ccttgcacgg ttcaattcca 1440
gcagatatat gtgactcagg caatttagct gtcctccaac ttgatggaaa ttcatttgaa 1500
gggaatattc cgtccgagat tggaaattgt agctctcttt acttgctgag ttcgtctcac 1560
aataatttga caggttcaat tccaaagtcc atggcaaagc taaacaagct caaaatcctc 1620
aagctggaat ttaatgagct tagtggagag ataccaatgg agcttggaat gctgcagagt 1680
cttcttgctg tgaacatatc ctacaacagg ctcacaggca ggcttcctac aagtagcata 1740
tttcaaaact tggacaaaag ttcattggag ggaaacctgg gtctttgctc acccttgttg 1800
aagggtccat gtaagatgaa tgtccccaaa ccactagtgc ttgacccaaa tgcctataac 1860
aaccaaataa gtcctcaaag gcaaagaaac gaatcatccg agtctggcca ggtccatcgc 1920
cacaggttcc ttagtgtatc tgctattgta gcaatatctg catcctttgt cattgtttta 1980
ggagtgattg ctgttagcct acttaatgtt tctgtaagga gaaggctaac atttgtggat 2040
aatgctttgg aaagcatgtg ctcaagctcc tcaagatcag gaagtccagc cacaggaaag 2100
cttatcctgt tcgattccca ttcctcacct gattggatca gcaatcctga gtccttgctc 2160
aacaaggcat ctgagattgg agaaggagtc tttggaacac tctacaaggt tccattggga 2220
tcacaaggta gaatggtagc aatcaagaag cttatatcct caaacataat ccaatatcca 2280
gaagactttg atagggaagt tagaatccta gggaaagcaa ggcacccaaa tctaattgca 2340
ttgaaaggat actattggac tcctcaatta cagcttttag tgaccgagtt tgccccaaat 2400
ggtagcttgc aagccaagct acatgaaagg cttccttcaa gtcctcctct ttcttgggct 2460
ataaggttca aaatcttgct tggaacagca aaggggcttg ctcatttgca ccactctttc 2520
cgtccgccga tcatccacta caacataaag ccaagtaaca ttttgcttga cgaaaattac 2580
aatgccaaga tctcggattt cgggttggct cggcttctga caaagctgga caggcatgtg 2640
atgagcaaca ggtttcagag tgcattagga tatgtggcac cagaattagc atgccagagc 2700
ttaagggtca atgagaaatg tgatgtctac ggttttggag tgatgatcct tgagctggtg 2760
acaggtagga ggccagtgga gtatggagaa gacaatgtgc tgatactgaa tgaccatgtg 2820
agggtgttgc ttgagcatgg taatgtgttg gagtgtgtgg atcaaagcat gagtgagtat 2880
ccagaagatg aggtgttgcc tgttctgaag ctagcaatgg tatgcacctc tcaaattcct 2940
tctagcaggc ctactatggc tgaagtggtg caaatactgc aggtcattaa aaccccggtt 3000
cctcaaagga tggaagtgtt ttga 3024
<210> 3
<211> 1016
<212> PRT
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 3
Met Gly Lys Gln Arg Arg Thr Met Ile Ser Phe Thr Leu Phe Leu Thr
1 5 10 15
Leu Thr Met Met Ser Ser Leu Ile Asn Gly Asp Thr Asp Ser Ile Gln
20 25 30
Leu Asn Asp Asp Val Leu Gly Leu Ile Val Phe Lys Ser Asp Leu Asn
35 40 45
Asp Pro Phe Ser His Leu Glu Ser Trp Thr Glu Asp Asp Asn Thr Pro
50 55 60
Cys Ser Trp Ser Tyr Val Lys Cys Asn Pro Lys Thr Ser Arg Val Ile
65 70 75 80
Glu Leu Ser Leu Asp Gly Leu Ala Leu Thr Gly Lys Ile Asn Arg Gly
85 90 95
Ile Gln Lys Leu Gln Arg Leu Lys Val Leu Ser Leu Ser Asn Asn Asn
100 105 110
Phe Thr Gly Asn Ile Asn Ala Leu Ser Asn Asn Asn His Leu Gln Lys
115 120 125
Leu Asp Leu Ser His Asn Asn Leu Ser Gly Gln Ile Pro Ser Ser Leu
130 135 140
Gly Ser Ile Thr Ser Leu Gln His Leu Asp Leu Thr Gly Asn Ser Phe
145 150 155 160
Ser Gly Thr Leu Ser Asp Asp Leu Phe Asn Asn Cys Ser Ser Leu Arg
165 170 175
Tyr Leu Ser Leu Ser His Asn His Leu Glu Gly Gln Ile Pro Ser Thr
180 185 190
Leu Phe Arg Cys Ser Val Leu Asn Ser Leu Asn Leu Ser Arg Asn Arg
195 200 205
Phe Ser Gly Asn Pro Ser Phe Val Ser Gly Ile Trp Arg Leu Glu Arg
210 215 220
Leu Arg Ala Leu Asp Leu Ser Ser Asn Ser Leu Ser Gly Ser Ile Pro
225 230 235 240
Leu Gly Ile Leu Ser Leu His Asn Leu Lys Glu Leu Gln Leu Gln Arg
245 250 255
Asn Gln Phe Ser Gly Ala Leu Pro Ser Asp Ile Gly Leu Cys Pro His
260 265 270
Leu Asn Arg Val Asp Leu Ser Ser Asn His Phe Ser Gly Glu Leu Pro
275 280 285
Arg Thr Leu Gln Lys Leu Lys Ser Leu Asn His Phe Asp Val Ser Asn
290 295 300
Asn Leu Leu Ser Gly Asp Phe Pro Pro Trp Ile Gly Asp Met Thr Gly
305 310 315 320
Leu Val His Leu Asp Phe Ser Ser Asn Glu Leu Thr Gly Lys Leu Pro
325 330 335
Ser Ser Ile Ser Asn Leu Arg Ser Leu Lys Asp Leu Asn Leu Ser Glu
340 345 350
Asn Lys Leu Ser Gly Glu Val Pro Glu Ser Leu Glu Ser Cys Lys Glu
355 360 365
Leu Met Ile Val Gln Leu Lys Gly Asn Asp Phe Ser Gly Asn Ile Pro
370 375 380
Asp Gly Phe Phe Asp Leu Gly Leu Gln Glu Met Asp Phe Ser Gly Asn
385 390 395 400
Gly Leu Thr Gly Ser Ile Pro Arg Gly Ser Ser Arg Leu Phe Glu Ser
405 410 415
Leu Ile Arg Leu Asp Leu Ser His Asn Ser Leu Thr Gly Ser Ile Pro
420 425 430
Gly Glu Val Gly Leu Phe Ile His Met Arg Tyr Leu Asn Leu Ser Trp
435 440 445
Asn His Phe Asn Thr Arg Val Pro Pro Glu Ile Glu Phe Leu Gln Asn
450 455 460
Leu Thr Val Leu Asp Leu Arg Asn Ser Ala Leu Ile Gly Ser Val Pro
465 470 475 480
Ala Asp Ile Cys Glu Ser Gln Ser Leu Gln Ile Leu Gln Leu Asp Gly
485 490 495
Asn Ser Leu Thr Gly Ser Ile Pro Glu Gly Ile Gly Asn Cys Ser Ser
500 505 510
Leu Lys Leu Leu Ser Leu Ser His Asn Asn Leu Thr Gly Pro Ile Pro
515 520 525
Lys Ser Leu Ser Asn Leu Gln Glu Leu Lys Ile Leu Lys Leu Glu Ala
530 535 540
Asn Lys Leu Ser Gly Glu Ile Pro Lys Glu Leu Gly Asp Leu Gln Asn
545 550 555 560
Leu Leu Leu Val Asn Val Ser Phe Asn Arg Leu Ile Gly Arg Leu Pro
565 570 575
Leu Gly Asp Val Phe Gln Ser Leu Asp Gln Ser Ala Ile Gln Gly Asn
580 585 590
Leu Gly Ile Cys Ser Pro Leu Leu Arg Gly Pro Cys Thr Leu Asn Val
595 600 605
Pro Lys Pro Leu Val Ile Asn Pro Asn Ser Tyr Gly Asn Gly Asn Asn
610 615 620
Met Pro Gly Asn Arg Ala Ser Gly Gly Ser Gly Thr Phe His Arg Arg
625 630 635 640
Met Phe Leu Ser Val Ser Val Ile Val Ala Ile Ser Ala Ala Ile Leu
645 650 655
Ile Phe Ser Gly Val Ile Ile Ile Thr Leu Leu Asn Ala Ser Val Arg
660 665 670
Arg Arg Leu Ala Phe Val Asp Asn Ala Leu Glu Ser Ile Phe Ser Gly
675 680 685
Ser Ser Lys Ser Gly Arg Ser Leu Met Met Gly Lys Leu Val Leu Leu
690 695 700
Asn Ser Arg Thr Ser Arg Ser Ser Ser Ser Ser Gln Glu Phe Glu Arg
705 710 715 720
Asn Pro Glu Ser Leu Leu Asn Lys Ala Ser Arg Ile Gly Glu Gly Val
725 730 735
Phe Gly Thr Val Tyr Lys Ala Pro Leu Gly Glu Gln Gly Arg Asn Leu
740 745 750
Ala Val Lys Lys Leu Val Pro Ser Pro Ile Leu Gln Asn Leu Glu Asp
755 760 765
Phe Asp Arg Glu Val Arg Ile Leu Ala Lys Ala Lys His Pro Asn Leu
770 775 780
Val Ser Ile Lys Gly Tyr Phe Trp Thr Pro Asp Leu His Leu Leu Val
785 790 795 800
Ser Glu Tyr Ile Pro Asn Gly Asn Leu Gln Ser Lys Leu His Glu Arg
805 810 815
Glu Pro Ser Thr Pro Pro Leu Ser Trp Asp Val Arg Tyr Lys Ile Ile
820 825 830
Leu Gly Thr Ala Lys Gly Leu Ala Tyr Leu His His Thr Phe Arg Pro
835 840 845
Thr Thr Ile His Phe Asn Leu Lys Pro Thr Asn Ile Leu Leu Asp Glu
850 855 860
Lys Asn Asn Pro Lys Ile Ser Asp Phe Gly Leu Ser Arg Leu Leu Thr
865 870 875 880
Thr Gln Asp Gly Asn Thr Met Asn Asn Asn Arg Phe Gln Asn Ala Leu
885 890 895
Gly Tyr Val Ala Pro Glu Leu Glu Cys Gln Asn Leu Arg Val Asn Glu
900 905 910
Lys Cys Asp Val Tyr Gly Phe Gly Val Leu Ile Leu Glu Leu Val Thr
915 920 925
Gly Arg Arg Pro Val Glu Tyr Gly Glu Asp Ser Phe Val Ile Leu Ser
930 935 940
Asp His Val Arg Val Met Leu Glu Gln Gly Asn Val Leu Glu Cys Ile
945 950 955 960
Asp Pro Val Met Glu Glu Gln Tyr Ser Glu Asp Glu Val Leu Pro Val
965 970 975
Leu Lys Leu Ala Leu Val Cys Thr Ser Gln Ile Pro Ser Asn Arg Pro
980 985 990
Thr Met Ala Glu Ile Val Gln Ile Leu Gln Val Ile Asn Ser Pro Val
995 1000 1005
Pro His Arg Ile Met Asp Ser Phe
1010 1015
<210> 4
<211> 1007
<212> PRT
<213> Soybean (Glycine max)
<400> 4
Met Ser Ser Phe Gln Phe His Leu Arg Val Leu Ser Leu Leu Ile Ser
1 5 10 15
Val Ser Tyr Leu Leu Thr Cys Leu Gly Asn Asn Asp Ile Pro Val Gln
20 25 30
Leu Asn Asp Asp Val Leu Gly Leu Ile Val Phe Lys Ser Asp Leu Asp
35 40 45
Asp Pro Ser Ser Tyr Leu Ala Ser Trp Asn Glu Asp Asp Ala Asn Pro
50 55 60
Cys Ser Trp Gln Phe Val Gln Cys Asn Pro Glu Ser Gly Arg Val Ser
65 70 75 80
Glu Val Ser Leu Asp Gly Leu Gly Leu Ser Gly Lys Ile Gly Arg Gly
85 90 95
Leu Glu Lys Leu Gln His Leu Thr Val Leu Ser Leu Ser His Asn Ser
100 105 110
Leu Ser Gly Ser Ile Ser Pro Ser Leu Thr Leu Ser Asn Ser Leu Glu
115 120 125
Arg Leu Asn Leu Ser His Asn Ala Leu Ser Gly Ser Ile Pro Thr Ser
130 135 140
Phe Val Asn Met Asn Ser Ile Arg Phe Leu Asp Leu Ser Glu Asn Ser
145 150 155 160
Phe Ser Gly Pro Val Pro Glu Ser Phe Phe Glu Ser Cys Ser Ser Leu
165 170 175
His His Ile Ser Leu Ala Arg Asn Ile Phe Asp Gly Pro Ile Pro Gly
180 185 190
Ser Leu Ser Arg Cys Ser Ser Leu Asn Ser Ile Asn Leu Ser Asn Asn
195 200 205
Arg Phe Ser Gly Asn Val Asp Phe Ser Gly Ile Trp Ser Leu Asn Arg
210 215 220
Leu Arg Thr Leu Asp Leu Ser Asn Asn Ala Leu Ser Gly Ser Leu Pro
225 230 235 240
Asn Gly Ile Ser Ser Ile His Asn Phe Lys Glu Ile Leu Leu Gln Gly
245 250 255
Asn Gln Phe Ser Gly Pro Leu Ser Thr Asp Ile Gly Phe Cys Leu His
260 265 270
Leu Ser Arg Leu Asp Phe Ser Asp Asn Gln Leu Ser Gly Glu Leu Pro
275 280 285
Glu Ser Leu Gly Met Leu Ser Ser Leu Ser Tyr Phe Lys Ala Ser Asn
290 295 300
Asn His Phe Asn Ser Glu Phe Pro Gln Trp Ile Gly Asn Met Thr Asn
305 310 315 320
Leu Glu Tyr Leu Glu Leu Ser Asn Asn Gln Phe Thr Gly Ser Ile Pro
325 330 335
Gln Ser Ile Gly Glu Leu Arg Ser Leu Thr His Leu Ser Ile Ser Asn
340 345 350
Asn Lys Leu Val Gly Thr Ile Pro Ser Ser Leu Ser Ser Cys Thr Lys
355 360 365
Leu Ser Val Val Gln Leu Arg Gly Asn Gly Phe Asn Gly Thr Ile Pro
370 375 380
Glu Ala Leu Phe Gly Leu Gly Leu Glu Asp Ile Asp Leu Ser His Asn
385 390 395 400
Gly Leu Ser Gly Ser Ile Pro Pro Gly Ser Ser Arg Leu Leu Glu Thr
405 410 415
Leu Thr Asn Leu Asp Leu Ser Asp Asn His Leu Gln Gly Asn Ile Pro
420 425 430
Ala Glu Thr Gly Leu Leu Ser Lys Leu Arg Tyr Leu Asn Leu Ser Trp
435 440 445
Asn Asp Leu His Ser Gln Met Pro Pro Glu Phe Gly Leu Leu Gln Asn
450 455 460
Leu Thr Val Leu Asp Leu Arg Asn Ser Ala Leu His Gly Ser Ile Pro
465 470 475 480
Ala Asp Ile Cys Asp Ser Gly Asn Leu Ala Val Leu Gln Leu Asp Gly
485 490 495
Asn Ser Phe Glu Gly Asn Ile Pro Ser Glu Ile Gly Asn Cys Ser Ser
500 505 510
Leu Tyr Leu Leu Ser Ser Ser His Asn Asn Leu Thr Gly Ser Ile Pro
515 520 525
Lys Ser Met Ala Lys Leu Asn Lys Leu Lys Ile Leu Lys Leu Glu Phe
530 535 540
Asn Glu Leu Ser Gly Glu Ile Pro Met Glu Leu Gly Met Leu Gln Ser
545 550 555 560
Leu Leu Ala Val Asn Ile Ser Tyr Asn Arg Leu Thr Gly Arg Leu Pro
565 570 575
Thr Ser Ser Ile Phe Gln Asn Leu Asp Lys Ser Ser Leu Glu Gly Asn
580 585 590
Leu Gly Leu Cys Ser Pro Leu Leu Lys Gly Pro Cys Lys Met Asn Val
595 600 605
Pro Lys Pro Leu Val Leu Asp Pro Asn Ala Tyr Asn Asn Gln Ile Ser
610 615 620
Pro Gln Arg Gln Arg Asn Glu Ser Ser Glu Ser Gly Gln Val His Arg
625 630 635 640
His Arg Phe Leu Ser Val Ser Ala Ile Val Ala Ile Ser Ala Ser Phe
645 650 655
Val Ile Val Leu Gly Val Ile Ala Val Ser Leu Leu Asn Val Ser Val
660 665 670
Arg Arg Arg Leu Thr Phe Val Asp Asn Ala Leu Glu Ser Met Cys Ser
675 680 685
Ser Ser Ser Arg Ser Gly Ser Pro Ala Thr Gly Lys Leu Ile Leu Phe
690 695 700
Asp Ser His Ser Ser Pro Asp Trp Ile Ser Asn Pro Glu Ser Leu Leu
705 710 715 720
Asn Lys Ala Ser Glu Ile Gly Glu Gly Val Phe Gly Thr Leu Tyr Lys
725 730 735
Val Pro Leu Gly Ser Gln Gly Arg Met Val Ala Ile Lys Lys Leu Ile
740 745 750
Ser Ser Asn Ile Ile Gln Tyr Pro Glu Asp Phe Asp Arg Glu Val Arg
755 760 765
Ile Leu Gly Lys Ala Arg His Pro Asn Leu Ile Ala Leu Lys Gly Tyr
770 775 780
Tyr Trp Thr Pro Gln Leu Gln Leu Leu Val Thr Glu Phe Ala Pro Asn
785 790 795 800
Gly Ser Leu Gln Ala Lys Leu His Glu Arg Leu Pro Ser Ser Pro Pro
805 810 815
Leu Ser Trp Ala Ile Arg Phe Lys Ile Leu Leu Gly Thr Ala Lys Gly
820 825 830
Leu Ala His Leu His His Ser Phe Arg Pro Pro Ile Ile His Tyr Asn
835 840 845
Ile Lys Pro Ser Asn Ile Leu Leu Asp Glu Asn Tyr Asn Ala Lys Ile
850 855 860
Ser Asp Phe Gly Leu Ala Arg Leu Leu Thr Lys Leu Asp Arg His Val
865 870 875 880
Met Ser Asn Arg Phe Gln Ser Ala Leu Gly Tyr Val Ala Pro Glu Leu
885 890 895
Ala Cys Gln Ser Leu Arg Val Asn Glu Lys Cys Asp Val Tyr Gly Phe
900 905 910
Gly Val Met Ile Leu Glu Leu Val Thr Gly Arg Arg Pro Val Glu Tyr
915 920 925
Gly Glu Asp Asn Val Leu Ile Leu Asn Asp His Val Arg Val Leu Leu
930 935 940
Glu His Gly Asn Val Leu Glu Cys Val Asp Gln Ser Met Ser Glu Tyr
945 950 955 960
Pro Glu Asp Glu Val Leu Pro Val Leu Lys Leu Ala Met Val Cys Thr
965 970 975
Ser Gln Ile Pro Ser Ser Arg Pro Thr Met Ala Glu Val Val Gln Ile
980 985 990
Leu Gln Val Ile Lys Thr Pro Val Pro Gln Arg Met Glu Val Phe
995 1000 1005
<210> 5
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atacaccaaa tcgactctag aatgggcaaa cagagaagaa ccat 44
<210> 6
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atccttgtaa tccatcccgg ggaaactatc catgatacgg tggggaacag g 51
<210> 7
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atacaccaaa tcgactctag aatgagcagt tttcagtttc acttg 45
<210> 8
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atccttgtaa tccatcccgg gaaacacttc catcctttga g 41

Claims (3)

1. The nucleotide sequence is shown as SEQ ID NO.1 or SEQ ID NO.2TK1Application of receptor-like kinase gene in constructing phytophthora capsici crop variety.
2. Contains nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO.2TK1Application of a recombinant expression vector of receptor-like kinase genes in constructing phytophthora capsici crop varieties.
3. A method for constructing phytophthora capsici crop variety is characterized in that the nucleotide sequence is shown as SEQ ID NO.1 or SEQ ID NO.2TK1Receptor kinase-like gene or gene containing nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO.2TK1The recombinant expression vector of the receptor-like kinase gene is introduced into crop plants, and positive transformation plants are obtained through resistance screening, so that phytophthora capsici resistant crop varieties are obtained.
CN202011157754.XA 2020-10-26 2020-10-26 Two TK1 receptor kinase genes in arabidopsis thaliana and soybean and application thereof Active CN112442494B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108948161A (en) * 2018-07-11 2018-12-07 南京农业大学 The application of four plant NAC transcription factor NAC089 genes and its expression vector in soybean and Ben's tobacco
CN110713528A (en) * 2019-10-30 2020-01-21 中国科学院东北地理与农业生态研究所 Application of soybean phytophthora root rot resistant related gene GmLMM1

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108948161A (en) * 2018-07-11 2018-12-07 南京农业大学 The application of four plant NAC transcription factor NAC089 genes and its expression vector in soybean and Ben's tobacco
CN110713528A (en) * 2019-10-30 2020-01-21 中国科学院东北地理与农业生态研究所 Application of soybean phytophthora root rot resistant related gene GmLMM1

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