CN114908104B

CN114908104B - Application of rice OsHAK1 protein gene in improving rice blast resistance of rice

Info

Publication number: CN114908104B
Application number: CN202210566985.9A
Authority: CN
Inventors: 宁约瑟; 郝泽芸
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date: 2022-05-18
Filing date: 2022-05-18
Publication date: 2023-09-12
Anticipated expiration: 2042-05-18
Also published as: CN114908104A

Abstract

The application discloses application of a paddy potassium ion transport protein gene OsHAK1 in improving rice blast resistance, wherein a CDS nucleotide sequence of the paddy potassium ion transport protein gene OsHAK1 is shown as SEQ ID No. 1. Transgenic experiments prove that the over-expression of the gene improves the resistance of rice to rice blast germ. Therefore, the gene can be used as a target gene to be introduced into plants to improve the disease resistance of the plants so as to improve the plant varieties. The transferred potassium ion transport protein can also effectively improve the plant defensive ability by improving the potassium ion content of rice.

Description

Application of rice OsHAK1 protein gene in improving rice blast resistance of rice

Technical Field

The application belongs to the field of genetic engineering, and in particular relates to application of a rice OsHAK1 protein gene in improving rice blast resistance of rice.

Background

Potassium (K) ⁺ ) Is the most abundant cation in the plant body, and is one of three nutritional elements (nitrogen, phosphorus and potassium) necessary for plant growth and development. The content of potassium in the plant body is next to nitrogen and accounts for 2% -10% of the dry weight of the plant. Although K ⁺ Is not involved in the composition of organic matters in plants, but is involved in various physiological and biochemical reactions in plants, including the regulation of cell osmotic pressure, the balance of intracellular and extracellular charges, the activation of enzyme activity, the synthesis of proteins, the generation of light and actions, and the like. Research shows that insufficient potassium nutrition can affect the growth and development of crops and the yield and quality of crops.

The plant mainly absorbs potassium from the soil through the root system to meet the self requirement, and the concentration of potassium in the soil is only 0.1-1 mM in general, so that the plant absorbs potassium from the soil in the process of reverse concentration gradient, and in the process, the plant needs to rely on a potassium transport system formed by potassium ion channels, potassium ion transport proteins and the like on the surface of the root system. Whereas plants also need to rely on potassium transport systems to transfer potassium from root to stem and further to different tissues and cells after uptake of potassium nutrition from the soil. In this system, the potassium ion transporter family is able to cope with low potassium stress, at H ⁺ Reverse cell membrane electrochemical potential gradient under the condition of providing energy for ATPase hydrolysis ATP, and make extracellular K ⁺ And (3) transporting the cell into the cell.

Plants can regulate the transcription level of the gene of the potassium ion transporter protein and then regulate the absorption and transport of potassium. For example, KT/HAK/KUP-type transporters have been demonstrated to have high affinity potassium transport functions in plants, and low potassium stress can induce an increase in the expression level of genes encoding HAK1 transporters in various plants. In rice, osHAK1 can mediate both high-affinity potassium transport system and low-affinity potassium transport system, and its expression level can be induced by low potassium. Knockout of OsHAK1 in rice reduces potassium ion absorption and transport by rice, and overexpression of OsHAK1 enhances potassium ion absorption and transport by rice. Therefore, the plant potassium ion transporter OsHAK1 plays an indispensable role in regulating and controlling the potassium ion absorption and transport process of rice.

Based on the research of potassium ion biological functions and potassium ion absorption and transport systems, people also pay attention to important roles of potassium ions in plants in the disease-resistant process of the plants.

It is widely believed in agricultural production that high potassium conditions contribute to reduced incidence of plant disease and insect pests. Statistical data shows that the effect of potassium is different in different crops and different pests. Based on the statistics, potassium is significant in alleviating fungal diseases, while bacterial, insect, viral and nematode diseases are secondary. For example, spraying potassium chloride on wheat can effectively inhibit the germination of the leaf blight and powdery mildew spores so as to inhibit the development of diseases; the harm degree of the rice stem blight and leaf sheath spot can be reduced by applying the potash fertilizer in the soil. These studies indicate that potassium ions play an important role in plant innate immunity.

However, potassium ion transporters in rice have little research in regulating plant disease resistance response.

Disclosure of Invention

The technical problems to be solved by the application are as follows: provides a new application of a rice potassium ion transporter gene.

The technical scheme of the application is as follows: the application of the paddy potassium ion transporter gene OsHAK1 in improving the rice blast resistance of paddy rice is that the CDS nucleotide sequence of the paddy potassium ion transporter gene OsHAK1 is shown as SEQ ID No.1, and the nucleotide sequence coding the same protein as the nucleotide sequence shown as SEQ ID No.1 has the same application due to the degeneracy of codons.

Application of polypeptide coded by paddy potassium ion transporter gene OsHAK1 in improving paddy rice blast resistance is provided, and the amino acid sequence of the polypeptide is shown as SEQ ID No. 2.

An expression vector comprising a nucleic acid fragment encoding a polypeptide as set forth in SEQ ID No. 2. Further, the nucleic acid fragment is shown as SEQ ID No. 1.

A method for improving rice blast resistance of rice includes cloning potassium ion transport protein gene OsHAK1 of rice, constructing plant expression carrier, transferring said carrier into agrobacterium, inoculating to rice, selecting over-expression strain with resistance to rice blast and its offspring, and the CDS nucleotide sequence of potassium ion transport protein gene OsHAK1 of rice is shown in SEQ ID No.1 or the nucleotide sequence coding same protein as that of SEQ ID No. 1.

And (3) carrying out qRT-PCR verification on the obtained transgenic plant, and then carrying out disease resistance evaluation on rice. Transgenic rice plants grown to 5-8 weeks old were inoculated with the wells and the disease was investigated for 10-14 days. Consistent results indicate that transgenic plants show significant resistance to Pyricularia oryzae compared to the control.

Compared with the prior art, the application has the following beneficial effects:

transgenic experiments prove that the over-expression of the gene improves the resistance of rice to rice blast germ. Therefore, the gene can be used as a target gene to be introduced into plants to improve the disease resistance of the plants so as to improve the plant varieties. The transferred potassium ion transport protein can also effectively improve the plant defensive ability by improving the potassium ion content of rice.

Drawings

FIG. 1 is a map of pRHVcGFP expression vector;

FIG. 2 shows the analysis of the expression level of OsHAK1 in transgenic rice. WT represents wild type, osHAK1-OX-7-8, osHAK1-OX-13-1 and OsHAK1-OX-2-4 represent three overexpressing strains;

FIG. 3 shows that OsHAK1-OX transgenic plants enhanced disease resistance to physiological race RO1-1 of Pyricularia oryzae;

FIG. 4 shows that OsHAK1-OX transgenic plants enhanced disease resistance to physiological race RB22 of Pyricularia oryzae;

FIG. 5 shows that the area of lesions generated after inoculation of a physiological race RO1-1 of Pyricularia oryzae with OsHAK1-OX transgenic plants is significantly reduced;

FIG. 6 shows that the area of lesions generated after inoculation of a physiological race RB22 of Pyricularia oryzae with OsHAK1-OX transgenic plants is significantly reduced;

FIG. 7 shows that fungal biomass in the plaque area is significantly reduced after inoculation of a physiological race RO1-1 of Pyricularia oryzae with OsHAK1-OX transgenic plants;

FIG. 8 shows that the fungal biomass in the plaque area was significantly reduced after inoculation of the OsHAK1-OX transgenic plants with a physiological race of Pyricularia oryzae RB 22.

Detailed Description

The biological material used in the application:

the physiological species RO1-1 and RB22 of Nipponbare are conventional materials in the prior art, and are preserved in the laboratory.

pRHVcGFP vector: the laboratory is created, the plasmid map is shown in figure 1, and the vector sequence is shown in SEQ ID No. 5.

Example 1

1) Extraction of Total RNA

Selecting japonica rice variety Nipponbare, immediately taking leaf liquid nitrogen for freezing when seedling grows to about two weeks, and storing in-80deg.C refrigerator. Taking part of the leaves, grinding by using a mortar, transferring into a 1.5ml EP tube containing Trizol lysate, fully oscillating, extracting total RNA, and identifying the total RNA quality by electrophoresis.

2) Cloning of rice potassium ion transporter gene OsHAK1 and construction of plant expression vector

Designing two-end primers:

P1：5-ATAGGATCCATGTCGTCGGCGCTGGAGGT-3(SEQ ID No.3)

P2：5-CGCACTAGTGATTTCATACGTGATCCCAAC-3(SEQ ID No.4)

reverse transcription of the total RNA obtained in step 1) into a cDNA first strand, and PCR amplification with high-fidelity KOD enzyme using the cDNA first strand as a template, wherein the PCR procedure is as follows: the target fragment was recovered by pre-denaturing at 96℃for 3min, denaturing at 96℃for 30s, renaturation at 57℃for 30s, elongation at 68℃for 1min, and elongation at 68℃for 10min after 32 cycles. Meanwhile, pRHVcGFP vector was digested with restriction enzymes BamHI and SpeI, and then the digested vector fragment was recovered, osHAK1 was further cloned into expression vector pRHVcGFP (FIG. 1), and sequencing identification was performed to ensure correct reading frame of coding region in the expression vector.

3) Obtaining transgenic plants

Transferring the expression vector pRHVcGFP-OsHAK1 obtained in the step 2) into an agrobacterium strain EHA105, further transferring into a japonica rice variety Nipponbare, and performing qRT-PCR verification on the obtained transgenic plant to evaluate the disease resistance of the rice (figure 2).

Example 2

Identification of disease resistance of OsHAK1-OX transgenic lines:

punching and inoculating: when the transgenic rice grows to 5-8 weeks old, selecting two inverted leaves to perform punching inoculation on rice blast fungi (physiological rice blast fungi micro-seeds RO1-1 and RB 22), then placing the rice in a plant growth box to be dark-cultured for 24 hours, transferring the rice to 12 hours of illumination, and growing for 10-14 days under the condition of 12 hours of darkness; observing the disease spot expansion condition, investigating the disease condition, and counting the disease spot area and biomass, compared with the control, the transgenic rice plant with obvious resistance is the obtained rice blast resistant transgenic plant.

As a result, it was found that the OsHAK1 overexpressing transgenic plants had enhanced disease resistance against Pyricularia oryzae (FIG. 3, FIG. 4) as compared with the wild-type plants (Table 1, table 2) and relative fungal biomass (Table 3, table 4) were counted, and it was found that the disease area after the OsHAK1 overexpressing transgenic plants were inoculated with Pyricularia oryzae was only about 50% of the control (FIG. 5, FIG. 6), and the fungal biomass in the disease area was reduced to about 50% of the control (FIG. 7, FIG. 8).

TABLE 1 relative plaque area of transgenic lines and wild-type post-seed for the inoculation of physiological race RO1-1 of Pyricularia oryzae

TABLE 2 relative plaque area of leaves following inoculation of a physiological microspecies RB22 of Pyricularia oryzae by transgenic lines and wild type

TABLE 3 relative fungal biomass of transgenic lines and wild-type post-inoculation Pyricularia oryzae physiological race RO1-1 postemergent leaves

TABLE 4 relative fungal biomass of transgenic lines and wild type post-inoculation Pyricularia oryzae physiological race RB22 post-emergence leaves

The above examples merely illustrate specific embodiments of the application, which are described in more detail and are not to be construed as limiting the scope of the application. It should be noted that it is possible for a person skilled in the art to make several variants and modifications without departing from the technical idea of the application, which fall within the scope of protection of the application.

Sequence listing

<110> institute of plant protection of national academy of agricultural sciences

Application of <120> rice OsHAK1 protein gene in improving rice blast resistance of rice

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2379

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

atgtcgtcgg cgctggaggt ggagggctcg ggctcgccgg gcgtcgagcc ggcggcgacg 60

gcgacggcga gcaggctgaa gcggcacgac tcgctgttcg gagacgcgga gaaggtgagc 120

ggcgggaagc accacggggg gtcggcggtg agctgggcgg tgacgctgca cctggcgttc 180

cagagcgtgg gcatcatcta cggcgacatc ggcacgtcgc cgctgtacgt ctactccagc 240

acgttcccgg acggcatcgg ccaccgcgac gacctcgtcg gcgtcctctc cctcatcctc 300

tacaccctca tcatcatccc catgctcaag tacgtcttca tcgtcctcta cgccaacgac 360

aatggcgacg gtggcacgtt cgcgctctac tcgctgatct cccggtacgc caagatcagg 420

atgatcccga accagcaggc ggaggacgcc atggtgtcca actacagcat cgaggcgccc 480

agctcgcagc tgaggagggc gcagtgggtg aagcacaagc tcgagagcag ccgtgccgcc 540

aagatggcgc tcttcttcct caccatcctc ggcacctcca tggtcatggg cgacggcacc 600

ttgacccccg ccatctccgt gctctctgcg gtgagcggga tcagggagaa agctccaaac 660

ttgactcaga cgcaagtcgt gctgatctcg gtggccatcc tgttcatgct cttctccgtc 720

cagcggttcg gcaccgacaa ggtcggctac accttcgccc cgatcatctc ggtgtggttc 780

ctcctcatcg ccggcatcgg gctgtacaac ctcgtcgtcc acgagatcac catcctcaag 840

gccttcaatc cgtggtacat cgtgcagtac ttcaggagga acggcaagaa gggttgggtg 900

tctctcggtg gcgtcgtcct ctgtgtcaca ggcacagagg gaatgtttgc cgacctgggc 960

catttcaaca tcagggccgt gcagatcagc ttcaactgca tcctgttccc atcggtggcg 1020

ctctgctaca tcgggcaggc agcgtacctg aggaaattcc ctgagaatgt cagtgacacc 1080

ttctacaaat ccatcccagg gccgctgttc tggccgacgt tcatcgtcgc gatcctcgcc 1140

gccatcatcg ccagccaggc catgctctcc ggcgcgtttg ccatcctctc caaggcgctg 1200

tccctcgggt gcttgcccag ggtccgggtg atccacacct ctaagaagta cgaggggcag 1260

gtgtacatcc ccgaggtgaa cttcatgatg ggtctcgcca gcatcatcgt caccatcgcc 1320

ttcaggacca ccaccagcat cggcaacgcc tacgggatct gtgtggtgac gacgttcatg 1380

gtgacgacgc acctgatgac ggtggtgatg ctgctgatat ggaagaagca cctggtgttc 1440

atcctgctct tctactgcgt gttcgggttc acggaggtgg tgtacctgtc gtcgatcctg 1500

tccaagttcg tggacggcgg gtacctcccc ttctgcttcg ccatggtgct gatgacgatg 1560

atggcgacgt ggcactacgt ccacgtgcgg cgctactggt acgagctgga ccacatcgtc 1620

cccacggcgg agctggcgtc gctgctggag gagaacggcg gcgtgcggcg ggtccccggc 1680

gtgggcctcc tctacacgga gctcgtccag ggcatcccgc cgctgttccc gcgcctcgtc 1740

cgcaagatcc cctcggtcca cgccgtcttc gtcttcatct ccatcaagca cctccccatc 1800

ccgcacgtcg ccgccgccga gcgcttcctg ttccgccagg tcggcccccg cgcccgccgc 1860

gtcttccgct gcgtcgcccg ctacggctac accgacgccc tcgaggagcc ccgggagttc 1920

gccgccttcc tcgtcgacgg cctcaagatg ttcatccagg aggagtccgc cttcgctcct 1980

catcaagaga tgatcgatgc cgccgccgac gacgacgacg aggcggcggc gaggccgagg 2040

cggtcgacga gctcggccgt gcacagcgag gaggcgatcc aggcggcgtc gtcggggagg 2100

acgacggcca gcagcgtcca gctccaggcg ggcggcgagc cgccggcggc gatggacgtg 2160

gaggaggaga agcggctgat cgacagggag gtgggccgcg gcgtggtgta cctgatgggc 2220

gaggccaacg tgtcggcggg gcccaactcg tccatcctca agaggatcgc ggtgaactac 2280

atctacacct tcctgaggaa gaacctgaca gaagggcaca gggcactggc gattccaaac 2340

gatcagctgc tcaaggttgg gatcacgtat gaaatctag 2379

<210> 2

<211> 792

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Met Ser Ser Ala Leu Glu Val Glu Gly Ser Gly Ser Pro Gly Val Glu

1 5 10 15

Pro Ala Ala Thr Ala Thr Ala Ser Arg Leu Lys Arg His Asp Ser Leu

20 25 30

Phe Gly Asp Ala Glu Lys Val Ser Gly Gly Lys His His Gly Gly Ser

35 40 45

Ala Val Ser Trp Ala Val Thr Leu His Leu Ala Phe Gln Ser Val Gly

50 55 60

Ile Ile Tyr Gly Asp Ile Gly Thr Ser Pro Leu Tyr Val Tyr Ser Ser

65 70 75 80

Thr Phe Pro Asp Gly Ile Gly His Arg Asp Asp Leu Val Gly Val Leu

85 90 95

Ser Leu Ile Leu Tyr Thr Leu Ile Ile Ile Pro Met Leu Lys Tyr Val

100 105 110

Phe Ile Val Leu Tyr Ala Asn Asp Asn Gly Asp Gly Gly Thr Phe Ala

115 120 125

Leu Tyr Ser Leu Ile Ser Arg Tyr Ala Lys Ile Arg Met Ile Pro Asn

130 135 140

Gln Gln Ala Glu Asp Ala Met Val Ser Asn Tyr Ser Ile Glu Ala Pro

145 150 155 160

Ser Ser Gln Leu Arg Arg Ala Gln Trp Val Lys His Lys Leu Glu Ser

165 170 175

Ser Arg Ala Ala Lys Met Ala Leu Phe Phe Leu Thr Ile Leu Gly Thr

180 185 190

Ser Met Val Met Gly Asp Gly Thr Leu Thr Pro Ala Ile Ser Val Leu

195 200 205

Ser Ala Val Ser Gly Ile Arg Glu Lys Ala Pro Asn Leu Thr Gln Thr

210 215 220

Gln Val Val Leu Ile Ser Val Ala Ile Leu Phe Met Leu Phe Ser Val

225 230 235 240

Gln Arg Phe Gly Thr Asp Lys Val Gly Tyr Thr Phe Ala Pro Ile Ile

245 250 255

Ser Val Trp Phe Leu Leu Ile Ala Gly Ile Gly Leu Tyr Asn Leu Val

260 265 270

Val His Glu Ile Thr Ile Leu Lys Ala Phe Asn Pro Trp Tyr Ile Val

275 280 285

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

290 295 300

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

305 310 315 320

His Phe Asn Ile Arg Ala Val Gln Ile Ser Phe Asn Cys Ile Leu Phe

325 330 335

Pro Ser Val Ala Leu Cys Tyr Ile Gly Gln Ala Ala Tyr Leu Arg Lys

340 345 350

Phe Pro Glu Asn Val Ser Asp Thr Phe Tyr Lys Ser Ile Pro Gly Pro

355 360 365

Leu Phe Trp Pro Thr Phe Ile Val Ala Ile Leu Ala Ala Ile Ile Ala

370 375 380

Ser Gln Ala Met Leu Ser Gly Ala Phe Ala Ile Leu Ser Lys Ala Leu

385 390 395 400

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

405 410 415

Tyr Glu Gly Gln Val Tyr Ile Pro Glu Val Asn Phe Met Met Gly Leu

420 425 430

Ala Ser Ile Ile Val Thr Ile Ala Phe Arg Thr Thr Thr Ser Ile Gly

435 440 445

Asn Ala Tyr Gly Ile Cys Val Val Thr Thr Phe Met Val Thr Thr His

450 455 460

Leu Met Thr Val Val Met Leu Leu Ile Trp Lys Lys His Leu Val Phe

465 470 475 480

Ile Leu Leu Phe Tyr Cys Val Phe Gly Phe Thr Glu Val Val Tyr Leu

485 490 495

Ser Ser Ile Leu Ser Lys Phe Val Asp Gly Gly Tyr Leu Pro Phe Cys

500 505 510

Phe Ala Met Val Leu Met Thr Met Met Ala Thr Trp His Tyr Val His

515 520 525

Val Arg Arg Tyr Trp Tyr Glu Leu Asp His Ile Val Pro Thr Ala Glu

530 535 540

Leu Ala Ser Leu Leu Glu Glu Asn Gly Gly Val Arg Arg Val Pro Gly

545 550 555 560

Val Gly Leu Leu Tyr Thr Glu Leu Val Gln Gly Ile Pro Pro Leu Phe

565 570 575

Pro Arg Leu Val Arg Lys Ile Pro Ser Val His Ala Val Phe Val Phe

580 585 590

Ile Ser Ile Lys His Leu Pro Ile Pro His Val Ala Ala Ala Glu Arg

595 600 605

Phe Leu Phe Arg Gln Val Gly Pro Arg Ala Arg Arg Val Phe Arg Cys

610 615 620

Val Ala Arg Tyr Gly Tyr Thr Asp Ala Leu Glu Glu Pro Arg Glu Phe

625 630 635 640

Ala Ala Phe Leu Val Asp Gly Leu Lys Met Phe Ile Gln Glu Glu Ser

645 650 655

Ala Phe Ala Pro His Gln Glu Met Ile Asp Ala Ala Ala Asp Asp Asp

660 665 670

Asp Glu Ala Ala Ala Arg Pro Arg Arg Ser Thr Ser Ser Ala Val His

675 680 685

Ser Glu Glu Ala Ile Gln Ala Ala Ser Ser Gly Arg Thr Thr Ala Ser

690 695 700

Ser Val Gln Leu Gln Ala Gly Gly Glu Pro Pro Ala Ala Met Asp Val

705 710 715 720

Glu Glu Glu Lys Arg Leu Ile Asp Arg Glu Val Gly Arg Gly Val Val

725 730 735

Tyr Leu Met Gly Glu Ala Asn Val Ser Ala Gly Pro Asn Ser Ser Ile

740 745 750

Leu Lys Arg Ile Ala Val Asn Tyr Ile Tyr Thr Phe Leu Arg Lys Asn

755 760 765

Leu Thr Glu Gly His Arg Ala Leu Ala Ile Pro Asn Asp Gln Leu Leu

770 775 780

Lys Val Gly Ile Thr Tyr Glu Ile

785 790

<210> 3

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ataggatcca tgtcgtcggc gctggaggt 29

<210> 4

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

cgcactagtg atttcatacg tgatcccaac 30

<210> 5

<211> 9381

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

atgcaggtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga 60

acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa 120

cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa 180

tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc 240

gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat 300

gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt 360

atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc 420

caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc 480

ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt 540

cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat 600

gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca 660

ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac 720

gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag 780

gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt 840

tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc 900

gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg 960

acttgacggg acggcgcaag ctcatgacca aaatccctta acgtgagttt tcgttccact 1020

gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 1080

taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 1140

aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 1200

ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 1260

catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 1320

ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 1380

ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 1440

agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 1500

taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 1560

atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 1620

cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 1680

ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 1740

accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 1800

gcgagtcagt gagcgaggaa gcggaagagc gcccggcgcg ccgaatgaac gccaagagga 1860

acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac cgaagagatc 1920

gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt ctcaaccgtg 1980

cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg gccggccagc 2040

ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt tgagtaaaac 2100

agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca aatacgcaag 2160

gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc aagacgacca 2220

tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg ttagtcgatt 2280

ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa ccgctaaccg 2340

ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc cggcgcgact 2400

tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg atcaaggcag 2460

ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc accgccgacc 2520

tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa gcggcctttg 2580

tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag gcgctggccg 2640

ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac ccaggcactg 2700

ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc cgcgaggtcc 2760

aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta aagagaaaat 2820

gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca gcaaggctgc 2880

aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc agttgccggc 2940

ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca ttaccgagct 3000

gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa atgagtagat 3060

gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc accgacgccg 3120

tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc tgggttgcct 3180

gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga gcggtcgcaa 3240

accatccggc ccggtacaaa tcggcgcggc gctgggtgat gacctggtgg agaagttgaa 3300

ggccgcgcag gccgcccagc ggcaacgcat cgaggcagaa gcacgccccg gtgaatcgtg 3360

gcaagcgtcc gctgatcgaa tccgcaaaga atcccggcaa ccgccggcag ccggtgcgcc 3420

gtcgattagg aagccgccca agggcgacga gcaaccagat tttttcgttc cgatgctcta 3480

tgacgtgggc acccgcgata gtcgcagcat catggacgtg gccgttttcc gtctgtcgaa 3540

gcgtgaccga cgagctggcg aggtgatccg ctacgagctt ccagacgggc acgtagaggt 3600

ttccgcaggg ccggccggca tggccagtgt gtgggattac gacctggtac tgatggcggt 3660

ttcccatcta accgaatcca tgaaccgata ccgggaaggg aagggagaca agcccggccg 3720

cgtgttccgt ccacacgttg cggacgtact caagttctgc cggcgagccg atggcggaaa 3780

gcagaaagac gacctggtag aaacctgcat tcggttaaac accacgcacg ttgccatgca 3840

gcgtacgaag aaggccaaga acggccgcct ggtgacggta tccgagggtg aagccttgat 3900

tagccgctac aagatcgtaa agagcgaaac cgggcggccg gagtacatcg agatcgagct 3960

agctgattgg atgtaccgcg agatcacaga aggcaagaac ccggacgtgc tgacggttca 4020

ccccgattac tttttgatcg atcccggcat cggccgtttt ctctaccgcc tggcacgccg 4080

cgccgcaggc aaggcagaag ccagatggtt gttcaagacg atctacgaac gcagtggcag 4140

cgccggagag ttcaagaagt tctgtttcac cgtgcgcaag ctgatcgggt caaatgacct 4200

gccggagtac gatttgaagg aggaggcggg gcaggctggc ccgatcctag tcatgcgcta 4260

ccgcaacctg atcgagggcg aagcatccgc cggttcctaa tgtacggagc agatgctagg 4320

gcaaattgcc ctagcagggg aaaaaggtcg aaaaggtctc tttcctgtgg atagcacgta 4380

cattgggaac ccaaagccgt acattgggaa ccggaacccg tacattggga acccaaagcc 4440

gtacattggg aaccggtcac acatgtaagt gactgatata aaagagaaaa aaggcgattt 4500

ttccgcctaa aactctttaa aacttattaa aactcttaaa acccgcctgg cctgtgcata 4560

actgtctggc cagcgcacag ccgaagagct gcaaaaagcg cctagtttac ccgccaatat 4620

atcctgtcag gcgcgccccg atctagtaac atagatgaca ccgcgcgcga taatttatcc 4680

tagtttgcgc gctatatttt gttttctatc gcgtattaaa tgtataattg cgggactcta 4740

atcataaaaa cccatctcat aaataacgtc atgcattaca tgttaattat tacatgctta 4800

acgtaattca acagaaatta tatgataatc atcgcaagac cggcaacagg attcaatctt 4860

aagaaacttt attgccaaat gtttgaacga tctactcact tagcggccgt tacttgtaca 4920

gctcgtccat gccgagagtg atcccggcgg cggtcacgaa ctccagcagg accatgtgat 4980

cgcgcttctc gttggggtct ttgctcaggg cggactgggt gctcaggtag tggttgtcgg 5040

gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca 5100

cgctgccgtc ctcgatgttg tggcggatct tgaagttcac cttgatgccg ttcttctgct 5160

tgtcggccat gatatagacg ttgtggctgt tgtagttgta ctccagcttg tgccccagga 5220

tgttgccgtc ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc 5280

cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag aagatggtgc 5340

gctcctggac gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt 5400

cggggtagcg gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg 5460

gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg taggtggcat 5520

cgccctcgcc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg tccagctcga 5580

ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccata gcggccgcac 5640

tagtaagctt ggtaccgagc tcacccgggg atcccactgg atctggatat cctgcataag 5700

taacaccaaa caacagggtg agcatcgaca aaagaaacag taccaagcaa ataaatagcg 5760

tatgaaggca gggctaaaaa aatccacata tagctgctgc atatgccatc atccaagtat 5820

atcaagatca aaataattat aaaacatact tgtttattat aatagatagg tactcaaggt 5880

tagagcatat gaatagatgc tgcatatgcc atcatgtata tgcatcagta aaacccacat 5940

caacatgtat acctatccta gatcgatatt tccatccatc ttaaactcgt aactatgaag 6000

atgtatgaca cacacataca gttccaaaat taataaatac accaggtagt ttgaaacagt 6060

attctactcc gatctagaac gaatgaacga ccgcccaacc acaccacatc atcacaacca 6120

agcgaacaaa aagcatctct gtatatgcat cagtaaaacc cgcatcaaca tgtataccta 6180

tcctagatcg atatttccat ccatcatctt caattcgtaa ctatgaatat gtatggcaca 6240

cacatacaga tccaaaatta ataaatccac caggtagttt gaaacagaat tctactccga 6300

tctagaacga ccgcccaacc agaccacatc atcacaacca agacaaaaaa aagcatgaaa 6360

agatgacccg acaaacaagt gcacggcata tattgaaata aaggaaaagg gcaaaccaaa 6420

ccctatgcaa cgaaacaaaa aaaatcatga aatcgatccc gtctgcggaa cggctagagc 6480

catcccagga ttccccaaag agaaacactg gcaagttagc aatcagaacg tgtctgacgt 6540

acaggtcgca tccgtgtacg aacgctagca gcacggatct aacacaaaca cggatctaac 6600

acaaacatga acagaagtag aactaccggg ccctaaccat ggaccggaac gccgatctag 6660

agaaggtaga gagggggggg gggggaggac gagcggcgta ccttgaagcg gaggtgccga 6720

cgggtggatt tgggggagat ctggttgtgt gtgtgtgcgc tccgaacaac acgaggttgg 6780

ggaaagaggg tgtggagggg gtgtctattt attacggcgg gcgaggaagg gaaagcgaag 6840

gagcggtggg aaaggaatcc cccgtagctg ccggtgccgt gagaggagga ggaggccgcc 6900

tgccgtgccg gctcacgtct gccgctccgc cacgcaattt ctggatgccg acagcggagc 6960

aagtccaacg gtggagcgga actctcgaga ggggtccaga ggcagcgaca gagatgccgt 7020

gccgtctgct tcgcttggcc cgacgcgacg ctgctggttc gctggttggt gtccgttaga 7080

ctcgtcgacg gcgtttaaca ggctggcatt atctactcga aacaagaaaa atgtttcctt 7140

agttttttta atttcttaaa gggtatttgt ttaattttta gtcactttat tttattctat 7200

tttatatcta aattattaaa taaaaaaact aaaatagagt tttagttttc ttaatttaga 7260

ggctaaaata gaataaaata gatgtactaa aaaaattagt ctataaaaac cattaaccct 7320

aaaccctaaa tggatgtact aataaaatgg atgaagtatt atataggtga agctatttgc 7380

aaaaaaaaag gagaacacat gcacactaaa aagataaaac tgtagagtcc tgttgtcaaa 7440

atactcaatt gtcctttaga ccatgtctaa ctgttcattt atatgattct ctaaaacact 7500

gatattattg tagtactata gattatatta ttcgtagagt aaagtttaaa tatatgtata 7560

aagatagata aactgcactt caaacaagtg tgacaaaaaa aatatgtggt aattttttat 7620

aacttagaca tgcaatgctc attatctcta gagaggggca cgaccgggtc acgctgcact 7680

gcagtaattc gggggatctg gattttagta ctggattttg gttttaggaa ttagaaattt 7740

tattgataga agtattttac aaatacaaat acatactaag ggtttcttat atgctcaaca 7800

catgagcgaa accctatagg aaccctaatt cccttatctg ggaactactc acacattatt 7860

atggagaaac tcgagcttgt cgatcgacag atcccggtcg gcatctactc tatttctttg 7920

ccctcggacg agtgctgggg cgtcggtttc cactatcggc gagtacttct acacagccat 7980

cggtccagac ggccgcgctt ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg 8040

atcggacgat tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa 8100

ccaagctctg atagagttgg tcaagaccaa tgcggagcat atacgcccgg agtcgtggcg 8160

atcctgcaag ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc atacaagcca 8220

accacggcct ccagaagaag atgttggcga cctcgtattg ggaatccccg aacatcgcct 8280

cgctccagtc aatgaccgct gttatgcggc cattgtccgt caggacattg ttggagccga 8340

aatccgcgtg cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc atcagctcat 8400

cgagagcctg cgcgacggac gcactgacgg tgtcgtccat cacagtttgc cagtgataca 8460

catggggatc agcaatcgcg catatgaaat cacgccatgt agtgtattga ccgattcctt 8520

gcggtccgaa tgggccgaac ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca 8580

tagcctccgc gaccggttgt agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg 8640

tgacaccctg tgtacggcgg gagatgcaat aggtcaggct ctcgctaaac tccccaatgt 8700

caagcacttc cggaatcggg agcgcggccg atgcaaagtg ccgataaaca taacgatctt 8760

tgtagaaacc atcggcgcag ctatttaccc gcaggacata tccacgccct cctacatcga 8820

agctgaaagc acgagattct tcgccctccg agagctgcat caggtcggag acgctgtcga 8880

acttttcgat cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt ttcattgtcc 8940

tctccaaatg aaatgaactt ccttatatag aggaagggtc ttgcgaagga tagtgggatt 9000

gtgcgtcatc ccttacgtca gtggagatgt cacatcaatc cacttgcttt gaagacgtgg 9060

ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg gggtccatct ttgggaccac 9120

tgtcggcaga gagatcttga atgatagcct ttcctttatc gcaatgatgg catttgtagg 9180

agccaccttc cttttctact gtcctttcga tgaagtgaca gatagctggg caatggaatc 9240

cgaggaggtt tcccgaaatt atcctttgtt gaaaagtctc aatagccctt tggtcttctg 9300

agactgtatc tttgacattt ttggagtaga ccagagtgtc gtgctccacc atgtttgttt 9360

acaccacaat atatcctgcc a 9381

Claims

1. Rice potassium ion transporter geneOsHAK1Application of potassium ion transfer in improving rice blast resistance of riceProtein transporting geneOsHAK1The CDS nucleotide sequence of (C) is shown as SEQ ID No.1 or the sequence of the same protein encoded by the nucleotide sequence shown as SEQ ID No. 1.

2. Rice potassium ion transporter geneOsHAK1The coded polypeptide has the amino acid sequence shown in SEQ ID No.2 and is applied to improving rice blast resistance of rice.

3. A method for improving rice blast resistance of rice is characterized by comprising the following steps of

OsHAK1

Cloning and constructing a plant expression vector, transferring the expression vector into agrobacterium, inoculating to rice, and selecting a super-expression strain with rice blast resistance and a progeny thereof, wherein the rice potassium ion transporter gene

OsHAK1

The CDS nucleotide sequence of (C) is shown as SEQ ID No.1 or the sequence of the same protein encoded by the nucleotide sequence shown as SEQ ID No. 1.