CN110452293B

CN110452293B - Rice pest-resistant regulatory protein and coding gene and application thereof

Info

Publication number: CN110452293B
Application number: CN201910772793.1A
Authority: CN
Inventors: 李怡峰; 张振飞; 肖汉祥; 李燕芳; 张扬
Original assignee: Plant Protection Research Institute Guangdong Academy of Agricultural Sciences
Current assignee: Plant Protection Research Institute Guangdong Academy of Agricultural Sciences
Priority date: 2019-08-21
Filing date: 2019-08-21
Publication date: 2022-03-29
Anticipated expiration: 2039-08-21
Also published as: CN110452293A; JP2021029248A

Abstract

The invention discloses a rice insect-resistant regulatory protein Os04g05560, and a coding gene and application thereof. The reduction or knockout of the expression of the protein coding gene can lead the pest-susceptible plant to have high pest resistance, thereby being capable of cultivating the plant with high resistant plant hopper. The protein and the coding gene thereof can be applied to plant genetic improvement.

Description

Rice pest-resistant regulatory protein and coding gene and application thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a rice insect-resistant regulatory protein, and a coding gene and application thereof.

Background

With the introduction of human beings into the post-genome era, the full development of functional genome research has become the leading field of life science research. Rice is considered as a model plant because of its relative ease of transgenic technology and co-linearity with the genomes of other gramineous crops. At present, the fine genetic map and the physical map of the rice genome are completed, the functional genes of rice are further researched, and the method has great significance for the development of social economy and the research of biology.

At present, more than half of people in the world use rice as staple food. The problem of food safety is a challenge for people all over the world. The dwarfing breeding in the 50 th and 60 th of the 20 th century and the two technological revolution of the hybrid rice cultivation in the 70 th of the century greatly improve the rice yield. However, in recent decades, rice is threatened by large-area pest and disease damage. Brown planthopper is a main pest in rice production in China, and adult insects and nymphs of brown planthopper suck rice juice through mouth needling to cause yellow leaves or withering, so that yield is reduced or the rice is not harvested. According to the records of Chinese agricultural yearbook, brown planthopper insect pests occur nationwide in 1966, 1969, 1973, 1977, 1983 and 2003, and are extremely large nationwide in 1987, 1991, 2005, 2006 and 2007, the damage area reaches more than 50% of the total area of rice, and serious loss is caused to rice production in China. And the harm of brown planthopper is mostly generated in the mature grain filling stage of rice, and the pollution to paddy is also very serious problem when a large amount of insecticide is used.

The cultivation of the anti-brown planthopper rice variety by using the anti-brown planthopper gene is the most economic and effective method in the comprehensive prevention and control of brown planthopper. The research results of the International Rice Research Institute (IRRI) and the rice production practice in southeast asia show that even rice varieties with only moderate resistance level can sufficiently control the brown planthopper population below the level causing harm, so that the actual harm and yield loss to rice are avoided. Therefore, the mining of the brown planthopper resistance gene of the rice and the application of the gene in a rice breeding project are fundamental measures for preventing and treating the brown planthopper of the rice.

Since the 60's of the 20 th century, research on resistance inheritance and breeding of brown planthoppers has begun, but with the development of new biotypes (or new nociceptive types), insect-resistant varieties are at risk of shortened life span and loss of resistance. For example, the international rice institute introduced the variety IR26 with the gene Bph1 in 1973, and after 2-3 years, the virulent biotype 2 appeared; 1977 and 1978, the varieties IR36 and IR42 with the Bph2 resistance gene were deduced, and as a result, new biotypes of brown planthopper appeared in some countries successively in 1982, and corresponding new resistant varieties IR56 and IR64 had to be cultivated again in 1983. Several korean rice varieties carrying different anti-plant hopper genes were tested by Seo in 2006, among which the insect resistance of rice plants of chenogchenog byo (containing Bph1 gene), ASD7 and M63 (containing Bph2 gene) was reduced, but the rice plants of gayabyo (containing both Bph1 and Bph2 genes) were also very good (Seo et al, 2009). The level of harm of Ptb33 (containing Bph2 and Bph3 genes at the same time) is low, the damage level is 2.5, and the variety shows insect resistance (Zhang et al, 2011).

Therefore, it is of great significance to continue to deeply screen and research the resistance source material, search for new resistance genes, locate and clone the related genes, and research and develop new rice varieties with high resistance gene material.

Disclosure of Invention

The invention aims to provide a rice insect pest resistance regulation protein, a coding gene and application thereof.

The technical scheme adopted by the invention is as follows:

the protein Os04g05560 is applied as rice insect-resistant regulation protein, and the amino acid sequence of the protein Os04g05560 is as follows:

a) SEQ ID NO: 1;

or b) SEQ ID NO: 1 through substitution and/or deletion and/or addition of one or more amino acids,

wherein, SEQ ID NO: 1 is:

MACLQLQVLLLLACLLLDAPHLSSAAATVPTPPFSFNFDFSNMSTYKPDDLRFEGNATVHGSFVDLTCNAYGLDISQCTAGRMSYNHPVPFYDQTTKEVASFSTQFTFKIIVPRFNNDKEKGDGMAFFLARYPSRMPPDSGGGSLGLITNNNYSSFGPDQFVSVEFDTYNNTWEQPKQTGDHMGININTVTFSTNTTSVSSFSPNESMMKASITFDSKTSMLVASLQYTGNYSNYAPVNVSAKLPDPTTLLPSEVAVGFSAATGAAFELHQIHSWSFNSTIAAPVQKDHKKAIAVGVSIGGGLILVLLVWSILSWWKWRKTNREFDKGTRGACRFNYHRLAAATNHFSMDNRIGAGTFGEVHKGFLTQLGREVAVKKILRESRAGNKDFFDEVQTISRAKQKNLVELLGWGMKGSSIIDFVMCWRRQKNTDLFLVYEFVDNGNLHMHLYEKEALLSWRIRYKIVKGIISALVYLHHDRHPYILHRDIKPSNILLDKNFNARLADFGLSRTADNGTIQSSMVVGTENYLDPECRKTGKFNRSSDVFSFGLVLLEIACKKDENSYAQVWERYIDKTLMQAADDRLQGAFDKRQMERVIVLGLWCCQPNIEMRPTMEKAMDFLESDGPLPKLAKPEITSSSAPSN。

further, the insect pest refers to rice planthopper; preferably, the rice planthopper refers to at least one of brown planthopper, sogatella furcifera and laodelphax striatellus.

The coding gene of the protein Os04g05560 is applied as a rice anti-planthopper regulation gene.

Further, the nucleotide sequence of the coding gene is shown as SEQ ID NO: as shown in figure 2, the first and second,

further, the nucleotide sequence of the cDNA of the coding gene is SEQ ID NO: 3, respectively.

A method for improving the pest resistance of rice comprises the following steps: the expression level of the protein Os04g05560 in rice is reduced, and the amino acid sequence of the protein Os04g05560 is as follows: a) SEQ ID NO: 1; or b) SEQ ID NO: 1 by substitution and/or deletion and/or addition of one or more amino acids.

Furthermore, the reduction of the expression level of the protein Os04g05560 is realized by knocking out or reducing the expression level of the coding gene of the protein Os04g05560, and the nucleotide sequence of the coding gene is shown as SEQ ID NO: 2, respectively.

Further, insect pests refer to rice planthoppers; preferably, the rice planthopper refers to at least one of brown planthopper, sogatella furcifera and laodelphax striatellus.

A cultivation method of pest-resistant rice comprises the following steps: the expression level of the protein Os04g05560 in the rice is reduced to improve the insect resistance of the rice, wherein the amino acid sequence of the protein Os04g05560 is as follows: a) SEQ ID NO: 1; or b) SEQ ID NO: 1 by substitution and/or deletion and/or addition of one or more amino acids.

The invention has the beneficial effects that:

the invention provides a novel rice insect-resistant regulatory protein Os04g05560 and a coding gene thereof. The reduction or knockout of the expression of the protein coding gene can lead the pest-susceptible plant to have high pest resistance, thereby being capable of cultivating the plant with high resistant plant hopper. The protein and the coding gene thereof can be applied to plant genetic improvement.

Detailed Description

The present invention will be described in further detail with reference to examples. It will also be understood that the following examples are included merely for purposes of further illustrating the invention and are not to be construed as limiting the scope of the invention, as the invention extends to insubstantial modifications and adaptations of the invention following in the light of the principles set forth herein. The specific process parameters and the like of the following examples are also only one example of suitable ranges, and the skilled person can make a selection within the suitable ranges through the description herein, and are not limited to the specific data of the following examples.

Example 1

By using a gene editing technology, the Os04g05560 gene is knocked out in a gene knockout experiment (knock-out), so that the pest-susceptible rice variety Nipponbare obtains high pest resistance.

Construction of Rice Gene knockout vector

Based on the cDNA sequence of the Os04g05560 gene, the sequence at the front end is selected as a target sequence, a gRNA (guide RNA) sequence (the sequence is shown as follows, but the target sequence and the corresponding gRNA sequence are not limited to the sequence) is designed and synthesized, and the gRNA sequence fragment is recombined into a pBWA (V) H vector containing a hygromycin resistance label. With this CRISPR/Cas9 genome editing vector system, one or more than 3-fold bases in the target sequence are mutated (deleted or added one base to the target sequence). The cDNA sequence of the Os04g05560 gene is subjected to frame shift mutation, and the expressed product is not the original amino acid product, so that the aim of knocking out the Os04g05560 gene is fulfilled.

gRNA sequence 1: 5'-GACCTCACCTGCAACGCATA-3' (SEQ ID NO: 4);

target sequence 1: 5'-GACCTCACCTGCAACGCATATGG-3' (SEQ ID NO: 5).

gRNA sequence 2: 5'-GCGGATGTCGTACAATCACC-3' (SEQ ID NO: 6);

target sequence 2: 5'-GCGGATGTCGTACAATCACCCGG-3' (SEQ ID NO: 7).

Second, obtaining rice gene knockout seedling by genetic transformation

1) The mature embryo of the insect-susceptible rice Nipponbare is used as a material for inducing callus: placing the cultured agrobacterium tumefaciens (EHA105) bacterial liquid in a centrifuge tube, centrifuging, taking the supernatant, and preparing an agrobacterium tumefaciens suspension; picking out the callus growing to a certain size, and placing the callus into an agrobacterium tumefaciens suspension for infection; the callus was placed on co-culture medium.

2) Screening: taking out the callus; transferring the aired callus to a screening culture medium for primary screening; the initial callus with resistant callus was transduced into new medium for a second selection.

3) Induced differentiation and rooting of resistant calli: and (4) selecting the resistant callus, transferring the resistant callus into a culture dish filled with a differentiation culture medium, sealing with a sealing film, and placing the resistant callus into a constant-temperature culture chamber for waiting for differentiation into seedlings. And transferring to a rooting culture medium for strengthening the seedlings after the seedlings grow to about 1 cm.

4) PCR detection of hygromycin (Hyg) resistance gene: the hygromycin resistance gene specific primer is used for detecting whether the rice seedling contains the gene or not by amplification through a conventional PCR method. If the positive seedling is contained, the positive seedling is the transformation positive seedling.

Resistance gene specific primers:

Hyg-f：5'-ACGGTGTCGTCCATCACAGTTTGCC-3'(SEQ ID NO：8)；

Hyg-r：5'-TTCCGGAAGTGCTTGACATTGGGA-3'(SEQ ID NO：9)。

5) gene knockout detection of positive seedlings: and designing a detection primer near the target for PCR, sequencing, and detecting the gene knockout condition (whether a successfully knocked-out homozygous seedling is obtained). Obtaining homozygous seedlings successfully knocked out of the Os04g05560 gene of the insect-susceptible rice Nipponbare.

Third, rice gene knock-out seedling insect resistance identification

The insect resistance of homozygous seedlings with the Os04g05560 gene knockout of the insect-susceptible rice Nipponbare to brown planthopper is identified by a seedling stage identification method.

The results show that the survival rate of the susceptible receptor variety Nipponbare is 0 percent, the survival rate of the Os04g05560 gene knockout homozygous seedling is 100 percent, and the resistance level is 0-1 level (high resistance level).

Example 2

Comparison of the Effect of Os04g05560 on the existing Gene against Brown planthopper

After the gene Os04g05560 of the insect-susceptible rice variety Nipponbare (resistance level 9) is knocked out, the resistance level is obviously improved to 0-1 (high resistance level).

The applicant carried out the seedling identification of the previous resistant varieties and found that the insect resistance of the varieties is seriously lost. Currently, the average resistance rating of Mudgo (containing Bph1) is 5.4, the average resistance rating of ASD7 (containing Bph2) is 8.89, the average resistance rating of Rathu Heenati (containing Bph3) is 4.61, and the average resistance rating of Babawee (containing Bph4) is 8.14.

Comparison shows that the rice planthopper-resistant gene Os04g05560 has good prospect when used for rice breeding. By a molecular breeding method or a genetic engineering method, the reduction or knockout of the expression of the protein coding gene can lead to insect-susceptible plants to have high insect-resistant function, thereby being capable of cultivating the rice with high resistant to the planthopper.

Example 3

Effect of Os04g05560 against Sogatella furcifera

After the Os04g05560 gene of the insect-susceptible rice variety Nipponbare (resistance level 9) is knocked out, the resistance level of the insect-susceptible rice variety Nipponbare to the white-backed planthopper is identified by a seedling stage identification method.

Therefore, the gene Os04g05560 for regulating and controlling rice planthopper resistance has a good effect when being applied to breeding of the rice planthopper resistance.

SEQUENCE LISTING

<110> institute for plant protection of academy of agricultural sciences of Guangdong province

<120> rice insect pest resistance regulation protein and coding gene and application thereof

<130>

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 642

<212> PRT

<213> Rice

<400> 1

Met Ala Cys Leu Gln Leu Gln Val Leu Leu Leu Leu Ala Cys Leu Leu

1 5 10 15

Leu Asp Ala Pro His Leu Ser Ser Ala Ala Ala Thr Val Pro Thr Pro

20 25 30

Pro Phe Ser Phe Asn Phe Asp Phe Ser Asn Met Ser Thr Tyr Lys Pro

35 40 45

Asp Asp Leu Arg Phe Glu Gly Asn Ala Thr Val His Gly Ser Phe Val

50 55 60

Asp Leu Thr Cys Asn Ala Tyr Gly Leu Asp Ile Ser Gln Cys Thr Ala

65 70 75 80

Gly Arg Met Ser Tyr Asn His Pro Val Pro Phe Tyr Asp Gln Thr Thr

85 90 95

Lys Glu Val Ala Ser Phe Ser Thr Gln Phe Thr Phe Lys Ile Ile Val

100 105 110

Pro Arg Phe Asn Asn Asp Lys Glu Lys Gly Asp Gly Met Ala Phe Phe

115 120 125

Leu Ala Arg Tyr Pro Ser Arg Met Pro Pro Asp Ser Gly Gly Gly Ser

130 135 140

Leu Gly Leu Ile Thr Asn Asn Asn Tyr Ser Ser Phe Gly Pro Asp Gln

145 150 155 160

Phe Val Ser Val Glu Phe Asp Thr Tyr Asn Asn Thr Trp Glu Gln Pro

165 170 175

Lys Gln Thr Gly Asp His Met Gly Ile Asn Ile Asn Thr Val Thr Phe

180 185 190

Ser Thr Asn Thr Thr Ser Val Ser Ser Phe Ser Pro Asn Glu Ser Met

195 200 205

Met Lys Ala Ser Ile Thr Phe Asp Ser Lys Thr Ser Met Leu Val Ala

210 215 220

Ser Leu Gln Tyr Thr Gly Asn Tyr Ser Asn Tyr Ala Pro Val Asn Val

225 230 235 240

Ser Ala Lys Leu Pro Asp Pro Thr Thr Leu Leu Pro Ser Glu Val Ala

245 250 255

Val Gly Phe Ser Ala Ala Thr Gly Ala Ala Phe Glu Leu His Gln Ile

260 265 270

His Ser Trp Ser Phe Asn Ser Thr Ile Ala Ala Pro Val Gln Lys Asp

275 280 285

His Lys Lys Ala Ile Ala Val Gly Val Ser Ile Gly Gly Gly Leu Ile

290 295 300

Leu Val Leu Leu Val Trp Ser Ile Leu Ser Trp Trp Lys Trp Arg Lys

305 310 315 320

Thr Asn Arg Glu Phe Asp Lys Gly Thr Arg Gly Ala Cys Arg Phe Asn

325 330 335

Tyr His Arg Leu Ala Ala Ala Thr Asn His Phe Ser Met Asp Asn Arg

340 345 350

Ile Gly Ala Gly Thr Phe Gly Glu Val His Lys Gly Phe Leu Thr Gln

355 360 365

Leu Gly Arg Glu Val Ala Val Lys Lys Ile Leu Arg Glu Ser Arg Ala

370 375 380

Gly Asn Lys Asp Phe Phe Asp Glu Val Gln Thr Ile Ser Arg Ala Lys

385 390 395 400

Gln Lys Asn Leu Val Glu Leu Leu Gly Trp Gly Met Lys Gly Ser Ser

405 410 415

Ile Ile Asp Phe Val Met Cys Trp Arg Arg Gln Lys Asn Thr Asp Leu

420 425 430

Phe Leu Val Tyr Glu Phe Val Asp Asn Gly Asn Leu His Met His Leu

435 440 445

Tyr Glu Lys Glu Ala Leu Leu Ser Trp Arg Ile Arg Tyr Lys Ile Val

450 455 460

Lys Gly Ile Ile Ser Ala Leu Val Tyr Leu His His Asp Arg His Pro

465 470 475 480

Tyr Ile Leu His Arg Asp Ile Lys Pro Ser Asn Ile Leu Leu Asp Lys

485 490 495

Asn Phe Asn Ala Arg Leu Ala Asp Phe Gly Leu Ser Arg Thr Ala Asp

500 505 510

Asn Gly Thr Ile Gln Ser Ser Met Val Val Gly Thr Glu Asn Tyr Leu

515 520 525

Asp Pro Glu Cys Arg Lys Thr Gly Lys Phe Asn Arg Ser Ser Asp Val

530 535 540

Phe Ser Phe Gly Leu Val Leu Leu Glu Ile Ala Cys Lys Lys Asp Glu

545 550 555 560

Asn Ser Tyr Ala Gln Val Trp Glu Arg Tyr Ile Asp Lys Thr Leu Met

565 570 575

Gln Ala Ala Asp Asp Arg Leu Gln Gly Ala Phe Asp Lys Arg Gln Met

580 585 590

Glu Arg Val Ile Val Leu Gly Leu Trp Cys Cys Gln Pro Asn Ile Glu

595 600 605

Met Arg Pro Thr Met Glu Lys Ala Met Asp Phe Leu Glu Ser Asp Gly

610 615 620

Pro Leu Pro Lys Leu Ala Lys Pro Glu Ile Thr Ser Ser Ser Ala Pro

625 630 635 640

Ser Asn

<210> 2

<211> 2542

<212> DNA

<213> Rice

<400> 2

atggcctgcc tgcagctgca ggttcttctc ctcctcgcct gcctgctgct tgatgctcct 60

catcttagta gtgctgcagc cacagttcca acaccaccct tctccttcaa cttcgacttc 120

tccaacatgt ccacatacaa gccggatgac ttgagatttg agggcaacgc gactgtgcac 180

ggcagcttcg tcgacctcac ctgcaacgca tatgggctgg acatctctca gtgcacggct 240

gggcggatgt cgtacaatca cccggtgccc ttctacgacc aaaccaccaa ggaggttgcc 300

agcttctcca cacagttcac cttcaagatt attgtaccaa gatttaacaa cgacaaggag 360

aagggagatg gtatggcttt cttccttgca cgttacccat caagaatgcc tccagattca 420

ggcggcggca gccttggtct catcaccaac aacaactata gtagctttgg cccggaccag 480

ttcgtatccg tcgagtttga cacgtacaac aacacctggg agcagcctaa gcagactggt 540

gaccatatgg gcatcaacat caacacagtc actttttcga ctaacacaac aagtgtatcc 600

agcttcagcc cgaatgagag catgatgaag gcgtccatca cctttgacag caagacctcg 660

atgctggtcg cctctctgca gtataccggt aattattcta actatgcccc tgttaacgtc 720

agtgcgaaac tcccagatcc tacgaccttg ctcccgtcgg aggtggcagt gggattttct 780

gcagccaccg gggcagcctt cgagctccat caaatacact catggtcttt caactcaact 840

attgctgccc cggtacaaaa aggtatatat gcagttacta catctcttga attgattgtt 900

ggattaatgc attgtactcg ttggaggtac ttctgacgca gcaattgaca ccttagtagt 960

ttgtattttt tttgaatgaa tttgtaatgc agcaattgac accgatctgc cgtaacatcg 1020

atgcagatca caagaaagcc attgccgtag gggtctcaat tggaggaggt ctcatattgg 1080

tgctcctggt gtggtctatt ctctcatggt ggaagtggag aaaaacaaac cgtgaatttg 1140

acaagggaac tcgtggggct tgtcggttca actaccaccg cttggctgct gcaacaaacc 1200

atttttcaat ggataatagg attggagcag gtacgttcgg tgaagtacac aaaggtttct 1260

tgacacaatt gggccgtgaa gtggctgtta agaagatctt gagggaatcc agagcaggaa 1320

acaaggactt ttttgacgaa gtccagacca tcagtagagc aaagcagaag aatcttgttg 1380

aacttcttgg ttggggaatg aagggcagct cgatcattga tttcgtcatg tgctggcgta 1440

ggcagaagaa caccgacctt ttcctggtat atgaatttgt ggataacggc aacctacata 1500

tgcacctgta tgagaaggag gccctgctgt catggagaat aaggtacatg ctgcgagctg 1560

agcaatataa tttatattca aattttaaga acctagctag gtattattat attactaccg 1620

cacacataac cccaaaaatt attgtctaat aatgttttac aaaactccac catgtccatg 1680

ttgagcataa tttttctaaa aataaaatat agattgtata tgaaaattgt aattactgat 1740

ttcttgacta aggttgatag tttaactttt cactgcttta aaaaaattat tttagatatg 1800

tttaaattaa atgacatttc aataatacct atatatgggt tttctgccac atagacccta 1860

aaacatgttg tttcctggaa gtaactattt tgaatatgga agtttgaaaa aacgagctag 1920

taatgttgac aatcattatg tacgttcctc ggaacaagaa aatgattatg catgcaaatg 1980

ttgtcttttc aggtataaaa tagtgaaggg cattatcagc gctcttgttt accttcacca 2040

tgatagacat ccatacattc ttcacaggga tatcaaaccg agcaacatac tcttggacaa 2100

aaatttcaat gctagattag cagatttcgg gctgtcgaga actgccgaca atggaacaat 2160

acaatcgtca atggttgtag gaacggaaaa ttacttggat ccagagtgca ggaagacggg 2220

aaagttcaac cgtagctcag atgtcttcag cttcgggctc gttctgctag agattgcttg 2280

caagaaagac gagaatagct atgcacaagt ctgggaaagg tacatcgaca aaactctgat 2340

gcaggctgct gacgataggt tacaaggtgc gttcgataag aggcagatgg agcgtgtgat 2400

cgtcttgggg ctgtggtgtt gtcagcctaa tattgagatg cgacctacga tggagaaagc 2460

aatggatttc ctggagagtg atgggccgtt gcctaaactg gccaaacccg aaattacctc 2520

ctcaagtgcg ccaagcaatt ga 2542

<210> 3

<211> 1929

<212> DNA

<213> Rice

<400> 3

atggcctgcc tgcagctgca ggttcttctc ctcctcgcct gcctgctgct tgatgctcct 60

catcttagta gtgctgcagc cacagttcca acaccaccct tctccttcaa cttcgacttc 120

tccaacatgt ccacatacaa gccggatgac ttgagatttg agggcaacgc gactgtgcac 180

ggcagcttcg tcgacctcac ctgcaacgca tatgggctgg acatctctca gtgcacggct 240

gggcggatgt cgtacaatca cccggtgccc ttctacgacc aaaccaccaa ggaggttgcc 300

agcttctcca cacagttcac cttcaagatt attgtaccaa gatttaacaa cgacaaggag 360

aagggagatg gtatggcttt cttccttgca cgttacccat caagaatgcc tccagattca 420

ggcggcggca gccttggtct catcaccaac aacaactata gtagctttgg cccggaccag 480

ttcgtatccg tcgagtttga cacgtacaac aacacctggg agcagcctaa gcagactggt 540

gaccatatgg gcatcaacat caacacagtc actttttcga ctaacacaac aagtgtatcc 600

agcttcagcc cgaatgagag catgatgaag gcgtccatca cctttgacag caagacctcg 660

atgctggtcg cctctctgca gtataccggt aattattcta actatgcccc tgttaacgtc 720

agtgcgaaac tcccagatcc tacgaccttg ctcccgtcgg aggtggcagt gggattttct 780

gcagccaccg gggcagcctt cgagctccat caaatacact catggtcttt caactcaact 840

attgctgccc cggtacaaaa agatcacaag aaagccattg ccgtaggggt ctcaattgga 900

ggaggtctca tattggtgct cctggtgtgg tctattctct catggtggaa gtggagaaaa 960

acaaaccgtg aatttgacaa gggaactcgt ggggcttgtc ggttcaacta ccaccgcttg 1020

gctgctgcaa caaaccattt ttcaatggat aataggattg gagcaggtac gttcggtgaa 1080

gtacacaaag gtttcttgac acaattgggc cgtgaagtgg ctgttaagaa gatcttgagg 1140

gaatccagag caggaaacaa ggactttttt gacgaagtcc agaccatcag tagagcaaag 1200

cagaagaatc ttgttgaact tcttggttgg ggaatgaagg gcagctcgat cattgatttc 1260

gtcatgtgct ggcgtaggca gaagaacacc gaccttttcc tggtatatga atttgtggat 1320

aacggcaacc tacatatgca cctgtatgag aaggaggccc tgctgtcatg gagaataagg 1380

tataaaatag tgaagggcat tatcagcgct cttgtttacc ttcaccatga tagacatcca 1440

tacattcttc acagggatat caaaccgagc aacatactct tggacaaaaa tttcaatgct 1500

agattagcag atttcgggct gtcgagaact gccgacaatg gaacaataca atcgtcaatg 1560

gttgtaggaa cggaaaatta cttggatcca gagtgcagga agacgggaaa gttcaaccgt 1620

agctcagatg tcttcagctt cgggctcgtt ctgctagaga ttgcttgcaa gaaagacgag 1680

aatagctatg cacaagtctg ggaaaggtac atcgacaaaa ctctgatgca ggctgctgac 1740

gataggttac aaggtgcgtt cgataagagg cagatggagc gtgtgatcgt cttggggctg 1800

tggtgttgtc agcctaatat tgagatgcga cctacgatgg agaaagcaat ggatttcctg 1860

gagagtgatg ggccgttgcc taaactggcc aaacccgaaa ttacctcctc aagtgcgcca 1920

agcaattga 1929

<210> 4

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 4

gacctcacct gcaacgcata 20

<210> 5

<211> 23

<212> DNA

<213> Artificial Synthesis

<400> 5

gacctcacct gcaacgcata tgg 23

<210> 6

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 6

gcggatgtcg tacaatcacc 20

<210> 7

<211> 23

<212> DNA

<213> Artificial Synthesis

<400> 7

gcggatgtcg tacaatcacc cgg 23

<210> 8

<211> 25

<212> DNA

<213> Artificial Synthesis

<400> 8

acggtgtcgt ccatcacagt ttgcc 25

<210> 9

<211> 24

<212> DNA

<213> Artificial Synthesis

<400> 9

ttccggaagt gcttgacatt ggga 24

Claims

1. The application of the substance for reducing the expression quantity of the protein Os04g05560 in insect pest resistance of rice, wherein the amino acid sequence of the protein Os04g05560 is shown as SEQ ID NO: 1 is shown in the specification;

the insects are at least one of brown planthopper and sogatella furcifera;

the substance for reducing the expression quantity of the protein Os04g05560 is gRNA, and the sequence of the gRNA is shown in SEQ ID NO: 4 and SEQ ID NO: and 6.

2. The application of a substance for knocking out the coding gene of the protein Os04g05560 of claim 1 in resisting the plant hoppers of rice;

the plant hopper is at least one of brown plant hopper and sogatella furcifera;

the substance for knocking out the coding gene of the protein Os04g05560 of claim 1 is gRNA, and the sequence of the gRNA is shown in SEQ ID NO: 4 and SEQ ID NO: and 6.

3. Use according to claim 2, characterized in that: the nucleotide sequence of the coding gene is shown as SEQ ID NO: 2, respectively.

4. A method for improving the pest resistance of rice comprises the following steps: the expression level of the protein Os04g05560 in rice is reduced, and the amino acid sequence of the protein Os04g05560 is shown as SEQ ID NO: 1 is shown in the specification; the insects are at least one of brown planthopper and sogatella furcifera.

5. The method of claim 4, wherein: the reduction of the expression level of the protein Os04g05560 is realized by knocking out the coding gene of the protein Os04g05560 or reducing the expression level of the coding gene, and the nucleotide sequence of the coding gene is shown as SEQ ID NO: 2, respectively.

6. A cultivation method of pest-resistant rice comprises the following steps: the expression level of the protein Os04g05560 in the rice is reduced to improve the insect resistance of the rice, wherein the amino acid sequence of the protein Os04g05560 is shown as SEQ ID NO: 1 is shown in the specification;

the insects are at least one of brown planthopper and sogatella furcifera.

7. The cultivation method as claimed in claim 6, wherein: the reduction of the expression level of the protein Os04g05560 is realized by knocking out the coding gene of the protein Os04g05560 or reducing the expression level of the coding gene, and the nucleotide sequence of the coding gene is shown as SEQ ID NO: 2, respectively.