CN107937363B

CN107937363B - Rice spike top degeneration related protein kinase and coding gene thereof

Info

Publication number: CN107937363B
Application number: CN201711257204.3A
Authority: CN
Inventors: 彭永彬; 侯飞雪; 白鹊; 徐培洲; 吴先军; 张红宇; 陈晓琼; 廖泳祥; 顾朝剑; 韩小行
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2017-12-04
Filing date: 2017-12-04
Publication date: 2020-06-09
Anticipated expiration: 2037-12-04
Also published as: CN107937363A

Abstract

The invention discloses a rice panicle top degeneration-related protein, namely OsCIPK31 protein, the known function of which is protein kinase for regulating and controlling stress response; the invention also discloses a gene for coding the related protein and application of the gene to control the rice ear top degeneration. The invention provides a new way for researching the rice ear top degradation mechanism and finally solving the ear top degradation problem; in particular, the ear top degenerated mutant and the cloned gene related by the invention are both from indica rice, and can be used for reducing the yield reduction loss of the indica rice caused by ear top degenerated and improving the yield of the indica rice; the ear tip degeneration character is controlled by a single recessive gene, and the identification and selection of transgenosis or filial generation are simple, so that the method is convenient to apply in breeding.

Description

Rice spike top degeneration related protein kinase and coding gene thereof

Technical Field

The invention belongs to the field of rice genetic engineering, and particularly relates to rice ear tip degeneration-related protein kinase, a coding gene of the protein kinase, and a new application of the protein.

Background

The ear top degradation refers to the phenomenon that the development of the top glumes of the rice is stopped and gradually degraded in the ear development process in the booting stage. The phenomenon of top degradation of the spike occurs in rice varieties occasionally, and the glumes at the top of the spike and the glumes at the top of the primary branch appear to be developmentally arrested and excessively decline when the phenomenon occurs. This results in a large reduction in the size of the ears and the number of grains per ear, resulting in a reduction in the number of grains per ear and a serious impact on the overall yield of rice. This phenomenon is susceptible to environmental conditions such as temperature and humidity during ear differentiation, which makes phenotypic identification difficult or unstable, thus increasing the difficulty of research. The mechanism causing the phenomenon is not clear at present, and is a problem to be solved urgently in rice production.

The breeding of the variety with the resistance to the top degradation of the spike is the most economic and effective method for controlling the degradation of the top of the spike, but the pathogenic mechanism of the degradation of the spike is not clear, the reports on the control of the degradation trait genes of the top degradation of the spike are few, the source-resistant materials of the degradation of the top of the spike of the rice are few, and the breeding of the variety with the resistance to the top degradation of the spike is difficult. The current reports of genes related to the ear tip degeneration mainly focus on QTL loci: 3 major QTLs associated with floret abortion at the base of the pre-flowering ear of rice were found on

chromosomes

1, 10 and 11 (Yamagishi J et al, therapeutic and Applied Genetics, 2004, 109 (8): 1555-; regulating and controlling a stem and spike length related gene SP1(Li S, etc. The Plant Journal, 2009, 58 (4): 592-605) in an inflorescence structure; 5 spike-top degenerated QTL loci are found on

chromosomes

1, 2, 4, 7 and 9, wherein the effect of the ds-9 locus of the chromosome 9 on the rice spike-top degeneration is relatively large (Xuhuashan et al, Proc. crops, 2007, 33 (6): 979-; there is an epistatic interaction between a QTL located on chromosome 3 and a QTL located on chromosome 9 (Tan C et al Plant Breeding, 2011, 130: 177-184). In addition, the number of genes cloned in the ear top degeneracy map is small: spd-hp73 (Yuansan, et al, university of Zhejiang, 2013, 39 (3): 267 and 273). The reported quantitative traits for controlling the rice panicle top degeneration are mostly controlled by multiple genes, and the obtained cloned genes are few, so that the problem of rice panicle top degeneration is limited to be solved.

OsCIPK31 is a calcineurin B subunit interacting protein kinase gene and is a typical serine/threonine protein kinase mediating calcium signals. During rice germination and seedling growth, stress responses are involved by modulating stress response genes (Hai-long Piao et al Molecules and Cells, 2010, 30 (1): 19-27). Through retrieval, no report about the effect of OsCIPK31 on development of rice panicle parts, particularly on degeneration of the panicle tops is found.

Disclosure of Invention

The inventor unexpectedly discovers a spike top degenerated mutant material of an indica rice background, named paa1019, when EMS (ethyl methane sulfonate) mutagenesis is carried out on indica rice variety Yixiang 1B, researches show that the rice spike top degenerated character is controlled by a single recessive gene, and further researches show that the phenomenon of degenerated decline of the top glumes of the rice spike can be caused after point mutation of a wild normal gene. The present invention has been accomplished on the basis of the above-mentioned unexpected findings.

The invention aims to provide a rice spike top degeneration-related protein.

Another object of the present invention is to provide a gene encoding the above-mentioned related protein.

The OsCIPK31 protein as the third object of the invention is applied to controlling the degradation property of the rice ear top.

The application of the OsCIPK31 gene of the fourth purpose of the invention in controlling the rice ear top degradation property.

The fifth object of the present invention is to provide an expression vector containing OsCIPK31 gene.

The sixth purpose of the invention is to provide a target sequence for knocking out OsCIPK31 gene.

The seventh purpose of the invention is to provide a rice panicle top degeneration gene.

The eighth purpose of the invention is to provide a method for improving the ear top degenerated rice variety.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention provides a rice panicle top degeneration-related protein, namely OsCIPK31 protein, which consists of an amino acid sequence shown in SEQ ID NO. 1.

The known function of the OsCIPK31 protein is a protein kinase that regulates stress responses.

The gene for coding the related protein, namely the OsCIPK31 gene, consists of a nucleotide sequence shown in SEQ ID NO. 2.

The invention also provides application of the OsCIPK31 protein in controlling the rice ear top degradation property; the OsCIPK31 protein consists of an amino acid sequence shown in SEQ ID NO. 1.

The invention also provides application of the OsCIPK31 gene in controlling the rice ear top degradation property; wherein the OsCIPK31 gene consists of a nucleotide sequence shown in SEQ ID NO. 2.

The invention also provides an expression vector containing the OsCIPK31 gene.

The invention also provides a rice panicle top degeneration gene, which is obtained by carrying out gene editing on the OsCIPK31 gene, wherein the gene editing is carried out by a CRISPR/CAS9 system.

The rice panicle top degeneration gene consists of nucleotide sequences shown by SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6.

The rice panicle top degeneration gene is preferably composed of a nucleotide sequence shown by SEQ ID NO. 6.

The invention also provides a target sequence for knocking out the OsCIPK31 gene, which consists of the nucleotide sequence shown in SEQ ID NO. 7.

The invention also provides sgRNA for knocking out OsCIPK31 gene, and the target sequence of the sgRNA consists of the nucleotide sequence shown in SEQ ID NO. 7.

A method for improving a spike-top degenerated rice variety comprises the step of transforming an OsCIPK31 gene into the spike-top degenerated rice variety by a transgenic method.

The invention has the advantages or beneficial effects that: (1) the invention provides a new function of the OsCIPK31 gene in the aspect of controlling the rice ear top degeneration, and provides a new way for researching the rice ear top degeneration mechanism and finally solving the ear top degeneration problem; the ear top degenerated mutant and the cloned gene are both from indica rice and can be used for reducing yield loss of the indica rice caused by ear top degenerated. (2) The ear tip degeneration character is controlled by a single recessive gene, and the identification and selection of transgenosis or filial generation are simple, so that the method is convenient to apply in breeding. (3) According to the invention, the OsCIPK31 gene is introduced into the ear top degenerating mutant receptor material, so that the transgenic material for recovering the normal ear phenotype is successfully obtained, and the method is simple and high in efficiency.

Drawings

FIG. 1 is a photograph of plants at heading stage Yixiang 1B and paa 1019; wherein 1 is Yixiang 1B; 2 is paa 1019.

FIG. 2 is a photograph of ears at heading stage Yixiang 1B and paa 1019; wherein 1 is Yixiang 1B; 2 is paa 1019.

FIG. 3 is a bar graph of the length of the main spike of Yixiang 1B and paa 1019; wherein 1 is Yixiang 1B, and 2 is paa 1019.

FIG. 4 is a bar graph of the number of single ear grains of Yixiang 1B and paa 1019; wherein 1 is Yixiang 1B, and 2 is paa 1019.

FIG. 5 is a photograph of a Tulip blue or Evans blue stain of Janus 1B and paa1019 glume flowers; wherein 1 is tyrant 1B dyed with taloxanil, 2 is paa1019 dyed with taloxanil, 3 is tyrant 1B dyed with evans blue, and 4 is paa1019 dyed with evans blue.

FIG. 6 is DAB staining photographs of young ears at the early stage of booting ears and ears at the later stage of booting ears; wherein 1 is the young ear at the early stage of the booting ear of Yixiang 1B, 2 is the young ear at the later stage of the booting ear of Yixiang 1B, 3 is the young ear at the early stage of the booting ear of paa1019, and 4paa 1019.

FIG. 7 is an electrophoretogram of linked molecular markers on chromosome 2 screened from a F2 population constructed by hybridization of paa1019 and 02428 (japonica rice); wherein 1 is a dominant pool, 2 is a recessive pool, and the molecular marker used is RM 7; 3 is a dominant pool, 4 is a recessive pool, and the molecular marker used is RM 251; 5 is dominant pool, 6 is recessive pool, using molecular marker os 3-46.6; 7 is a dominant pool, 8 is a recessive pool, and the using molecular marker is os 3-50.8;

FIG. 8 is a partial electropherogram of one of the linked markers in gene mapping; wherein 1 is paa1019 band type, 2 is 02428 (japonica rice) band type, and lanes 3-15, 17, 19-24 are recessive single plant band types; 16. 18 is of the single exchange band type.

FIG. 9 photographs of plants of the knockout line; wherein 1 is Yixiang 1B, 2 is paa1019, 3, 4 and 5 are independent knock-out strains KO1019-11, KO1019-26 and KO1019-29 respectively.

FIG. 10 is a photograph of ears of a knock-out strain; 1 is Yixiang 1B, 2 is paa1019, 3, 4 and 5 are knock-out strains KO1019-11, KO1019-26 and KO1019-29 respectively.

FIG. 11 is a bar graph of the head degeneration rate of the knockout strain; wherein 1 is Yixiang 1B, 2 is paa1019, and 3, 4 and 5 are knock-out strains KO1019-11, KO1019-26 and KO1019-29 respectively.

FIG. 12 is a photograph of a plant showing complementation of a transgenic plant line; wherein 1 is Yixiang 1B, 2 is paa1019, 3, 4 and 5 are transgenic positive strains CE1019-1, CE1019-2 and CE1019-3 respectively.

FIG. 13 is a photograph of ears of a complementation verification transgenic plant line; wherein 1 is Yixiang 1B, 2 is paa1019, 3, 4 and 5 are transgenic positive strains CE1019-1, CE1019-2 and CE1019-3 respectively.

Detailed Description

The present invention is further illustrated and described by the following examples, which are not intended to limit the scope of the invention in any way. Unless otherwise specified, the methods used in the examples are conventional methods; the medicines or reagents used are all conventional reagents.

The test materials used in the examples below, spike top degenerated mutant paa1019, Yixiang 1B (indica) and 02428 (japonica) were obtained from the laboratory of heterosis utilization of the Rice institute of Sichuan university. The ear top degenerated mutant paa1019 is obtained by screening from an EMS (ethyl methane sulfonate) mutation library constructed by Yixiang 1B, the phenotype of the mutant is that the ear top glumes are degenerated and withered, and the degeneration phenomenon appears in the ear-bearing stage of young ears till the ear emergence stage. After backcrossing with Yixiang 1B for multiple generations in Wenjiang river and Hainan Ling water in Sichuan, the degraded phenotype of the ear can be stably inherited.

EXAMPLE 1 genetic analysis test for Top of ear degeneration trait of mutant paa1019 of the present invention

The method comprises the following steps:

the ear top degenerating mutant paa1019 and Yixiang 1B were planted in the Wenjiang test field of Sichuan university of agriculture, Rice research institute. Paa1019 and Yixiang 1B are used as parents for hybridization to respectively construct F₂A population; separately observing the parents and F₁And F₂Ear phenotype of the population and statistics of paa 1019F after crossing with Yixiang 1B₁And F₂The segregation ratio of the population.

Results (see fig. 1 and 2) mutant paa1019 exhibited a top-degenerated phenotype starting from the ear at the booting stage compared to wild-type xiangxiang 1B, before development of ears was indistinguishable between wild-type and mutant regardless of apical meristem primordia, shoot primordia, and spikelet primordia. As the development of the ear matures, the phenomenon of tip degeneration tends to be severe. Mutant paa1019 and F of Yixiang 1B₁The generation plants have normal ear phenotype and growth period, which indicates that paa1019 ear top degenerating shape is controlled by recessive gene; in addition, by Chi Fang test (see Table 1), F₂The segregation ratio of the wild type and the ear top degenerated phenotype in the generation population is 3:1, which meets the segregation ratio controlled by the Mendelian single recessive gene, and indicates that the ear top degenerated state of the mutant paa1019 is controlled by the single recessive nuclear gene.

TABLE 1 genetic analysis test results for Top-degenerating traits in mutant paa1019

Combination of	Generation of generation	Total number of plants	Recessive individual plant	Separation ratio	Chi fang check
						Paa 1019/Yixiang 1B	F2	605	147	3.12：1	χ² _(3：1)＝0.765

Note: chi shape² _0.05,1＝3.36

Example 2 cell Activity assay of degenerate ears of mutant paa1019 of the present invention

Contrast test for detecting paa1019 dead cells in degenerated ear by trypan blue staining solution

(1) Preparing trypan blue dye solution: prepared by adding 2.5mg trypan blue into 1ml of the lactophenol solution. Wherein the lactol solution contains 25% (mass volume ratio) of lactol, 23% of water-soluble phenol, 25% of glycerin and 27% of sterile water. The prepared trypan blue dye solution needs to be stored under the condition of keeping out of the sun and low temperature.

(2) Paa1019 ears growing in heading period in a climatic chamber and contemporaneous normal ears suitable for 1B are stained in boiling trypan blue staining solution for 10min, the ears are taken out and placed at room temperature for 12h, then decolorized in 2.5mg/ml chloral hydrate solution for 3-4 days, and then cell death in the ears is observed by using a stereomicroscope and photographed.

As a result, it was noted that incense 1B was hardly stained (see FIG. 5-1), while the degenerate glume flower of paa1019 (FIG. 5-2) was stained dark blue, indicating that paa1019 degenerate ear cells were programmed to die.

(II) assay for detecting paa1019 cell viability in degenerated ears by Evans blue staining

The degraded spike of the spike top of the mutant paa1019 growing in the heading stage in a climatic chamber and the synchronous normal spike of the optimized fragrance 1B are soaked in 0.25% (W/V) Evans blue solution for 24h, then the spike is taken out, the blue dye solution on the surface is thoroughly cleaned by water, the surface water is absorbed, and then the spike is placed into a boiled decolorizing solution (the decolorizing solution comprises absolute ethyl alcohol: glycerol: 9: 1) for 30min, and the chlorophyll is removed until the bottom color of the glume shell is close to white. The decolorized glume flowers of Yixiang 1B, paa1019 were observed under a microscope and photographed.

As a result, it is preferred that glume flowers of incense 1B are substantially non-stained (see FIGS. 5-3), while degenerated glume flowers of paa1019 are stained dark blue (see FIGS. 5-4), indicating that the degenerated ear cells of paa1019 are programmed to die.

(III) peroxide content test for detecting paa1019 spike cells by DAB staining

(1) Preparing DAB working solution: DAB working solution was prepared according to the instruction of DA1010DAB color development kit (20X) solarbio (Beijing Sorbo technologies, Ltd.).

(2) Respectively soaking young ears at the early stage of the booting ear and ears at the later stage of the booting ear of the Yixiang 1B and paa1019 in DAB working solution, and incubating for 3-10 minutes at room temperature in a dark place until the color development reaches the expected depth. And removing DAB dyeing working solution, washing with distilled water for 2-3 times, and terminating the reaction. The color development was observed with a stereoscope and recorded by photographing.

Compared with the wild type Yixiang 1B, the hydrogen peroxide accumulation of the mutant paa1019 ear is higher than that of the wild type ear (see FIGS. 6-3 and 6-4) in both early stage of pregnancy (see FIG. 6-1) and late stage of pregnancy (see FIG. 6-2), which indicates that the ear tip degeneration phenotype of paa1019 may be related to the accumulation of peroxide.

Example 3 localization of candidate Gene for ear degeneration trait of mutant paa1019 of the present invention

(1) Construction of Gene mapping populations and genomic DNA extraction

F obtained by hybridizing mutant paa1019 and japonica rice 02428₂In the population, 200 single leaves (implicit) with obvious ear degeneration mutation phenotype and 10 leaves (explicit) with normal ear phenotype are selected, and 10 leaves are respectively selectedDNA is extracted by equal mixing, and two progeny pools, namely a recessive pool and a dominant pool, are constructed and used for polymorphism detection and primary positioning primer screening between mutant paa1019 and 02428 genomes. The DNA of the leaf is extracted by an improved CTAB method.

(2) Screening paa1019 for polymorphic primers between 1019 and 02428 parents

Using paa1019 or 02428 genome DNA as template, using 543 pairs of SSR primers (the primer sequences are shown in the description in the specification) equally distributed on 12 rice chromosomeshttp://www.gramene.org/bd/markers) Performing PCR amplification, wherein the PCR reaction system (20uL) comprises: 0.2uL of Tap enzyme (5U/. mu.L), 2uL of primer (10 mmol/. mu.L), 2uL of dNTP (2.5 mmol/. mu.L), 2uL of DNA template (20-100 ng/. mu.L), 2uL of 10 XBuffer (25mM), ddH₂O11.8 uL. PCR reaction procedure: 5min at 95 ℃; 35 cycles of 95 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 45 s; 7min at 72 ℃ and 5min at 12 ℃. The obtained amplification product is electrophoresed for about 0.5h-1h under the conditions of 3.0% agarose gel and constant pressure of 180-200V, imaged by a gel imaging system and stored for recording. As a result, primer 102 pair having a polymorphism between the genomes of mutant paa1019 and 02428 was selected.

The selected polymorphic primers were then used to test the progeny pools constructed in (1), and F constructed using mutants paa1019 and 02428₂Recessive individual plants in the population are subjected to gene primary positioning;

(3) construction of linkage map

The individuals with the band pattern of mutant paa1019 were marked as 0, the individuals with the band pattern of heterozygous were marked as 1, the individuals with the band pattern of 02428 were marked as 2, the individuals without band were marked as 3, and F was identified by software MAPMARKER 3.0.0₂And (3) carrying out linkage analysis on the separation data of the molecular marker and the mutation character in the separation population, and converting the recombination value into a genetic map distance (cM).

(4) Preliminary localization result of candidate gene

Using the selected 102 polymorphic primers at F ₂200 recessive individuals in the population are screened, and as a result (see fig. 7 and fig. 8), the two SSR markers RM251 and Os3-65.4 at the long-arm end of chromosome 3 are found to have a linkage relation with candidate genes.

(5) Whole genome re-sequencing analysis of SNP sites (BSA method)

Mutant paa1019 was hybridized with Yixiang 1B, F₁Selfing, from F ₂25 plant leaves with remarkable mutation phenotype are taken and equally mixed to form a recessive progeny pool for extracting DNA, and the DNA with Yixiang 1B is sent to NozaoyionBiotech Co. And after the library is constructed and the quality is qualified, carrying out Illumina HiSeq TM PE150 sequencing. And comparing the finally obtained sequencing data with a reference genome (9311 genome is used as a reference, and download addresses ftp:// ftp. ensimblegenes. org/pub/plantats/release-26/fasta/oryzae _ indica/dna /), and analyzing the existing SNP difference by using bioinformatics to determine candidate genes.

The alignment results found 16 candidate sites, consistent with non-synonymous mutations located in the exon regions and with SNP-index values > 80%, and analysis of the primary localization interval before recombination found that there was one and only one SNP candidate site between RM5748 and os 3-50.8. This site is located at the exon-intron junction, where the mutation results in a change in the manner in which the transcript is spliced, preferably 1B relative to wild type, resulting in the last two bases of the first exon and the entire deletion of the second exon except for the last two bases (see SEQ ID No. 6). The segment belongs to LOC _ Os03g20380, namely a known OsCIPK31 gene, belongs to a calcineurin-like protein kinase family (CIPKs), and OsCIPK31 has a regulation function in a plant stress response link and has a certain regulation function in the stress response of rice to ABA in a seedling stage (Hai-long Piao et al, Molecule and Cells, 2010, 30 (1): 19-27).

Example 4 test for verification of candidate Gene knockout

Coli DH5 α and Agrobacterium EHA105 strains used in this experiment were purchased from all-grass Biotechnology Ltd.

1. CRISPR/CAS9-OsCIPK31 gene knockout vector construction

A nucleotide sequence of an OsCIPK31(LOC _ Os03g20380) gene in Yixiang 1B is used as a template, a proper region is selected, 1 knockout target site is designed, and a CRISPR/CAS9-OsCIPK31 vector is constructed by utilizing a BWA (V) H-CAS9 vector reference kit (Hangzhou Baige biology company). The specific construction process is as follows:

(1) designing a target sequence for knocking out OsCIPK31 gene, wherein the target sequence is as follows:

5’-AACTAATGGAAGGTTGAAGG-3’(SEQ ID NO.7)。

(2) the following adapter primers were designed and synthesized to form gRNA target sequences:

F:5’-CAGTGGTCTCAGGCAACTAATGGAAGGTTGAAGG-3’，

R:5’-CAGTGGTCTCAGGCAACCTGAAAGTATCTGACTT-3’。

(3) preparation of primer dimer

And (3) adding water to dissolve the primer pair synthesized in the step (2) to 10 mu M, mixing according to the following reaction system, heating for 3 minutes at 95 ℃ in a PCR instrument, and then slowly reducing to 20 ℃ at about 0.2 ℃/second to obtain a primer dimer. The reaction system is as follows: annealing Buffer18ul, gRNA target primer 1ul each, add ddH₂O, make up to 20 ul.

(4) And constructing the primer dimer into a BWA (V) H vector. Mixing the components on ice according to the following reaction system, uniformly mixing, reacting at 20 ℃ for 1 hour, and transforming escherichia coli for later use to obtain an expression vector containing elements such as a promoter, a target sequence, gRAN and the like, thereby obtaining the CRISPR/CAS9-OsCIPK31 gene knockout vector. The reaction system comprises the following steps: BWA (V) 2ul of H vector, 1ul of Oligo dimer, 1ul of enzyme mixture, and ddH₂O, make up to 10 ul.

2 transformation of Escherichia coli

(1) Taking a pipe of prepared escherichia coli competent cells out of a refrigerator at the temperature of-80 ℃, and putting the escherichia coli competent cells on ice for thawing;

(2) adding 100 mu L of competent cell suspension into every 100ng of ligation product (namely CRISPR/CAS9-OsCIPK31 gene knockout vector constructed in the step 1), uniformly mixing, and placing on ice for 30 min;

(3) heat shock is carried out for 30s at 42 ℃, and the mixture is quickly taken out and immediately placed on ice for 2 min;

(4) adding 500 mu L of LB liquid culture medium without antibiotics, culturing at 37 ℃ and 200rpm for about 1 hour to activate bacteria liquid;

(5) centrifuging the activated bacterial liquid at 5000rpm for 1min, pouring out most of supernatant under the aseptic condition, gently sucking and pumping the mixed precipitate by using a pipette gun, sucking 100 mu L of the mixed precipitate, transferring the bacterial liquid on a super clean bench and coating the bacterial liquid on an LB screening plate containing kanamycin;

(6) placing the LB solid culture medium plate coated with the bacterial liquid for about 10 minutes from the front side upwards, inverting the culture medium coated with the plate after the bacterial liquid is completely absorbed by the LB solid culture medium, and culturing in a thermostat at 37 ℃ overnight;

(7) and selecting a single colony, and carrying out PCR detection on the bacterial liquid by using a P20380-1 primer. The P20380-1 primer pair is as follows:

P20380-1F:5’-ACCTGAAAGTATCTGACTT-3’，

P20380-1R:5’-ATACGAAGTTATGACTGCGACCGA-3’。

wherein the PCR reaction program: 5min at 95 ℃; 30s at 95 ℃, 30s at 56 ℃, 30s at 72 ℃ and 35 cycles; 72 ℃ for 10min and 12 ℃ for 1 min.

(8) The positive clone was added to 3ml of LB medium containing kanamycin (50mg/L), and cultured at 37 ℃ for about 10 hours at 200rpm in a shaker, and the resulting culture was then stored to extract a plasmid.

3. The E.coli Plasmid was extracted according to the instructions of the OMEGA Plasmid Extraction Kit, and the extracted Plasmid DNA was collected in a clean centrifuge tube and stored at-20 ℃.

4. Determination of plasmid sequence and sequence analysis

The positive clone plasmid was sent to Chengdu science and technology Co., Ltd for sequencing. And (3) carrying out sequence alignment on the sequencing result by using DNAMAN software, and confirming the correctness of the gRNA sequence. Finally, the CRISPR/CAS9-OsCIPK31 plasmid is obtained.

5. Agrobacterium transformation

(1) Chemical transformation method of agrobacterium

According to one plasmid: 50ul of competent cells were taken out at-80 ℃ and thawed quickly; adding 0.4-1 ug of the constructed CRISPR/CAS9-OsCIPK31 plasmid into 50ul of competent cells, and standing for 30 minutes on ice; freezing in liquid nitrogen for 2 minutes; water bath at 37 deg.C for 2min to melt cells; immediately adding 5 times volume of LB liquid culture medium without antibiotics, and shake culturing at 28 deg.C and 170rpm for 2-3 hr; centrifuging at 7000rpm for 2min, and suspending the cells in 100ul of LB liquid medium; coating on rifampicin and cana resistant plate, blow drying, and culturing at 28 deg.C for 2-3 days; carrying out PCR detection on bacteria liquid by using a hygromycin molecular marker P9150-1 primer, adding glycerol serving as a protective agent into a positive agrobacterium monoclonal capable of amplifying a target strip, and storing at-80 ℃ for later use.

(2) Agrobacterium impregnation method for transforming rice

(a) Induction of callus: sterilizing Nipponbare seeds with 75% alcohol for 1min, rinsing with sterile water for 3 times, rinsing with 40% sodium hypochlorite for 30min, rinsing with sterile water for 5 times, placing in a culture dish with filter paper, draining, inoculating onto NMB culture medium with tweezers, and culturing at 28 deg.C under illumination for 7 days. Subcultured every 7 days. After 2-3 subcultures, good calli grown from the seeds were picked and subcultured on NMB medium at 28 ℃ for 4 days in the dark.

(b) Activation of agrobacterium strain: adding 30ul of Agrobacterium stored at-80 ℃ in (1) into 3mL of YEP liquid medium containing rifampicin and kanamycin, and performing shake culture at 28 ℃ for 14 h; then 1mL of the suspension is taken to be put into 50mLYEP liquid culture medium containing rifampicin and kanamycin, and the suspension is subjected to shaking culture for 4 hours at the temperature of 28 ℃ to obtain activated agrobacterium liquid.

(c) Co-culture transformation: centrifuging the activated bacteria liquid of (b) at 5000rpm to collect thallus, resuspending thallus with AAM liquid culture medium 30mL containing 100 μ M/L acetosyringone, soaking the callus selected in (a) in the bacteria liquid for 20min, sucking off the excess bacteria liquid, spreading on co-culture solid culture medium, and dark culturing at 28 deg.C for 2 d.

(d) Callus degerming culture and callus resistance screening: washing the callus after co-culture for 2d with sterile water until the water is clear, then shaking with sterile water containing cefamycin (500mg/L) for 30min for sterilization, thoroughly sucking the callus with sterile filter paper or absorbent paper, and then inoculating on a selective culture medium for about 3 weeks.

(e) Differentiation and rooting of transgenic plants: inoculating the newly grown resistant callus in the step (d) to a differentiation culture medium, culturing for 1-2 months by illumination, then transferring the grown seedlings with the height of about 3cm to a rooting culture medium for rooting culture, taking leaves to extract DNA when the seedlings grow to about 10cm, identifying positive plant seedlings by using a P20380-2 primer for amplifying the full-length DNA of a target gene, and finally obtaining 3 transgenic positive plants which are respectively named as KO1019-11, KO1019-26 and KO 1019-29.

(f) And (4) hardening seedlings indoors for 2-3 days, and transplanting the positive transgenic plants into a field.

6. Detection of transgenic Rice

(1) Extracting positive transgenic rice DNA by using an improved CTAB method, and amplifying a partial sequence of a knockout target gene in a transgenic plant by using a P20380-2 primer pair according to the following PCR conditions. The P20380-2 primer pair is as follows:

P20380-2F：5'-AAACTTATGAAAGGATTGCCCGAT-3'，

P20380-2R：5'-AAAGGATTACCCCACAAGACCAGA-3'。

wherein the PCR reaction system (25 uL): tap enzyme (5U/. mu.L) 0.5ul, Primer (10 mmol/. mu.L) 2ul, dNTP (2.5 mmol/. mu.L) 0.5ul, DNA (20-100 ng/. mu.L) 2ul, 2 XBuffer (25mM)12.5ul, ddH₂O7.5 ul. The PCR reaction program is: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 56 ℃ for 5s, and 72 ℃ for 1 min; 72 ℃ for 10min and 12 ℃ for 1 min.

(2) Recovery and sequencing of PCR products

The target DNA fragment was recovered according to the Omega Gel Extraction Kit product manual, and 2. mu.L of the Gel recovered product was put in 1% agarose Gel for electrophoresis detection and then sent to the company for sequencing.

Results (see fig. 9 and 10)3 independent transgenic positive line leaves all showed spike-top degenerated phenotype; the degeneration rates of the transgenic positive lines (i.e. the knockout lines) and mutant paa1019 were identical (see FIG. 11). Compared with a negative control sequencing result, 3 transgenic knockout strains are found to be mutated in a CDS coding region of an OsCIPK31 gene and have insertion and replacement of bases (see SEQ ID NO. 3-5). The knockout test proves that the OsCIPK31 gene is a gene for controlling the ear tip degeneration character.

Example 5paa1019 functional complementation transgene validation test

pBWA (V) BII-GUS-OsCIPK31 Gene complementation vector construction

1. Amplification of target Gene

(1) The nucleotide sequence of OsCIPK31(LOC _ Os03g20380) gene in Yixiang 1B is taken as a template, a sequence for amplifying the full length of OsCIPK31 is designed and amplified by selecting a prediction promoter region, an exon region, an intron region and a terminator region, and the specific construction process is as follows:

the following primers were designed and synthesized to amplify the sequence for the full-length sequence of OsCIPK 31:

P20380-3F:5'-cagtCACCTGCaaaatagactccatcctcctctgctcct-3'，

P20380-3R:5'-cagtCACCTGCaaaacgacccatcctctcctccctcctc-3'。

wherein the PCR reaction system (50 ul): KOD enzyme 1ul, 2 XKOD Buffer 25ul, dNTP mix 10ul, OsCIPK31(+)1ul, OsCIPK31(-)1ul, template 1ul, and distilled water 11 ul. Reaction procedure for PCR: 10min at 98 ℃; circulating for 32 times at 98 deg.C for 30s, 58 deg.C for 30s, and 72 deg.C for 7min for 30 s; 72 ℃ for 15min and 14 ℃ for 10 min.

The amplification product was recovered by using an agarose gel DNA recovery kit of TaKaRa according to the procedures provided. The size of the detected amplification product is 6720bp, and the sequencing sequence is the full-length sequence of the target gene OsCIPK 31.

2. pBWA (V) BII-GUS-OsCIPK31 recombinant vector construction and identification

(1) Enzyme digestion connection reaction system:

(a) no-load enzyme digestion system (10 ul): pBWA (V) BII-GUS 2.5ul, Eco31I 0.5ul, T4Buffer1ul, distilled water to make up to 10 ul. Reaction conditions are as follows: the enzyme is cut for 1h at 37 ℃ in a water bath kettle at constant temperature.

Wherein the empty vector is pBWA (V) BII-GUS K; s: 35s promoter

(b) The target fragment digestion system (10 ul): the recovered product in step 1 was 3ul, AarI 0.5ul, T4Buffer1ul, and distilled water was made up to 10 ul. Reaction conditions are as follows: ligation was performed at 37 ℃ for 2 h.

(c) Ligation reaction system (10 ul): (a) 10ul of the cleavage product in (b), and 0.5ul of T4 ligase. Reaction conditions are as follows: ligation was performed at 37 ℃ for 2 h. The obtained product is pBWA (V) BII-GUS-OsCIPK31 recombinant plasmid.

(2) pBWA (V) transformation of BII-GUS-OsCIPK31 recombinant plasmid

(a) Taking a tube of 200. mu.L of Escherichia coli competent cell DH5a and 5. mu.L of the recombinant plasmid obtained in the step 2, (1) (c), and carrying out ice bath for 30 min;

(b) quickly placing in a constant temperature water bath kettle at 42 deg.C, thermally shocking for 90s, and ice-cooling for 2 min;

(c) adding 500 μ L LB liquid culture medium, mixing;

(d) culturing at 37 deg.C and 200rpm for 45min to restore normal growth state of cells;

(e) uniformly coating the bacterial liquid on an LB solid culture medium flat plate;

(f) after 30min, the cells were incubated overnight in a 37 ℃ incubator.

(3) pBWA (V) bacterial examination of BII-GUS-OsCIPK31 recombinant plasmid:

bacteria assay reaction system (20 ul): 2 XMix 10ul, detection primer P20380-4F 1ul, detection primer P20380-4R 1ul, and distilled water 8 ul.

Wherein the detection primer P20380-4F: 5'-gcctcggaaggttgtaac-3' the flow of the air in the air conditioner,

detection primer P20380-4R: 5'-gagactctgtatgaactg-3' are provided.

Bacterial detection size 1209bp

PCR reaction procedure: 5min at 95 ℃; 30s at 95 ℃, 30s at 55 ℃, 1min at 72 ℃ for 35s, and 32 cycles; 72 ℃ for 15min and 14 ℃ for 10 min.

(4) If the bacteria detection is correct, shake bacteria, extract plasmid, and verify enzyme digestion (Eco32I enzyme)

(a) The pBWA (V) BII-GUS-OsCIPK31 recombinant plasmid was extracted, and the quality of the extracted plasmid was checked by agarose gel electrophoresis at a concentration of 1%.

(b) Restriction enzyme identification of recombinant plasmid (Eco32I enzyme)

Digestion reaction system (10. mu.L): 10 XGreen Buffer1u L, Eco32I 0.5.5 u L, pBWA (V) BII-GUS-OsCIPK31 recombinant plasmid 3u L, sterile distilled water 5.5 u L. Reaction conditions are as follows: incubate in incubator at 37 ℃ for 30 min. The cleavage products were detected by electrophoresis on a 1% agarose gel. The detection result has correct sequence.

3. Agrobacterium impregnation method for transforming rice

The pBWA (V) BII-GUS-OsCIPK31 recombinant plasmid was transformed into Agrobacterium EHA105, and the mutant material paa1019 was transformed by Agrobacterium-mediated transformation. The specific procedure was according to the transformation scheme in example 4.

4. Detection of transgenic rice positive plants

The positive plants are screened by using a BIpR herbicide resistance marker in a recombinant vector pBWA (V) BII-GUS-OsCIPK31, namely, the herbicide is added into a culture medium, and the transgenic plant seedlings growing on the culture medium are good positive plants and inhibited negative plants. Finally, 3 positive strains were identified, which were designated paa1019-1, paa1019-2, paa1019-3, respectively. Results compared with mutant paa1019, ears of 3 lines of transgenic positive plants are normally developed, and the ear degeneration mutation phenotype is recovered (see fig. 12 and fig. 13). The test results show that OsCIPK31 is a gene for controlling the ear tip degeneration trait of the mutant paa 1019.

Sequence listing

<110> Sichuan university of agriculture

<120> rice panicle top degeneration related protein kinase and coding gene thereof

<130>2017S1116INH

<141>2017-12-04

<160>7

<170>SIPOSequenceListing 1.0

<210>1

<211>449

<212>PRT

<213>Oryza sativa

<400>1

Met Tyr Arg Ala Lys Arg Ala Ala Leu Ser Pro Lys Val Lys Arg Arg

1 5 10 15

Val Gly Lys Tyr Glu Leu Gly Arg Thr Ile Gly Glu Gly Thr Phe Ala

20 25 30

Lys Val Arg Phe Ala Lys Asn Thr Glu Asn Asp Glu Pro Val Ala Ile

35 40 45

Lys Ile Leu Asp Lys Glu Lys Val Gln Lys His Arg Leu Val Glu Gln

50 55 60

Ile Arg Arg Glu Ile Cys Thr Met Lys Leu Val Lys His Pro Asn Val

65 70 75 80

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

85 90 95

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

100 105 110

Gly Arg Leu Lys Glu Glu Glu Ala Arg Lys Tyr Phe Gln Gln Leu Ile

115 120 125

Asn Ala Val Asp Tyr Cys His Ser Arg Gly Val Tyr His Arg Asp Leu

130 135 140

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

145 150 155 160

Asp Phe Gly Leu Ser Ala Leu Thr Glu Gln Val Lys Ala Asp Gly Leu

165 170 175

Leu His Thr Thr Cys Gly Thr Pro Asn Tyr Val Ala Pro Glu Val Ile

180 185 190

Glu Asp Arg Gly Tyr Asp Gly Ala Ala Ala Asp Ile Trp Ser Cys Gly

195 200 205

Val Ile Leu Tyr Val Leu Leu Ala Gly Phe Leu Pro Phe Glu Asp Asp

210 215 220

Asn Ile Ile Ala Leu Tyr Lys Lys Ile Ser Glu Ala Gln Phe Thr Cys

225 230 235 240

Pro Ser Trp Phe Ser Thr Gly Ala Lys Lys Leu Ile Thr Arg Ile Leu

245 250 255

Asp Pro Asn Pro Thr Thr Arg Ile Thr Ile Ser Gln Ile Leu Glu Asp

260 265 270

Pro Trp Phe Lys Lys Gly Tyr Lys Pro Pro Val Phe Asp Glu Lys Tyr

275 280 285

Glu Thr Ser Phe Asp Asp Val Asp Ala Ala Phe Gly Asp Ser Glu Asp

290 295 300

Arg His Val Lys Glu Glu Thr Glu Asp Gln Pro Thr Ser Met Asn Ala

305 310 315 320

Phe Glu Leu Ile Ser Leu Asn Gln Ala Leu Asn Leu Asp Asn Leu Phe

325 330 335

Glu Ala Lys Lys Glu Tyr Lys Arg Glu Thr Arg Phe Thr Ser Gln Cys

340 345 350

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

355 360 365

Gly Phe Asp Ile Gln Lys Lys Asn Tyr Lys Met Arg Met Glu Asn Leu

370 375 380

Lys Ala Gly Arg Lys Gly Asn Leu Asn Val Ala Thr Glu Val Phe Gln

385 390 395 400

Val Ala Pro Ser Leu His Val Val Glu Leu Lys Lys Ala Lys Gly Asp

405 410 415

Thr Leu Glu Phe Gln Lys Phe Tyr Arg Thr Leu Ser Thr Gln Leu Lys

420 425 430

Asp Val Val Trp Lys Cys Asp Gly Glu Val Glu Gly Asn Gly Ala Ala

435 440 445

Ala

<210>2

<211>1347

<212>DNA

<213>Oryza sativa

<400>2

atgtataggg ctaagagggc tgcattatct ccaaaggtga agcgccgtgt agggaagtat 60

gagctcgggc gcaccattgg agaaggaacc tttgcaaagg tccggtttgc gaagaacact 120

gaaaatgacg aaccagttgc tatcaaaatc cttgacaagg agaaggttca gaagcacaga 180

ttggttgaac agattaggcg tgaaatttgt actatgaagt tagtaaagca tcctaatgtt 240

gttcggctgt tcgaggtcat gggaagtaaa gcaagaattt tcattgttct ggaatatgtt 300

actggaggag agctctttga aatcattgca actaatggaa ggttgaagga ggaggaagca 360

cgaaaatact ttcaacaact tatcaatgca gttgactact gccacagtag gggtgtgtac 420

cacagagact tgaagttaga aaatttgctg cttgatgctt ctggaaacct gaaagtatct 480

gactttggtt tgagtgcttt aaccgagcaa gtgaaggctg acggtttgct gcacacgaca 540

tgtggaactc ctaattatgt tgctccagag gtgattgagg acagaggcta tgatggggca 600

gctgcagata tctggtcttg tggggtaatc ctttatgttc tgcttgctgg gtttttacca 660

tttgaggatg acaacatcat tgctctttat aaaaagatct ctgaagctca gtttacctgt 720

ccctcttggt tttctactgg agctaagaag ctgatcacca gaattctgga tcccaaccct 780

acaactagga tcaccatttc tcaaatactg gaagatcctt ggttcaaaaa gggttacaaa 840

ccgcctgtat ttgacgagaa atatgaaact agttttgacg atgtcgatgc tgcttttgga 900

gactccgaag accggcatgt caaagaagaa actgaagatc agcctacctc tatgaacgcg 960

tttgaactca tttcactgaa tcaggcactg aatctggaca atttgttcga ggcaaaaaag 1020

gagtataaaa gagagacaag attcacatca caatgtcctc caaaagaaat tatcaccaag 1080

attgaagaag ctgcaaagcc acttggattt gatattcaaa agaaaaatta caagatgcgc 1140

atggagaacc tgaaagcagg tagaaaaggc aatctcaatg ttgcaactga ggttttccaa 1200

gtagctccat ccttacatgt ggttgagctc aagaaggcaa agggggacac tctggagttc 1260

caaaagttct acagaaccct gtcgacccag ctcaaggacg tggtctggaa gtgcgacggc 1320

gaggtcgaag gcaacggcgc cgcggcg 1347

<210>3

<211>1347

<212>DNA

<213>Artificial Sequence

<220>

<221>mutation

<222>(330)..(344)

<223> knockout of OsCIPK31 sequence of strain KO1019-11

<400>3

atgtataggg ctaagagggc tgcattatct ccaaaggtga agcgccgtgt agggaagtat 60

gagctcgggc gcaccattgg agaaggaacc tttgcaaagg tccggtttgc gaagaacact 120

gaaaatgacg aaccagttgc tatcaaaatc cttgacaagg agaaggttca gaagcacaga 180

ttggttgaac agattaggcg tgaaatttgt actatgaagt tagtaaagca tcctaatgtt 240

gttcggctgt tcgaggtcat gggaagtaaa gcaagaattt tcattgttct ggaatatgtt 300

actggaggag agctctttga aatcattgcc aatgacagat gatggaagga ggaggaagca 360

cgaaaatact ttcaacaact tatcaatgca gttgactact gccacagtag gggtgtgtac 420

cacagagact tgaagttaga aaatttgctg cttgatgctt ctggaaacct gaaagtatct 480

gactttggtt tgagtgcttt aaccgagcaa gtgaaggctg acggtttgct gcacacgaca 540

tgtggaactc ctaattatgt tgctccagag gtgattgagg acagaggcta tgatggggca 600

gctgcagata tctggtcttg tggggtaatc ctttatgttc tgcttgctgg gtttttacca 660

tttgaggatg acaacatcat tgctctttat aaaaagatct ctgaagctca gtttacctgt 720

ccctcttggt tttctactgg agctaagaag ctgatcacca gaattctgga tcccaaccct 780

acaactagga tcaccatttc tcaaatactg gaagatcctt ggttcaaaaa gggttacaaa 840

ccgcctgtat ttgacgagaa atatgaaact agttttgacg atgtcgatgc tgcttttgga 900

gactccgaag accggcatgt caaagaagaa actgaagatc agcctacctc tatgaacgcg 960

tttgaactca tttcactgaa tcaggcactg aatctggaca atttgttcga ggcaaaaaag 1020

gagtataaaa gagagacaag attcacatca caatgtcctc caaaagaaat tatcaccaag 1080

attgaagaag ctgcaaagcc acttggattt gatattcaaa agaaaaatta caagatgcgc 1140

atggagaacc tgaaagcagg tagaaaaggc aatctcaatg ttgcaactga ggttttccaa 1200

gtagctccat ccttacatgt ggttgagctc aagaaggcaa agggggacac tctggagttc 1260

caaaagttct acagaaccct gtcgacccag ctcaaggacg tggtctggaa gtgcgacggc 1320

gaggtcgaag gcaacggcgc cgcggcg 1347

<210>4

<211>1348

<212>DNA

<213>Artificial Sequence

<220>

<221>mutation

<222>(346)..(347)

<223> knockout of OsCIPK31 sequence of strain KO1019-26

<400>4

atgtataggg ctaagagggc tgcattatct ccaaaggtga agcgccgtgt agggaagtat 60

gagctcgggc gcaccattgg agaaggaacc tttgcaaagg tccggtttgc gaagaacact 120

gaaaatgacg aaccagttgc tatcaaaatc cttgacaagg agaaggttca gaagcacaga 180

ttggttgaac agattaggcg tgaaatttgt actatgaagt tagtaaagca tcctaatgtt 240

gttcggctgt tcgaggtcat gggaagtaaa gcaagaattt tcattgttct ggaatatgtt 300

actggaggag agctctttga aatcattgca actaatggaa ggttgatagg aggaggaagc 360

acgaaaatac tttcaacaac ttatcaatgc agttgactac tgccacagta ggggtgtgta 420

ccacagagac ttgaagttag aaaatttgct gcttgatgct tctggaaacc tgaaagtatc 480

tgactttggt ttgagtgctt taaccgagca agtgaaggct gacggtttgc tgcacacgac 540

atgtggaact cctaattatg ttgctccaga ggtgattgag gacagaggct atgatggggc 600

agctgcagat atctggtctt gtggggtaat cctttatgtt ctgcttgctg ggtttttacc 660

atttgaggat gacaacatca ttgctcttta taaaaagatc tctgaagctc agtttacctg 720

tccctcttgg ttttctactg gagctaagaa gctgatcacc agaattctgg atcccaaccc 780

tacaactagg atcaccattt ctcaaatact ggaagatcct tggttcaaaa agggttacaa 840

accgcctgta tttgacgaga aatatgaaac tagttttgac gatgtcgatg ctgcttttgg 900

agactccgaa gaccggcatg tcaaagaaga aactgaagat cagcctacct ctatgaacgc 960

gtttgaactc atttcactga atcaggcact gaatctggac aatttgttcg aggcaaaaaa 1020

ggagtataaa agagagacaa gattcacatc acaatgtcct ccaaaagaaa ttatcaccaa 1080

gattgaagaa gctgcaaagc cacttggatt tgatattcaa aagaaaaatt acaagatgcg 1140

catggagaac ctgaaagcag gtagaaaagg caatctcaat gttgcaactg aggttttcca 1200

agtagctcca tccttacatg tggttgagct caagaaggca aagggggaca ctctggagtt 1260

ccaaaagttc tacagaaccc tgtcgaccca gctcaaggac gtggtctgga agtgcgacgg 1320

cgaggtcgaa ggcaacggcg ccgcggcg 1348

<210>5

<211>1347

<212>DNA

<213>Artificial Sequence

<220>

<221>mutation

<222>(328)..(347)

<223> knock-out OsCIPK31 gene sequence of strain KO1019-29

<400>5

atgtataggg ctaagagggc tgcattatct ccaaaggtga agcgccgtgt agggaagtat 60

gagctcgggc gcaccattgg agaaggaacc tttgcaaagg tccggtttgc gaagaacact 120

gaaaatgacg aaccagttgc tatcaaaatc cttgacaagg agaaggttca gaagcacaga 180

ttggttgaac agattaggcg tgaaatttgt actatgaagt tagtaaagca tcctaatgtt 240

gttcggctgt tcgaggtcat gggaagtaaa gcaagaattt tcattgttct ggaatatgtt 300

actggaggag agctctttga aatcattaag gccaattaag gaaaggtgga ggaggaagca 360

cgaaaatact ttcaacaact tatcaatgca gttgactact gccacagtag gggtgtgtac 420

cacagagact tgaagttaga aaatttgctg cttgatgctt ctggaaacct gaaagtatct 480

gactttggtt tgagtgcttt aaccgagcaa gtgaaggctg acggtttgct gcacacgaca 540

tgtggaactc ctaattatgt tgctccagag gtgattgagg acagaggcta tgatggggca 600

gctgcagata tctggtcttg tggggtaatc ctttatgttc tgcttgctgg gtttttacca 660

tttgaggatg acaacatcat tgctctttat aaaaagatct ctgaagctca gtttacctgt 720

ccctcttggt tttctactgg agctaagaag ctgatcacca gaattctgga tcccaaccct 780

acaactagga tcaccatttc tcaaatactg gaagatcctt ggttcaaaaa gggttacaaa 840

ccgcctgtat ttgacgagaa atatgaaact agttttgacg atgtcgatgc tgcttttgga 900

gactccgaag accggcatgt caaagaagaa actgaagatc agcctacctc tatgaacgcg 960

tttgaactca tttcactgaa tcaggcactg aatctggaca atttgttcga ggcaaaaaag 1020

gagtataaaa gagagacaag attcacatca caatgtcctc caaaagaaat tatcaccaag 1080

attgaagaag ctgcaaagcc acttggattt gatattcaaa agaaaaatta caagatgcgc 1140

atggagaacc tgaaagcagg tagaaaaggc aatctcaatg ttgcaactga ggttttccaa 1200

gtagctccat ccttacatgt ggttgagctc aagaaggcaa agggggacac tctggagttc 1260

caaaagttct acagaaccct gtcgacccag ctcaaggacg tggtctggaa gtgcgacggc 1320

gaggtcgaag gcaacggcgc cgcggcg 1347

<210>6

<211>1284

<212>DNA

<213>Artificial Sequence

<220>

<221>mutation

<222>(190)..(253)

<223> OsCIPK31 sequence of mutant paa1019

<400>6

atgtataggg ctaagagggc tgcattatct ccaaaggtga agcgccgtgt agggaagtat 60

gagctcgggc gcaccattgg agaaggaacc tttgcaaagg tccggtttgc gaagaacact 120

gaaaatgacg aaccagttgc tatcaaaatc cttgacaagg agaaggttca gaagcacaga 180

ttggttgaac aggtcatggg aagtaaagca agaattttca ttgttctgga atatgttact 240

ggaggagagc tctttgaaat cattgcaact aatggaaggt tgaaggagga ggaagcacga 300

aaatactttc aacaacttat caatgcagtt gactactgcc acagtagggg tgtgtaccac 360

agagacttga agttagaaaa tttgctgctt gatgcttctg gaaacctgaa agtatctgac 420

tttggtttga gtgctttaac cgagcaagtg aaggctgacg gtttgctgca cacgacatgt 480

ggaactccta attatgttgc tccagaggtg attgaggaca gaggctatga tggggcagct 540

gcagatatct ggtcttgtgg ggtaatcctt tatgttctgc ttgctgggtt tttaccattt 600

gaggatgaca acatcattgc tctttataaa aagatctctg aagctcagtt tacctgtccc 660

tcttggtttt ctactggagc taagaagctg atcaccagaa ttctggatcc caaccctaca 720

actaggatca ccatttctca aatactggaa gatccttggt tcaaaaaggg ttacaaaccg 780

cctgtatttg acgagaaata tgaaactagt tttgacgatg tcgatgctgc ttttggagac 840

tccgaagacc ggcatgtcaa agaagaaact gaagatcagc ctacctctat gaacgcgttt 900

gaactcattt cactgaatca ggcactgaat ctggacaatt tgttcgaggc aaaaaaggag 960

tataaaagag agacaagatt cacatcacaa tgtcctccaa aagaaattat caccaagatt 1020

gaagaagctg caaagccact tggatttgat attcaaaaga aaaattacaa gatgcgcatg 1080

gagaacctga aagcaggtag aaaaggcaat ctcaatgttg caactgaggt tttccaagta 1140

gctccatcct tacatgtggt tgagctcaag aaggcaaagg gggacactct ggagttccaa 1200

aagttctaca gaaccctgtc gacccagctc aaggacgtgg tctggaagtg cgacggcgag 1260

gtcgaaggca acggcgccgc ggcg 1284

<210>7

<211>23

<212>DNA

<213>Artificial Sequence

<220>

<221>misc_binding

<222>(1)..(23)

<223> target sequence for knocking out OsCIPK31 gene

<400>7

aactaatgga aggttgaagg agg 23

Claims

The application of OsCIPK31 protein in controlling the degradation property of the top of rice ears; it is characterized in that the protein consists of an amino acid sequence shown in SEQ ID NO. 1.
2, the application of the OsCIPK31 gene in controlling the rice spike top degradation property; the OsCIPK31 gene is characterized by consisting of a nucleotide sequence shown in SEQ ID NO. 2.