CN107164401A - A kind of method and application that rice Os PIL15 mutant is prepared based on CRISPR/Cas9 technologies - Google Patents

Abstract

The present invention relates to a kind of method and application that rice Os PIL15 mutant is prepared based on CRISPR/Cas9 technologies, belong to field of plant genetic.The present invention is oriented editor according to CRISPR/Cas9 technical principles to paddy rice phytochrome interaction factor OsPIL15, design OsPIL15 mutation target spots, build CRISPR/Cas9 gRNA (pBUN411 gRNA) expression vector, japonica rice variety Nipponbare is imported using agrobacterium-mediated transformation, positive transgenic plant is obtained with herbicide resistance markers' screening, mutation individual plant is analyzed and identified using enzyme cutting method and PCR sequencing PCR.A set of paddy rice ospil15 mutant new germ plasms with significant application value are obtained, Mutant progeny seed grain length pole is dramatically increased, average amplification can be applied to rice high yield, stable yields breeding up to 4.79%.

Description

A method for preparing rice OsPIL15 mutants based on CRISPR/Cas9 technology and application

technical field

The invention relates to a method and application for preparing a rice OsPIL15 mutant based on CRISPR/Cas9 technology, and belongs to the technical field of plant genetic engineering.

Background technique

Gene editing technology refers to the ability to "edit" target genes to achieve knockout and insertion of specific DNA fragments. In recent years, a genome-directed editing technology called CRISPR/Cas9 (clustered regularly interspaced shortpalindromic repeats/CRISPR-associated nuclease 9, Cas9) has attracted much attention. In early 2013, two articles in "Science" reported for the first time that Cas9 nuclease was used for genome editing in human and mouse cells (Mali et al., 2013; Cong et al., 2013), which has been widely recognized as the third-generation gene editing technology. extensive attention. CRISPR/Cas9 technology uses nuclease Cas9 protein to form a complex with single guide RNA (Single guide RNA, sgRNA). The sgRNA determines the specificity of the target sequence through complementary base pairing. spacers) complementary genomic DNA, causing double-strand DNA damage, and then introducing gene mutations through the NHEJ (non-homologous end joining) repair mechanism in vivo (Wiedenheft et al., 2012). With the establishment and application of the CRISPR/Cas9 system in human and animal cells, the CRISPR/Cas9 system has achieved targeted genome editing in plants such as Arabidopsis thaliana, tobacco, rice, wheat, corn, sorghum, tomato, and sweet orange ( Belhaj et al., 2015; Doudna et al., 2014).

Rice is one of the most important food crops in the world, providing food for more than half of the world's population. The stable and increased production of rice is of great strategic significance to ensure my country's food security. The yield of rice is mainly determined by the three factors of yield, the number of panicles, the number of grains per panicle and the weight of thousand grains, and the grain weight is the factor with the highest heritability among the three factors of yield. Grain weight is mainly determined by grain size and grain filling, and grain size is mainly controlled by grain length, grain width and grain thickness (Xing et al., 2015). At present, with the acceleration of mutant research and the application of biotechnology methods such as map-based cloning and QTL, many genes related to grain length, grain width and grain weight (GS3, GS5, GW2, GW5, GW7 and GW8) have been cloned one after another ( Song et al., 2007; Jianfeng et al., 2008; Mao et al., 2010; Li et al., 2011; Wang et al., 2012; Wang et al., 2015). Among them, GS3 is the main QTL controlling rice grain weight and grain length, and it functions as a negative regulator in regulating grain and organ size; GS5 encodes a serine carboxypeptidase, which is a quantity controlling rice grain width, filling degree and thousand-grain weight. Trait gene; GW2 encodes a circular E3 ubiquitin ligase, which negatively regulates grain width and grain weight; GW5 can control grain width and grain weight by affecting ubiquitination degradation pathway; GW7 can control rice grain by changing cell division pattern Shape and improve rice quality; GW8 is a transcription factor containing SBP domain, which can directly bind to the GW7 promoter and repress its expression, thereby controlling grain size and quality. The study of these genes provides an important theoretical basis for rice breeding, and some yield-related genes have been widely used in the breeding of high-yielding and stable-yielding rice varieties.

PIFs (Phytochrome-Interacting Factors) or PILs (Phytochrome Interacting Factor-Like) are a type of transcription factor in the bHLH transcription factor family, and their main characteristic is that they can interact with phytochrome to directly or indirectly regulate light response Gene. As a type of bHLH protein, all PIFs family proteins contain an APB (Active Phytochrome B-binding) or APA (Active Phytochrome A-binding) domain that interacts with phytochrome at the N-terminus and a bHLH-DNA binding structure at the C-terminus domain and nuclear localization domain (Shen et al., 2008; Khanna et al., 2004). In rice, six PIF transcription factors (OsPIL11-OsPIL16) were identified in the rice genome by homology analysis (Nakamura et al., 2007). Overexpression of OsPIL13 in rice can promote the elongation of rice internodes, whereas low expression can inhibit its internode elongation (Todaka et al., 2012), and the germination of rice seeds overexpressing OsPIL15 in the dark environment is inhibited (Zhou et al., 2014), OsPIL16 can negatively regulate PGL1 to regulate grain size (Heang et al., 2012). Therefore, the construction of the phytochrome interaction factor OsPIL15 mutant is of great significance for rice breeding fields such as seed weight gain and yield increase.

Contents of the invention

The object of the present invention is to provide a method for preparing rice OsPIL15 mutants based on CRISPR/Cas9 technology.

The present invention also provides the application of the mutant prepared by the above method in rice breeding.

In order to achieve the above object, the technical solution adopted in the present invention is:

A method for preparing rice OsPIL15 mutants based on CRISPR/Cas9 technology, comprising the steps of:

1) Selection of gRNA target sequence: the sequence is 5′-GACTTCTTCTCCGAGCTCCAGG-3′, the PAM sequence at the 3′ end of the sequence is AGG, and the restriction enzyme SacI recognition sequence is 5′-GAGCTC-3′;

2) Design of upstream and downstream primers for the gRNA oligonucleotide chain:

The upstream primer is sgRNA-F: 5′-GGCGGACTTCTTCTCCGAGCTCC-3′,

The downstream primer is sgRNA-R: 5′-AAACGGAGCTCGGAGAAGAAGTC-3′;

3) Construction of gRNA expression vector: Mix and anneal the upstream and downstream primers to obtain oligonucleotide double-stranded DNA; digest the pBUN411 plasmid with endonuclease BsaI to obtain a linear plasmid; use T4 ligase to combine the linear plasmid and Ligation of oligonucleotide double-stranded DNA to obtain the ligation product; transformation, screening, and verification of the ligation product to obtain the pBUN411-gRNA expression vector;

4) Introducing the pBUN411-gRNA expression vector into Agrobacterium to obtain CRISPR/Cas9-gRNA Agrobacterium; infecting rice callus with CRISPR/Cas9-gRNA Agrobacterium;

5) Inducing the callus obtained in step 4) to obtain regenerated shoots, screening to obtain transgene-positive plants, and identifying the rice OsPIL15 mutant.

The annealing in step 3) is annealing at 65° C. for 5 minutes.

The enzyme digestion system described in step 3) is: 2 μg of pBUN411 plasmid, 5 μL of 10×CutSmart Buffer, 10 U of Bsa I, and added ddH ₂ O to 50 μL.

The conditions for enzyme digestion in step 3) are: enzyme digestion at 37°C for 4 hours.

The system for T4 ligase ligation in step 3) is: 6 μL of linearized plasmid, 2 μL of oligonucleotide double-stranded DNA, 5 μL of 10×Buffer, and 1 μL of T4 DNA ligase.

The conditions for T4 ligase ligation in step 3) are: ligation at 4° C. for 12 hours.

In step 3), the specific operations of transforming, screening, and verifying the ligated product are as follows: transform the ligated product into Escherichia coli DH5α competent cells by the heat shock method, and spread the bacterial solution on a LB medium plate containing 50 mg/L of kanamycin After culturing overnight, single clones were picked and propagated by shaking; primers pBUN411-VF and pBUN411-VR (see Table 1 for primer sequences) were used for colony PCR verification, and the amplified fragment of 336bp was the correct pBUN411-gRNA expression vector.

The formula of the LB medium is (1L): tryptone 10g; yeast extract 5g; sodium chloride 10g; agar 15g.

In step 4), the pBUN411-gRNA expression vector was introduced into Agrobacterium agrobacterium EHA105 by heat shock method.

Step 5) is to obtain regenerated shoots by screening under the condition of herbicide.

Step 5) screening by using herbicide resistance gene primers;

The primers used are: Bar-F: AAGCACGGTCAACTTCCGTA;

Bar-R: GAAGTCCAGCTGCCAGAAAC.

In step 5), plants with 412bp amplified products of Bar-F and Bar-R were screened out for further identification.

Step 5) identifying by using primers on both sides of the gRNA target sequence;

The primers on both sides are OsPIL15-test-F: 5′-TGTTTTGTGTGTGCAGGTCC-3′;

OsPIL15-test-R: 5'-CGGGAGAAGAGCGAGAAGTT-3'.

Identification described in step 5) includes:

A: Using the DNA of the transgenic positive plant as a template, and OsPIL15-test-F and OsPIL15-test-R as detection primers, perform PCR amplification;

B: Digest the PCR amplification product with SacⅠ enzyme;

C: Use electrophoresis to separate the digested products. If the electrophoresis results are only two bands of 400bp and 273bp, it is an unmutated strain; otherwise, it is a mutant strain.

The PCR amplification system in step A is: 2×Taq MasterMix 25 μL, OsPIL15-test-F 2 μL, OsPIL15-test-R 2 μL, DNA template 1 μg, add ddH ₂ O to 50 μL.

The PCR amplification conditions in step A are: pre-denaturation at 94°C for 2min, denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, and final extension at 72°C for 10min, wherein denaturation, annealing and extension are 30 cycles.

The digestion system in step B is: 1 μg of PCR product, 2 μL of 10×L Buffer, 1 U of SacⅠ, and add ddH ₂ O to 20 μL.

The digestion conditions in step B are: digestion at 37°C overnight.

In step C, 2% agarose gel is used for electrophoresis separation.

Sequence the PCR amplification product of the transgene positive plant to determine whether it is a heterozygote with a homologous chromosome mutation, or a biallelic mutant in which two alleles have different mutations, or two alleles with the same mutation. Homozygous mutants for mutations, or single plants without mutations.

Application of the rice OsPIL15 mutant prepared by the method for preparing the rice OsPIL15 mutant in rice breeding.

Specifically, the homozygous mutant among the selfed offspring of the rice OsPIL15 mutant is selected for breeding for rice seed length. For example, select the biallelic mutants of the T ₀ generation to self-cross to obtain the T ₁ generation, and select homozygous mutants from the T ₁ generation and other subsequent generations for rice breeding.

The beneficial effects of the present invention are: the present invention performs directional editing on the rice phytochrome interaction factor OsPIL15 according to the technical principle of CRISPR/Cas9. Design the OsPIL15 mutation target, construct the CRISPR/Cas9-gRNA (pBUN411-gRNA) expression vector, use the Agrobacterium-mediated method to introduce the japonica rice variety Nipponbare, and obtain positive transgenic plants by screening with herbicide resistance markers, using enzyme digestion and sequencing methods Analysis and identification of mutant individual plants. A set of new rice ospil15 mutant germplasm with important application value was obtained by this method. Compared with wild-type Nipponbare, the grain length of the prepared new ospil15 mutant germplasm was significantly increased.

Aiming at the present situation of continuous population growth and increasing food demand, the present invention discusses the function of phytochrome interaction factor OsPIL15 in rice variety improvement, and provides an experimental basis for controlling rice grain length by regulating OsPIL15 gene expression. The successful implementation of the invention based on the CRISPR/Cas9 technology for the creation of the rice ospil15 mutant provides a simple and effective technical means for the rapid establishment of new rice strains with important application value in production such as grain enlargement. It has potential application value in increasing crop weight and increasing crop yield, and has important practical significance for high-yield and stable-yield breeding of rice.

Description of drawings

Figure 1 is a schematic diagram of the assembly of the gRNA target site and the CRISPR/Cas9-gRNA expression vector;

Figure 2 is an agarose gel result diagram of PCR products and enzyme-cleaved PCR products identifying gene-edited plants;

Figure 3 is a comparison chart of different ospil15 mutant mutant sequences and wild-type Nipponbare sequence analysis;

Figure 4 is a comparison of the grain length of the ospil15 mutant and the wild type Nipponbare;

Fig. 5 is an analysis diagram of the difference in grain length between the ospil15 mutant and the wild type Nipponbare.

detailed description

The present invention will be further described in detail below in conjunction with specific embodiments.

The primers in the following examples and comparative examples were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

Example 1

1. Selection of OsPIL15 gene gRNA target sequence and design of upstream and downstream primers of gRNA oligonucleotide chain

According to the exon sequence of the rice OsPIL15 gene (GenBank accession number AK102252.1), the gRNA target sequence 5'-GACTTCTTCTCCGAGCTCCAGG-3' was designed. The PAM sequence at the 3' end of the sequence is AGG, and the Cas9 protein will be 3-3' upstream of the AGG sequence. Cut the DNA at 4bp to form a smooth end. There is a restriction endonuclease SacI recognition sequence (5′-GAGCT ^▼ C-3′) at the cut point. The target site, PAM sequence and restriction site are shown in Figure 1- A: The schematic diagram of sgRNA and Cas9 protein targeting OsPIL15 gene.

Design the upstream and downstream primers of the gRNA oligonucleotide chain according to the sgRNA sequence. The primer sequences are shown in Table 1 for sgRNA-F and sgRNA-R; the first four bases in sgRNA-F and sgRNA-R are BsaI restriction endonucleases sticky end adapters.

2. Construction of CRISPR/Cas9-gRNA (pBUN411-gRNA) expression vector

1) Take an equal amount of gRNA oligonucleotide chain upstream and downstream primers (final concentration 10 μM) and mix, anneal at 65°C for 5 minutes, then gradually cool to room temperature to form complementary double-stranded DNA, which will be used for subsequent vector construction;

2) Digest the CRISPR/Cas9 vector pBUN411 with the restriction endonuclease BsaⅠ. The CRISPR/Cas9 vector pBUN411 was donated by Professor Chen Qijun of China Agricultural University (Xing et al., 2014) to linearize it. The 50 μL digestion system is as follows: pBUN411 Plasmid 2 μg, 10×CutSmart Buffer 5 μL, BsaⅠ10U (1 μL), add ddH ₂ O to 50 μL, digest at 37°C for 4 hours, then heat-treat at 65°C for 20 minutes to inactivate the enzyme; after purification, add the following ligation system: linear plasmid 6 μL, oligonucleotide Acid duplex 2 μL, 10×Buffer 5 μL, T4 DNA ligase 1 μL; ligation conditions: 4°C for 12 hours.

3) The ligation product was transformed into Escherichia coli DH5α competent cells by heat shock method, and the bacterial solution was spread on the LB medium plate containing 50 mg/L kanamycin, and after culturing overnight, single clones were picked and expanded by shaking. Colony PCR verification was performed using primers pBUN411-VF and pBUN411-VR (see Table 1 for primer sequences).

The PCR system is: 2×Taq MasterMix 5 μL; pBUN411-VF (10 μM) 0.4 μL; pBUN411-VR (10 μM) 0.4 μL; monoclonal template 1 μL; ddH ₂ O 3.2 μL. The PCR conditions were: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and final extension at 72°C for 10 min, in which denaturation, annealing and extension were 35 cycles.

The length of the pBUN411 empty vector amplified fragment is 1538bp, and it is ligated after digestion with BsaⅠ. The size of the target sequence inserted and amplified fragment should be 336bp (as shown in SEQ ID NO.17). Select the colony with the correct PCR fragment size and extract the plasmid, indicating that The pBUN411-gRNA expression vector was constructed successfully. The target sequence is driven by the promoter of the OsU3 gene, and the gene encoding the Cas9 protein is driven by the promoter of the maize ubiquitin gene (Ubi) (as shown in Figure 1-B: the linear structure between LB and RB of the CRISPR/Cas9-gRNA expression vector) .

Example 2

Agrobacterium-mediated genetic transformation of rice callus and detection of positive transgenic plants

Using the heat shock method, referring to the method reported by Hood et al. (Hood et al., 1993), the pBUN411-gRNA expression vector was introduced into Agrobacterium agrobacterium EHA105, and the rice variety Nipponbare (Oryza sativa ssp. .Japonica cv.Nipponbare) callus, transgenic rice was carried out with reference to the method reported by Nishimura et al. (Nishimura et al., 2006), and regenerated shoots were obtained by screening with herbicide (Basta). Genomic DNA of transgenic plants was extracted by SLS method, and positive screening of transgenic plants was carried out using herbicide-resistant gene (bar) primers Bar-F and Bar-R (see Table 1 for primer sequences) (the herbicide gene is located on the pBUN411-gRNA expression vector) .

The PCR system is: 2×Taq MasterMix 5 μL; Bar-F (10 μM) 0.4 μL; Bar-R (10 μM) 0.4 μL; DNA template 1 μl; ddH ₂ O 3.2 μL. The PCR conditions were: pre-denaturation at 94°C for 2min, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 30s, and final extension at 72°C for 10min, in which denaturation, annealing and extension were 35 cycles. The length of the PCR amplification product is 412bp, which is the positive transgenic plant.

Example 3

Screening and identification of T ₀ generation mutant of OsPIL15 gene

1) In order to detect the mutation of the target site in the positive transgenic plants of the T ₀ generation, design primers OsPIL15- test-F and OsPIL15-test-R (see Table 1 for the primer sequence), amplify the target site sequence with the DNA of a transgenic positive individual plant as a template, and the 50 μL amplification system is as follows: 2×Taq MasterMix 25 μL, OsPIL15-test-F 2 μL , OsPIL15-test-R 2μL, DNA template 1μg, add ddH ₂ O to 50μL; PCR conditions: pre-denaturation at 94°C for 2min, denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, final extension at 72°C for 10min, where Denaturation, annealing and extension were 35 cycles.

A part of the amplified products were separated by agarose gel electrophoresis. As shown in Figure 2-A, the size of the wild-type amplified fragment was 673bp, and the amplified fragments of individual plants 1, 2, 23 and 24 were significantly smaller than the wild-type bands, preliminarily indicating that these four individual plants may have large fragment deletions, and the PCR products of individual plants 1 and 24 have two bands, only one of which is significantly smaller than the wild-type band, so the two may have only one homology Chromosomes had large fragment deletions; the PCR products of individual plants 2 and 23 had only one band and were obviously less than 673bp. It can be preliminarily judged that both homologous chromosomes had large fragment deletions.

A part of the amplified product was digested with SacI restriction endonuclease, and the 20 μL system was as follows: 1 μg of PCR product, 2 μL of 10×L Buffer, SacI 1U (1 μL), added ddH ₂ O to 20 μL, and digested overnight at 37°C.

Use 2% agarose gel electrophoresis to detect the enzyme digestion results. If the PCR product can be completely cut, only two bands of 400bp and 273bp are obtained, indicating that the target site has not been mutated, and it is an unmutated individual plant; otherwise, it is a mutant plant. . If the PCR product is partially cut and three bands are obtained, two of which are 400bp and 273bp, and the other is around 673bp, it indicates that a homologous chromosome has a mutation and is a heterozygote; if the PCR product cannot be cut at all, it indicates that two Mutations in both homologous chromosomes may be biallelic mutants with different mutations in the two alleles or homozygous for the same mutation in the two alleles.

The detection results are shown in Figure 2-B, and the results showed that no mutation occurred in individual plants 4, 5, 6, 7, 10, 12, 15, 16, 18 and 25; the PCR products of individual plants 8, 9, 11 and 17 were detected by Partial digestion, forming three bands, which is a heterozygous mutation of a homologous chromosome; the PCR products of individual plants 1, 2, 3, 13, 14, 19, 20, 21, 22, 23 and 24 cannot be cut at all, then May be biallelic or homozygous.

The PCR products of mutants 1, 2, 3, 8, 9, 11, 13, 14, 17, 19, 20, 21, 22, 23 and 24 were sent for sequencing, and the sequencing results were compared with the wild-type sequence to analyze the mutants genotype. Comprehensive analysis of the sequencing results obtained the strain mutations, as shown in Figure 3, a total of 5 homozygous mutations (single plants 2, 3, 13, 14 and 23), 6 biallelic mutations (single plants 1, 19, 20, 21, 22 and 24) and 4 heterozygous mutants (single plants 8, 9, 11 and 17), including 10 different mutant genotypes.

Example 4

Grain phenotype analysis of _ospil15 homozygous mutant in T1 generation

The amino acid sequence of rice phytochrome interaction factor OsPIL15 is shown in SEQ ID NO.1, and the protein coding sequence is shown in SEQ ID NO.2.

The T ₀ generation mutants obtained in Example 3 were self-crossed for 1 generation to obtain the T ₁ generation ospil15 homozygous mutants, and the mutant lines 1, 21 and 22 were selected for grain phenotype analysis.

In the mutant ospil15-1, the coding sequence of the mutated rice phytochrome-interacting factor OsPIL15 is shown in SEQ ID NO.3; the amino acid sequence of the mutated rice phytochrome-interacting factor OsPIL15 is shown in SEQ ID NO.4.

In the mutant ospil15-21, the coding sequence of the mutated rice phytochrome-interacting factor OsPIL15 is shown in SEQ ID NO.5; the amino acid sequence of the mutated rice phytochrome-interacting factor OsPIL15 is shown in SEQ ID NO.6.

In the mutant ospil15-22, the coding sequence of the mutated rice phytochrome-interacting factor OsPIL15 is shown in SEQ ID NO.7; the amino acid sequence of the mutated rice phytochrome-interacting factor OsPIL15 is shown in SEQ ID NO.8.

The above three homozygous mutant lines were selected, and 30 mature harvested grains were randomly selected, and the grain length was measured using a rice appearance quality detector (JMWT12, Dongfu Jiuheng, Beijing), and the average value was obtained after 5 repetitions. The results are shown in Figures 4 and 5, the grain length of the three groups of ospil15 mutants was significantly increased, among which the mutant ospil15-22 had the highest increase of 5.69%, and the smallest increase of the mutant ospil15-21 also reached 3.88%.

The specific examples of the present invention have been described above, but the implementation of the present invention is not limited to the above specific examples. The changes, modifications, substitutions, combinations, and simplifications made by those skilled in the art within the scope of the claims shall all be equivalent replacements, and shall be included in the protection scope of the present invention.

Table 1 Primer sequence list

Primer name Primer sequence (5'-3') sgRNA-F GGCG GACTTCTTCTCCGAGCTCC sgRNA-R AAAC GGAGCTCGGAGAAGAAGTC pBUN411-VF CCATGAAGCCTTTCAGGACATGTA pBUN411-VR ACGCTGCAAACATGAGACGGAGAA Bar-F AAGCACGGTCAACTTCCGTA Bar-R GAAGTCCAGCTGCCAGAAAC OsPIL15-test-F TGTTTTGTGTGTGCAGGTCC OsPIL15-test-R CGGGAGAAGAGCGAGAAGTT

<110> Henan Agricultural University

<120> A method and application for preparing rice OsPIL15 mutants based on CRISPR/Cas9 technology

<160> 18

<170> PatentIn version 3.5

<211> 637

<212> PRT

<213> sequence

<221> Amino acid sequence of rice phytochrome interaction factor OsPIL15

<400> 1

Met Ser Asp Gly Asn Asp Phe Ala Glu Leu Leu Trp Glu Asn Gly

1 5 10 15

Gln Ala Val Val His Gly Arg Lys Lys His Pro Gln Pro Ala Phe

20 25 30

Pro Pro Phe Gly Phe Phe Gly Gly Thr Gly Gly Gly Gly Gly Gly

35 40 45

Ser Ser Ser Arg Ala Gln Glu Arg Gln Pro Gly Gly Ile Asp Ala

50 55 60

Phe Ala Lys Val Gly Gly Gly Phe Gly Ala Leu Gly MET Ala Pro

65 70 75

Ala Val His Asp Phe Ala Ser Gly Phe Gly Ala Thr Thr Gln Asp

80 85 90

Asn Gly Asp Asp Asp Thr Val Pro Trp Ile His Tyr Pro Ile Ile

95 100 105

Asp Asp Glu Asp Ala Ala Ala Pro Ala Ala Leu Ala Ala Ala Asp

110 115 120

Tyr Gly Ser Asp Phe Phe Ser Glu Leu Gln Ala Ala Ala Ala Ala

125 130 135

Ala Ala Ala Ala Ala Pro Pro Thr Asp Leu Ala Ser Leu Pro Ala

140 145 150

Ser Asn His Asn Gly Ala Thr Asn Asn Arg Asn Ala Pro Val Ala

155 160 165

Thr Thr Thr Thr Arg Glu Pro Ser Lys Glu Ser His Gly Gly Leu

170 175 180

Ser Val Pro Thr Thr Arg Ala Glu Pro Gln Pro Gln Pro Gln Leu

185 190 195

Ala Ala Ala Lys Leu Pro Arg Ser Ser Gly Ser Gly Gly Gly Glu

200 205 210

Gly Val MET Asn Phe Ser Leu Phe Ser Arg Pro Ala Val Leu Ala

215 220 225

Arg Ala Thr Leu Glu Ser Ala Gln Arg Thr Gln Gly Thr Asp Asn

230 235 240

Lys Ala Ser Asn Val Thr Ala Ser Asn Arg Val Glu Ser Thr Val

245 250 255

Val Gln Thr Ala Ser Gly Pro Arg Ser Ala Pro Ala Phe Ala Asp

260 265 270

Gln Arg Ala Ala Ala Trp Pro Pro Gln Pro Lys Glu MET Pro Phe

275 280 285

Ala Ser Thr Ala Ala Ala Pro MET Ala Pro Ala Val Asn Leu His

290 295 300

His Glu MET Gly Arg Asp Arg Ala Gly Arg Thr MET Pro Val His

305 310 315

Lys Thr Glu Ala Arg Lys Ala Pro Glu Ala Thr Val Ala Thr Ser

320 325 330

Ser Val Cys Ser Gly Asn Gly Ala Gly Ser Asp Glu Leu Trp Arg

335 340 345

Gln Gln Lys Arg Lys Cys Gln Ala Gln Ala Glu Cys Ser Ala Ser

350 355 360

Gln Asp Asp Asp Leu Asp Asp Glu Pro Gly Val Leu Arg Lys Ser

365 370 375

Gly Thr Arg Ser Thr Lys Arg Ser Arg Thr Ala Glu Val His Asn

380 385 390

Leu Ser Glu Arg Arg Arg Arg Arg Asp Arg Ile Asn Glu Lys MET Arg

395 400 405

Ala Leu Gln Glu Leu Ile Pro Asn Cys Asn Lys Ile Asp Lys Ala

410 415 420

Ser MET Leu Asp Glu Ala Ile Glu Tyr Leu Lys Thr Leu Gln Leu

425 430 435

Gln Val Gln MET MET Ser MET Gly Thr Gly Leu Cys Ile Pro Pro

440 445 450

MET Leu Leu Pro Thr Ala MET Gln His Leu Gln Ile Pro Pro MET

455 460 465

Ala His Phe Pro His Leu Gly MET Gly Leu Gly Tyr Gly MET Gly

470 475 480

Val Phe Asp MET Ser Asn Thr Gly Ala Leu Gln MET Pro Pro MET

485 490 495

Pro Gly Ala His Phe Pro Cys Pro MET Ile Pro Gly Ala Ser Pro

500 505 510

Gln Gly Leu Gly Ile Pro Gly Thr Ser Thr MET Pro MET Phe Gly

515 520 525

Val Pro Gly Gln Thr Ile Pro Ser Ser Ala Ser Ser Ser Val Pro Pro

530 535 540

Phe Ala Ser Leu Ala Gly Leu Pro Val Arg Pro Ser Gly Val Pro

545 550 555

Gln Val Ser Gly Ala MET Ala Asn MET Val Gln Asp Gln Gln Gln

560 565 570

Gly Ile Ala Asn Gln Gln Gln Gln Cys Leu Asn Lys Glu Ala Ile

575 580 585

Gln Gly Ala Asn Pro Gly Asp Ser Gln MET Gln Ile Ile MET Gln

590 595 600

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

605 610 615

Ser Ser Gln Phe Ser Asp Gly Thr Gly Lys Gly Thr Asn Ala Arg

620 625 630

Glu Arg Asp Gly Ala Glu Thr

635 637

<211> 1914

<212>DNA

<213> sequence

<221> Gene coding sequence of rice phytochrome interaction factor OsPIL15

<400> 2

atgtccgacg gcaacgactt cgccgagctg ctgtggggaga acggccaggc ggtggtgcac 60

gggaggaaga agcacccgca gccggccttc ccgccgttcg gcttcttcgg tggcaccggc 120

ggtggcggcg gcggcagcag tagtagagcc caggagaggc agcccggcgg catcgatgcg 180

ttcgccaagg tggggggcgg cttcggcgcc ttgggcatgg ctccggcggt gcacgacttc 240

gcttctggct tcggcgccac cacgcaggac aacggtgatg atgacaccgt tccgtggatc 300

cattacccca taattgacga tgaagacgcc gccgcccctg ctgctctcgc agcagcggac 360

tatggctccg acttcttctc cgagctccag gcggcggcgg ctgccgcggc ggccgccgcg 420

ccgccgaccg atctcgcctc tctgccagcc tccaatcaca acggcgccac caataacaga 480

aatgctccgg ttgccaccac caccaccagg gaaccctcca aggaaagcca cggcggcctg 540

tcggttccca ccacccgagc cgagccgcag ccgcagccac agctcgccgc agccaagctg 600

cctcggtcga gcggcagcgg cggcggcgag ggcgtgatga acttctcgct cttctcccgc 660

ccggccgtcc tggcgagggc gacgctggag agcgcgcaga ggacgcaggg caccgacaat 720

aaggcgtcca atgtcaccgc gagcaaccgc gtcgagtcga cggtcgtgca gacggcgagc 780

gggccaagga gcgcaccggc gttcgccgat cagagggcgg cggcgtggcc gccgcagccg 840

aaggagatgc cgttcgcgtc cacggcagcc gctcccatgg ccccggccgt taacctgcac 900

cacgagatgg gccgtgacag ggcaggccga accatgcctg tccacaaaac cgaggcgagg 960

aaggcacctg aggccacggt cgcgacatcg tcggtgtgct ccggcaacgg agctgggagt 1020

gacgagctgt ggcgccagca gaagcggaag tgccaggccc aggcagagtg ctcagctagc 1080

caagacgatg atcttgacga tgaacctgga gtattgagaa aatctggaac caggagcacg 1140

aaacgcagcc gcacagctga ggttcacaat ttatcagaaa ggaggagaag gcacaggatc 1200

aatgaaaaga tgcgcgctct gcaagaactc attcccaact gcaacaagat tgataaagcc 1260

tcgatgctgg atgaagctat agagtacctc aaaacccttc agcttcaagt acagatgatg 1320

tccatgggaa ctgggctgtg cattcctcca atgctattac caacagccat gcagcacttg 1380

caaattccac cgatggctca tttccctcat ctcggcatgg gattggggta cgggatgggc 1440

gtcttcgaca tgagcaacac tggagcactt cagatgccac ccatgcctgg tgctcacttt 1500

ccctgcccaa tgatcccagg tgcgtcacca caaggtcttg ggatccctgg cacaagcacc 1560

atgccaatgt ttggggttcc tgggcaaaca attccttcgt cagcgtctag tgtacccacca 1620

tttgcatctt tggctggtct tcctgttagg ccaagcgggg tccctcaagt atcaggcgcc 1680

atggctaaca tggtgcaaga ccagcaacaa ggcatagcga atcaacagca gcaatgtctg 1740

aacaaggaag ctatacaggg agcaaatcca ggtgattcac aaatgcagat catcatgcag 1800

ggtgacaacg agaattttag gataccctct tcagcccaaa caaaaagcag tcaattttca 1860

gatggtaccg gcaaggggac caacgctaga gagagagatg gggctgaaac ataa 1914

<211> 1848

<212>DNA

<213> sequence

<221> Gene coding sequence of rice phytochrome interaction factor OsPIL15 in mutant ospil15-1

<400> 3

atgtccgacg gcaacgactt cgccgagctg ctgtggggaga acggccaggc ggtggtgcac 60

gggaggaaga agcacccgca gccggccttc ccgccgttcg gcttcttcgg tggcaccggc 120

ggtggcggcg gcggcagcag tagtagagcc caggagaggc agcccggcgg catcgatgcg 180

ttcgccaagg tggggggcgg cttcggcgcc ttgggcatgg ctccggcggt gcacgacttc 240

gcttctggct tcggcgccac cacgcaggac aacggtgatg atgacaccgt tccgtggatc 300

cattacccca taattgacga tgaagacgcc gccgcccctg ctgctctcgc agcagcggac 360

tatggctccg actctctgcc agcctccaat cacaacggcg ccaccaataa cagaaatgct 420

ccggttgcca ccaccaccac cagggaaccc tccaaggaaa gccacggcgg cctgtcggtt 480

cccaccacccc gagccgagcc gcagccgcag ccacagctcg ccgcagccaa gctgcctcgg 540

tcgagcggca gcggcggcgg cgagggcgtg atgaacttct cgctcttctc ccgcccggcc 600

gtcctggcga gggcgacgct ggagagcgcg cagaggacgc agggcaccga caataaggcg 660

tccaatgtca ccgcgagcaa ccgcgtcgag tcgacggtcg tgcagacggc gagcgggcca 720

aggagcgcac cggcgttcgc cgatcagagg gcggcggcgt ggccgccgca gccgaaggag 780

atgccgttcg cgtccacggc agccgctccc atggccccgg ccgttaacct gcaccacgag 840

atgggccgtg acagggcagg ccgaaccatg cctgtccaca aaaccgaggc gaggaaggca 900

cctgaggcca cggtcgcgac atcgtcggtg tgctccggca acggagctgg gagtgacgag 960

ctgtggcgcc agcagaagcg gaagtgccag gcccaggcag agtgctcagc tagccaagac 1020

gatgatcttg acgatgaacc tggagtattg agaaaatctg gaaccaggag cacgaaacgc 1080

agccgcacag ctgaggttca caatttatca gaaaggagga gaagggacag gatcaatgaa 1140

aagatgcgcg ctctgcaaga actcattccc aactgcaaca agattgataa agcctcgatg 1200

ctggatgaag ctatagagta cctcaaaacc cttcagcttc aagtacagat gatgtccatg 1260

ggaactgggc tgtgcattcc tccaatgcta ttaccaacag ccatgcagca cttgcaaatt 1320

ccaccgatgg ctcatttccc tcatctcggc atgggattgg ggtacgggat gggcgtcttc 1380

gacatgagca acactggagc acttcagatg ccacccatgc ctggtgctca ctttccctgc 1440

ccaatgatcc caggtgcgtc accacaaggt cttgggatcc ctggcacaag caccatgcca 1500

atgtttgggg ttcctgggca aacaattcct tcgtcagcgt ctagtgtacc accatttgca 1560

tctttggctg gtcttcctgt taggccaagc ggggtccctc aagtatcagg cgccatggct 1620

aacatggtgc aagaccagca acaaggcata gcgaatcaac agcagcaatg tctgaacaag 1680

gaagctatac agggagcaaa tccaggtgat tcacaaatgc agatcatcat gcagggtgac 1740

aacgagaatt ttaggatacc ctcttcagcc caaacaaaaa gcagtcaatt ttcagatggt 1800

accggcaagg ggaccaacgc tagagagaga gatggggctg aaacataa 1848

<211>615

<212> PRT

<213> sequence

<221> Amino acid sequence of rice phytochrome interaction factor OsPIL15 in mutant ospil15-1

<400> 4

Met Ser Asp Gly Asn Asp Phe Ala Glu Leu Leu Trp Glu Asn Gly

1 5 10 15

Gln Ala Val Val His Gly Arg Lys Lys His Pro Gln Pro Ala Phe

20 25 30

Pro Pro Phe Gly Phe Phe Gly Gly Thr Gly Gly Gly Gly Gly Gly

35 40 45

Ser Ser Ser Arg Ala Gln Glu Arg Gln Pro Gly Gly Ile Asp Ala

50 55 60

Phe Ala Lys Val Gly Gly Gly Phe Gly Ala Leu Gly MET Ala Pro

65 70 75

Ala Val His Asp Phe Ala Ser Gly Phe Gly Ala Thr Thr Gln Asp

80 85 90

Asn Gly Asp Asp Asp Thr Val Pro Trp Ile His Tyr Pro Ile Ile

95 100 105

Asp Asp Glu Asp Ala Ala Ala Pro Ala Ala Leu Ala Ala Ala Asp

110 115 120

Tyr Gly Ser Asp Ser Leu Pro Ala Ser Asn His Asn Gly Ala Thr

125 130 135

Asn Asn Arg Asn Ala Pro Val Ala Thr Thr Thr Thr Thr Arg Glu Pro

140 145 150

Ser Lys Glu Ser His Gly Gly Leu Ser Val Pro Thr Thr Arg Ala

155 160 165

Glu Pro Gln Pro Gln Pro Gln Leu Ala Ala Ala Lys Leu Pro Arg

170 175 180

Ser Ser Gly Ser Gly Gly Gly Glu Gly Val MET Asn Phe Ser Leu

185 190 195

Phe Ser Arg Pro Ala Val Leu Ala Arg Ala Thr Leu Glu Ser Ala

200 205 210

Gln Arg Thr Gln Gly Thr Asp Asn Lys Ala Ser Asn Val Thr Ala

215 220 225

Ser Asn Arg Val Glu Ser Thr Val Val Gln Thr Ala Ser Gly Pro

230 235 240

Arg Ser Ala Pro Ala Phe Ala Asp Gln Arg Ala Ala Ala Trp Pro

245 250 255

Pro Gln Pro Lys Glu MET Pro Phe Ala Ser Thr Ala Ala Ala Pro

260 265 270

MET Ala Pro Ala Val Asn Leu His His Glu MET Gly Arg Asp Arg

275 280 285

Ala Gly Arg Thr MET Pro Val His Lys Thr Glu Ala Arg Lys Ala

290 295 300

Pro Glu Ala Thr Val Ala Thr Ser Ser Val Cys Ser Gly Asn Gly

305 310 315

Ala Gly Ser Asp Glu Leu Trp Arg Gln Gln Lys Arg Lys Cys Gln

320 325 330

Ala Gln Ala Glu Cys Ser Ala Ser Gln Asp Asp Asp Leu Asp Asp

335 340 345

Glu Pro Gly Val Leu Arg Lys Ser Gly Thr Arg Ser Thr Lys Arg

350 355 360

Ser Arg Thr Ala Glu Val His Asn Leu Ser Glu Arg Arg Arg Arg

365 370 375

Asp Arg Ile Asn Glu Lys MET Arg Ala Leu Gln Glu Leu Ile Pro

380 385 390

Asn Cys Asn Lys Ile Asp Lys Ala Ser MET Leu Asp Glu Ala Ile

395 400 405

Glu Tyr Leu Lys Thr Leu Gln Leu Gln Val Gln MET MET Ser MET

410 415 420

Gly Thr Gly Leu Cys Ile Pro Pro MET Leu Leu Pro Thr Ala MET

425 430 435

Gln His Leu Gln Ile Pro Pro MET Ala His Phe Pro His Leu Gly

440 445 450

MET Gly Leu Gly Tyr Gly MET Gly Val Phe Asp MET Ser Asn Thr

455 460 465

Gly Ala Leu Gln MET Pro Pro MET Pro Gly Ala His Phe Pro Cys

470 475 480

Pro MET Ile Pro Gly Ala Ser Pro Gln Gly Leu Gly Ile Pro Gly

485 490 495

Thr Ser Thr MET Pro MET Phe Gly Val Pro Gly Gln Thr Ile Pro

500 505 510

Ser Ser Ala Ser Ser Ser Val Pro Pro Phe Ala Ser Leu Ala Gly Leu

515 520 525

Pro Val Arg Pro Ser Gly Val Pro Gln Val Ser Gly Ala MET Ala

530 535 540

Asn MET Val Gln Asp Gln Gln Gln Gly Ile Ala Asn Gln Gln Gln

545 550 555

Gln Cys Leu Asn Lys Glu Ala Ile Gln Gly Ala Asn Pro Gly Asp

560 565 570

Ser Gln MET Gln Ile Ile MET Gln Gly Asp Asn Glu Asn Phe Arg

575 580 585

Ile Pro Ser Ser Ala Gln Thr Lys Ser Ser Gln Phe Ser Asp Gly

590 595 600

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

605 610 615

<211> 1915

<212>DNA

<213> sequence

<221> Gene coding sequence of rice phytochrome interaction factor OsPIL15 in mutant ospil15-21

<400> 5

atgtccgacg gcaacgactt cgccgagctg ctgtggggaga acggccaggc ggtggtgcac 60

gggaggaaga agcacccgca gccggccttc ccgccgttcg gcttcttcgg tggcaccggc 120

ggtggcggcg gcggcagcag tagtagagcc caggagaggc agcccggcgg catcgatgcg 180

ttcgccaagg tggggggcgg cttcggcgcc ttgggcatgg ctccggcggt gcacgacttc 240

gcttctggct tcggcgccac cacgcaggac aacggtgatg atgacaccgt tccgtggatc 300

cattacccca taattgacga tgaagacgcc gccgcccctg ctgctctcgc agcagcggac 360

tatggctccg acttcttctc cgagcttcca ggcggcggcg gctgccgcgg cggccgccgc 420

gccgccgacc gatctcgcct ctctgccagc ctccaatcac aacggcgcca ccaataacag 480

aaatgctccg gttgccacca ccaccaccag ggaaccctcc aaggaaagcc acggcggcct 540

gtcggttccc accacccgag ccgagccgca gccgcagcca cagctcgccg cagccaagct 600

gcctcggtcg agcggcagcg gcggcggcga gggcgtgatg aacttctcgc tcttctcccg 660

cccggccgtc ctggcgaggg cgacgctgga gagcgcgcag aggacgcagg gcaccgacaa 720

taaggcgtcc aatgtcaccg cgagcaaccg cgtcgagtcg acggtcgtgc agacggcgag 780

cgggccaagg agcgcaccgg cgttcgccga tcagagggcg gcggcgtggc cgccgcagcc 840

gaaggagatg ccgttcgcgt ccacggcagc cgctcccatg gccccggccg ttaacctgca 900

ccacgagatg ggccgtgaca gggcaggccg aaccatgcct gtccacaaaa ccgaggcgag 960

gaaggcacct gaggccacgg tcgcgacatc gtcggtgtgc tccggcaacg gagctgggag 1020

tgacgagctg tggcgccagc agaagcggaa gtgccaggcc caggcagagt gctcagctag 1080

ccaagacgat gatcttgacg atgaacctgg agtattgaga aaatctggaa ccaggagcac 1140

gaaacgcagc cgcacagctg aggttcacaa tttatcagaa aggaggagaa gggacaggat 1200

caatgaaaag atgcgcgctc tgcaagaact cattcccaac tgcaacaaga ttgataaagc 1260

ctcgatgctg gatgaagcta tagagtacct caaaaccctt cagcttcaag tacagatgat 1320

gtccatggga actgggctgt gcattcctcc aatgctatta ccaacagcca tgcagcactt 1380

gcaaattcca ccgatggctc atttccctca tctcggcatg ggattggggt acgggatggg 1440

cgtcttcgac atgagcaaca ctggagcact tcagatgcca cccatgcctg gtgctcactt 1500

tccctgccca atgatcccag gtgcgtcacc acaaggtctt gggatccctg gcacaagcac 1560

catgccaatg tttggggttc ctgggcaaac aattccttcg tcagcgtcta gtgtacccacc 1620

atttgcatct ttggctggtc ttcctgttag gccaagcggg gtccctcaag tatcaggcgc 1680

catggctaac atggtgcaag accagcaaca aggcatagcg aatcaacagc agcaatgtct 1740

gaacaaggaa gctatacagg gagcaaatcc aggtgattca caaatgcaga tcatcatgca 1800

gggtgacaac gagaatttta ggataccctc ttcagcccaa acaaaaagca gtcaattttc 1860

agatggtacc ggcaagggga ccaacgctag agagagagat ggggctgaaa cataa 1915

<211> 158

<212> PRT

<213> sequence

<221> Amino acid sequence of rice phytochrome interaction factor OsPIL15 in mutant ospil15-21

<400> 6

MET Ser Asp Gly Asn Asp Phe Ala Glu Leu Leu Trp Glu Asn Gly

1 5 10 15

Gln Ala Val Val His Gly Arg Lys Lys His Pro Gln Pro Ala Phe

20 25 30

Pro Pro Phe Gly Phe Phe Gly Gly Thr Gly Gly Gly Gly Gly Gly

35 40 45

Ser Ser Ser Arg Ala Gln Glu Arg Gln Pro Gly Gly Ile Asp Ala

50 55 60

Phe Ala Lys Val Gly Gly Gly Phe Gly Ala Leu Gly MET Ala Pro

65 70 75

Ala Val His Asp Phe Ala Ser Gly Phe Gly Ala Thr Thr Gln Asp

80 85 90

Asn Gly Asp Asp Asp Thr Val Pro Trp Ile His Tyr Pro Ile Ile

95 100 105

Asp Asp Glu Asp Ala Ala Ala Pro Ala Ala Leu Ala Ala Ala Asp

110 115 120

Tyr Gly Ser Asp Phe Phe Ser Glu Leu Pro Gly Gly Gly Gly Cys

125 130 135

Arg Gly Gly Arg Arg Ala Ala Asp Arg Ser Arg Leu Ser Ala Ser

140 145 150

Leu Gln Ser Gln Arg Arg His Gln

155 158

<211> 1910

<212>DNA

<213> sequence

<221> Gene coding sequence of rice phytochrome interaction factor OsPIL15 in mutant ospil15-22

<400> 7

atgtccgacg gcaacgactt cgccgagctg ctgtggggaga acggccaggc ggtggtgcac 60

gggaggaaga agcacccgca gccggccttc ccgccgttcg gcttcttcgg tggcaccggc 120

ggtggcggcg gcggcagcag tagtagagcc caggagaggc agcccggcgg catcgatgcg 180

ttcgccaagg tggggggcgg cttcggcgcc ttgggcatgg ctccggcggt gcacgacttc 240

gcttctggct tcggcgccac cacgcaggac aacggtgatg atgacaccgt tccgtggatc 300

cattacccca taattgacga tgaagacgcc gccgcccctg ctgctctcgc agcagcggac 360

tatggctccg acttcttctc ctccaggcgg cggcggctgc cgcggcggcc gccgcgccgc 420

cgaccgatct cgcctctctg ccagcctcca atcacaacgg cgccaccaat aacagaaatg 480

ctccggttgc caccaccacc accagggaac cctccaagga aagccacggc ggcctgtcgg 540

ttcccaccac ccgagccgag ccgcagccgc agccacagct cgccgcagcc aagctgcctc 600

ggtcgagcgg cagcggcggc ggcgagggcg tgatgaactt ctcgctcttc tcccgcccgg 660

ccgtcctggc gagggcgacg ctggagagcg cgcagaggac gcagggcacc gacaataagg 720

cgtccaatgt caccgcgagc aaccgcgtcg agtcgacggt cgtgcagacg gcgagcgggc 780

caaggagcgc accggcgttc gccgatcaga gggcggcggc gtggccgccg cagccgaagg 840

agatgccgtt cgcgtccacg gcagccgctc ccatggcccc ggccgttaac ctgcaccacg 900

agatgggccg tgacagggca ggccgaacca tgcctgtcca caaaaccgag gcgaggaagg 960

cacctgaggc cacggtcgcg acatcgtcgg tgtgctccgg caacggagct gggagtgacg 1020

agctgtggcg ccagcagaag cggaagtgcc aggcccaggc agagtgctca gctagccaag 1080

acgatgatct tgacgatgaa cctggagtat tgagaaaatc tggaaccagg agcacgaaac 1140

gcagccgcac agctgaggtt cacaatttat cagaaaggag gagaagggac aggatcaatg 1200

aaaagatgcg cgctctgcaa gaactcattc ccaactgcaa caagattgat aaagcctcga 1260

tgctggatga agctatagag tacctcaaaa cccttcagct tcaagtacag atgatgtcca 1320

tgggaactgg gctgtgcatt cctccaatgc tattaccaac agccatgcag cacttgcaaa 1380

ttccaccgat ggctcatttc cctcatctcg gcatggatt ggggtacggg atgggcgtct 1440

tcgacatgag caacactgga gcacttcaga tgccacccat gcctggtgct cactttccct 1500

gcccaatgat cccaggtgcg tcaccacaag gtcttgggat ccctggcaca agcaccatgc 1560

caatgtttgg ggttcctggg caaacaattc cttcgtcagc gtctagtgta ccaccatttg 1620

catctttggc tggtcttcct gttaggccaa gcggggtccc tcaagtatca ggcgccatgg 1680

ctaacatggt gcaagaccag caacaaggca tagcgaatca acagcagcaa tgtctgaaca 1740

aggaagctat acaggggagca aatccaggtg attcacaaat gcagatcatc atgcagggtg 1800

acaacgagaa ttttaggata ccctcttcag cccaaacaaa aagcagtcaa ttttcagatg 1860

gtaccggcaa ggggaccaac gctagagaga gagatggggc tgaaacataa 1910

<211> 210

<212> PRT

<213> sequence

<221> Amino acid sequence of rice phytochrome interaction factor OsPIL15 in mutant ospil15-22

<400> 8

MET Ser Asp Gly Asn Asp Phe Ala Glu Leu Leu Trp Glu Asn Gly

1 5 10 15

Gln Ala Val Val His Gly Arg Lys Lys His Pro Gln Pro Ala Phe

20 25 30

Pro Pro Phe Gly Phe Phe Gly Gly Thr Gly Gly Gly Gly Gly Gly

35 40 45

Ser Ser Ser Arg Ala Gln Glu Arg Gln Pro Gly Gly Ile Asp Ala

50 55 60

Phe Ala Lys Val Gly Gly Gly Phe Gly Ala Leu Gly MET Ala Pro

65 70 75

Ala Val His Asp Phe Ala Ser Gly Phe Gly Ala Thr Thr Gln Asp

80 85 90

Asn Gly Asp Asp Asp Thr Val Pro Trp Ile His Tyr Pro Ile Ile

95 100 105

Asp Asp Glu Asp Ala Ala Ala Pro Ala Ala Leu Ala Ala Ala Asp

110 115 120

Tyr Gly Ser Asp Phe Phe Ser Ser Arg Arg Arg Arg Arg Leu Pro Arg

125 130 135

Arg Pro Pro Arg Arg Arg Pro Ile Ser Pro Leu Cys Gln Pro Pro

140 145 150

Ile Thr Thr Ala Pro Pro Ile Thr Glu MET Leu Arg Leu Pro Pro

155 160 165

Pro Pro Pro Gly Asn Pro Pro Arg Lys Ala Thr Ala Ala Cys Arg

170 175 180

Phe Pro Pro Pro Glu Pro Ser Arg Ser Arg Ser His Ser Ser Pro

185 190 195

Gln Pro Ser Cys Leu Gly Arg Ala Ala Ala Ala Ala Ala Arg Ala

200 205 210

<211> 23

<212>DNA

<213> Artificial sequence

<221> sgRNA-F

<400> 9

ggcggacttc ttctccgagc tcc 23

<211> 23

<212>DNA

<213> Artificial sequence

<221> sgRNA-R

<400> 10

aaacggagct cggagaagaa gtc 23

<211> 24

<212>DNA

<213> Artificial sequence

<221> pBUN411-VF

<400> 11

ccatgaagcc tttcaggaca tgta 24

<211> 24

<212>DNA

<213> Artificial sequence

<221> pBUN411-VR

<400> 12

acgctgcaaa catgagacgg agaa 24

<211> 20

<212>DNA

<213> Artificial sequence

<221> Bar-F

<400> 13

aagcacggtc aacttccgta 20

<211> 20

<212>DNA

<213> Artificial sequence

<221> Bar-R

<400> 14

gaagtccagc tgccagaaac 20

<211> 20

<212>DNA

<213> Artificial sequence

<221> OsPIL15-test-F

<400> 15

tgttttgtgtgtgcaggtcc 20

<211> 20

<212>DNA

<213> Artificial sequence

<221> OsPIL15-test-R

<400> 16

cgggagaaga gcgagaagtt 20

<211> 336

<212>DNA

<213> sequence

<221> PCR amplification product sequence

<400> 17

ccatgaagcc tttcaggaca tgtattgcag tatgggccgg cccattacgc aattggacga 60

caacaaagac tagtattagt accacctcgg ctatccacat agatcaaagc tgatttaaaa 120

gagttgtgca gatgatccgt ggcgtcctcc accgcatcga tgtgttttag agctagaaat 180

agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 240

tttttttttc gttttgcatt gagttttctc cgtcgcatgt ttgcagtttt attttccgtt 300

ttgcattgaa atttctccgt ctcatgtttg cagcgt 336

<211> 3148

<212>DNA

<213> sequence

<221> Rice phytochrome interaction factor OsPIL15 gene full length

<400> 18

aggtcccccc acgccacagc ctcttatccc aacacgcggac caacagcgcc gccccgcccg 60

tcccccgctt caccttcggc ttcgacttcg gcttcgtttc ctcttctctt cgtctctccc 120

tccctccagc gaaagaaaga gagagctcac ctcgcgttgc ccggccggcc gcggaggaga 180

gcacggcttc gcattcggct ggagctctcg tctctgcact cggagtacag ctgcaaactc 240

ctccttcctt gatttcttca ccctgtctcc acggactgca cagatgtggg tgcaacgcga 300

tctttcgctg cctccggttt agctctccgg ttgattccga tcgaggaagc tgatgcatgt 360

gtttgtatat ggctcggtgt tttgtgtgtgtg caggtccgac ggcaacgact tcgccgagct 420

gctgtgggag aacggccagg cggtggtgca cgggaggaag aagcacccgc agccggcctt 480

cccgccgttc ggcttcttcg gtggcaccgg cggtggcggc ggcggcagca gtagtagagc 540

ccaggagagg cagcccggcg gcatcgatgc gttcgccaag gtggggggcg gcttcggcgc 600

cttgggcatg gctccggcgg tgcacgactt cgcttctggc ttcggcgcca ccacgcagga 660

caacggtgat gatgacaccg ttccgtggat ccattacccc ataattgacg atgaagacgc 720

cgccgcccct gctgctctcg cagcagcgga ctatggctcc gacttcttct ccgagctcca 780

ggcggcggcg gctgccgcgg cggccgccgc gccgccgacc gatctcgcct ctctgccagc 840

ctccaatcac aacggcgcca ccaataacag aaatgctccg gttgccacca ccaccaccag 900

ggaaccctcc aaggaaagcc acggcggcct gtcggttccc accacccgag ccgagccgca 960

gccgcagcca cagctcgccg cagccaagct gcctcggtcg agcggcagcg gcggcggcga 1020

gggcgtgatg aacttctcgc tcttctcccg cccggccgtc ctggcgaggg cgacgctgga 1080

gagcgcgcag aggacgcagg gcaccgacaa taaggcgtcc aatgtcaccg cgagcaaccg 1140

cgtcgagtcg acggtcgtgc agacggcgag cgggccaagg agcgcaccgg cgttcgccga 1200

tcagagggcg gcggcgtggc cgccgcagcc gaaggagatg ccgttcgcgt ccacggcagc 1260

cgctcccatg gccccggccg ttaacctgca ccacgagatg ggccgtgaca gggcaggccg 1320

aaccatgcct gtccacaaaa ccgaggcgag gaaggcacct gaggccacgg tcgcgacatc 1380

gtcggtgtgc tccggcaacg gagctgggag tgacgagctg tggcgccagc agaagcggaa 1440

gtgccaggcc caggcagagt gctcagctag ccaagacgat gtaagtaaat ggtatgagat 1500

agatatgcac tgcataacca gctgactata ccttcgctga ttctcatgat aaaaaactgg 1560

ttctattcag gatcttgacg atgaacctgg agtattgaga aaatctggaa ccaggagcac 1620

gaaacgcagc cgcacagctg aggttcacaa tttatcagaa agggtgagta gctcacatct 1680

tcagtgcatg gatcatcctg catccatttg cttcaaagtt cacatgtcag tgcattgatc 1740

atcctgcatc catttgcttc aatcccatga ctcgactcat gctgcaattt tattgactgt 1800

attgcaaccc aacaatcttt gcagaggaga agggacagga tcaatgaaaa gatgcgcgct 1860

ctgcaagaac tcattcccaa ctgcaacaag gtaaagataa gccattccat cgtcttgctc 1920

cctctgagat gcctctgaat gaacatttgg tcaattcagg catgctatgt tttgcagatt 1980

gataaagcct cgatgctgga tgaagctata gagtacctca aaacccttca gcttcaagta 2040

caggtacatt gaaactgcct tcgaacaaat gtaccatgat tgtcgggtga atatgtacat 2100

agatgcattg acaaggtgca gttgtcattg acacagatga tgtccatggg aactgggctg 2160

tgcattcctc caatgctatt accaacagcc atgcagcact tgcaaattcc accgatggct 2220

catttccctc atctcggcat gggattgggg tacgggatgg gcgtcttcga catgagcaac 2280

actggagcac ttcagatgcc acccatgcct ggtgctcact ttccctgccc aatgatccca 2340

ggtgcgtcac cacaaggtct tgggatccct ggcacaagca ccatgccaat gtttggggtt 2400

cctgggcaaa caattccttc gtcagcgtct agtgtaccac catttgcatc tttggctggt 2460

cttcctgtta ggccaagcgg ggtccctcaa gtatcaggcg ccatggctaa catggtgcaa 2520

gaccagcaac aaggcatagc gaatcaacag cagcaatgtc tgaacaagga agctatacag 2580

ggagcaaatc caggtgattc acaaatgcag atcatcatgc aggtactaat taaaaattaa 2640

caaatgatgt caagcgaata gaagacattt gctagtactt aagtgcatta cttactccag 2700

tttattttaa tattccaggg tgacaacgag aattttagga taccctcttc agcccaaaca 2760

aaaagcagtc aattttcaga tggtaccggc aaggggacca acgctagaga gagagatggg 2820

gctgaaacat aaagaaaggc cagtaggtgt aacttgactt tcatgctaaa tttgagatct 2880

accactgaaa tctgcagaat gttcctgtcc taaaaagatc aatggctgag gaatttcatg 2940

tacgaccaac taacaacttc cagatatgaa agttagcaat tcatactggg agaacttact 3000

tgagtatcaa gatccacgag gcagatgtat cagccaagat gccccaaaat tttgtgtaaa 3060

atgtagatgg tagaagatgc taccaggtta caagctgtaa ttcttgactt ccagtgcagt 3120

ttcctatgag tagtttttgc cctatctg 3148

Claims (9)

1. a kind of method that rice Os PIL15 mutant is prepared based on CRISPR/Cas9 technologies, it is characterised in that：Including as follows Step：

1) selection of gRNA target sequences：Sequence is 5 '-GACTTCTTCTCCGAGCTCCAGG-3 ', the PAM that the sequence 3 ' is held Sequence is AGG, and the recognition sequences of restriction enzyme Sac I are 5 '-GAGCTC-3 '；

2) design of gRNA oligonucleotide chains upstream and downstream primer：

Sense primer is sgRNA-F：5 '-GGCGGACTTCTTCTCCGAGCTCC-3 ',

Anti-sense primer is sgRNA-R：5′-AAACGGAGCTCGGAGAAGAAGTC-3′；

3) gRNA expression vector establishments：The upstream and downstream primer is mixed, annealed, oligonucleotides double-stranded DNA is obtained；Use restriction endonuclease The digestion pBUN411 plasmids of Bsa I, obtain linear plasmid；The linear plasmid and oligonucleotides double-stranded DNA are connected with T4 ligases, Obtain connection product；Connection product is converted, screen, verified, pBUN411-gRNA expression vectors are produced；

4) pBUN411-gRNA expression vectors are imported in Agrobacterium, obtains CRISPR/Cas9-gRNA Agrobacteriums；Use CRISPR/ Cas9-gRNA Agrobacteriums infect Rice Callus；

5) by step 4) callus induction that obtains obtains regrowth, and screening obtains transgenic positive plant, and identification produces water Rice OsPIL15 mutant.

2. the method according to claim 1 for preparing rice Os PIL15 mutant, it is characterised in that：Step 3) described in It is annealed into the 5min that annealed at 65 DEG C.

3. the method according to claim 1 for preparing rice Os PIL15 mutant, it is characterised in that：Step 5) be except Screening obtains regrowth under conditions of careless agent.

4. the method according to claim 3 for preparing rice Os PIL15 mutant, it is characterised in that：Step 5) it is to use Anti-herbicide gene primer is screened；

The primer is：Bar-F：5′-AAGCACGGTCAACTTCCGTA-3′；

Bar-R：5′-GAAGTCCAGCTGCCAGAAAC-3′.

5. the method according to claim 1 for preparing rice Os PIL15 mutant, it is characterised in that：Step 5) it is to use GRNA target sequences both sides primer is identified；

Both sides primer is OsPIL15-test-F：5′-TGTTTTGTGTGTGCAGGTCC-3′；

OsPIL15-test-R：5′-CGGGAGAAGAGCGAGAAGTT-3′.

6. the method according to claim 5 for preparing rice Os PIL15 mutant, it is characterised in that：The identification includes：

A：Using transgenic positive plant DNA as template, OsPIL15-test-F and OsPIL15-test-R are detection primer, are carried out PCR is expanded；

B：With the enzyme digestion pcr amplification products of Sac I；

C：It is unmutated strain if electrophoresis result is only 400bp and the bands of 273bp two with electrophoretic separation digestion products；Otherwise, it is Mutant strain.

7. the method according to claim 6 for preparing rice Os PIL15 mutant, it is characterised in that：By the transgenosis The pcr amplification product sequencing of positive plant, it is determined that being heterozygote, double allelic variant bodies, Mutants homozygous or list of not undergoing mutation Strain.

8. the application of the method for rice Os PIL15 mutant is prepared according to claim 1, it is characterised in that：The paddy rice Application of the OsPIL15 mutant in rice breeding.

9. application according to claim 8, it is characterised in that：Select the self progeny of the rice Os PIL15 mutant In Mutants homozygous be used for rice paddy seed grain length breeding.