CN113637688B

CN113637688B - Rice amylose content regulating gene OsACF1 and application thereof

Info

Publication number: CN113637688B
Application number: CN202111111365.8A
Authority: CN
Inventors: 赵国超; 李建粤; 王彤
Original assignee: Shanghai Normal University
Current assignee: Shanghai Normal University
Priority date: 2021-09-23
Filing date: 2021-09-23
Publication date: 2023-10-13
Anticipated expiration: 2041-09-23
Also published as: CN113637688A

Abstract

The invention relates to a rice amylose content gene OsACF1 and application thereof. The invention discovers a novel gene OsACF1 capable of regulating and controlling the content of amylose in rice. The amino acid sequence of the OsACF1 is shown as SEQ ID NO.1, and the application is as follows: by adopting a conventional method, gene knockout, change, inhibition or excessive OsACF1 gene is adopted, so that the expression level of the OsACF1 gene in the conventional rice variety is changed, and rice with different amylose contents is obtained. The rice with low amylose content prepared by the invention has no abnormal phenotype in the vegetative growth period of the rice, but the amylose content of the mature rice is reduced. If the gene is applied to rice breeding, the rice quality can be obviously improved, a new gene resource is provided for cultivating novel high-quality rice varieties, and the gene has important application in agricultural production. On the other hand, rice germplasm with high amylose content can be obtained by over-expressing the OsACF1 gene, which lays a foundation for cultivating high-resistance starch special rice.

Description

Rice amylose content regulating gene OsACF1 and application thereof

Technical Field

The invention relates to a gene sequence capable of controlling the amylose content of rice seed endosperm and application of the gene sequence.

Background

Rice is one of the world's important food crops, and about 50% of the world's population is taking rice as the main food, and rice is also the raw material for processing many foods. Because the genome of rice is small and the rice transformation system is very mature, the rice can be used as a model plant for monocotyledonous research.

The ratio of amylose to total starch is called Amylose Content (AC), and the level of amylose is an important factor in determining the quality of rice and its use. The difference of amylose content of rice of different rice varieties is large, indica rice has an amylose content of 20-30%, japonica rice has an amylose content of 15-22%, and glutinous rice amylose content is lower than 2%. Rice with too high amylose content has relatively poor rice quality and large expansibility, and is hard after meal formation; glutinous rice with amylose content lower than 2% is too soft and has poor elasticity; the soft rice with low amylose content (8% -12%) has the advantages of between waxy and sticky properties, good taste, sweet and refreshing taste, fluffy rice with hot and cold properties, soft texture, no retrogradation and no hardening after cooling. The current report also shows that the transparency of the rice has a certain relation with the content of amylose, and the content of the amylose of the rice of different types of rice is as follows from high to low: indica rice > japonica rice > soft rice > glutinous rice, and the transparency of the rice is from high to low: indica rice > polished round-grained rice > soft rice > glutinous rice (Zhang Zhengmao, modern food technology, 2015,31 (6): 190-197).

The Wx gene is a key enzyme gene for amylose synthesis and encodes Granule-binding starch synthase I (Granule-Bound Starch Synthase I, GBSSI) (Wang et al The Plant Journal,1995,7 (4): 613-622). Wx has two major alleles Wx ^a And Wx ^b Japonica rice has Wx ^b Allelic forms, most indica rice has Wx ^a Allelic type. Mutation of Wx gene can reduce amylose content of rice, so that softness of rice can be improved. When Wx is completely disabled, the amylose content of rice is less than 2%, such as glutinous rice. Weak mutations in Wx coding sequences, e.g. Wx ^mq Soft rice of the type having an amylose content of between 6 and 12% (Zhang et al, molecular Plant,2019, 12:1157-1166). In addition to the Wx gene, the Du1 gene has also been reported to regulate Wx directly ^b Splicing of pre-mRNA resulting in Wx ^b Splicing of the gene mRNA does not proceed smoothly, resulting in a decrease in amylose content (Zeng et al, plant Molecular Biology,2007, 65:501-509).

With the rapid development of economy, the living standard of people is improved, and the requirements of common people on the rice quality are improved year by year. The soft rice has the advantages of soft quality, elasticity, no retrogradation and no hardening after cooling, is popular with consumers and breeders at present, for example, soft rice variety Nanjing 46 and the like, the amylose content of the soft rice is between 8 and 12 percent, and the soft rice mainly has weak allele type of Wx genes, for example, wx ^mq And Wx ^hp Genotype.

Different areas of people have great differences in preference of the softness of rice. In order to create rice with different amylose content, recently, scientists accurately edit the Wx gene promoter and the 5' UTR region or replace key amino acids of Wx protein by using CRISPR/Cas9 gene editing technology, so as to obtain novel rice germplasm (AC between 0% and 12%) with different amylose content (Zeng et al, plant Biotechnology Journal,2020,18:2385-2387;Huang et al,Plant Biotechnology Journal,2020,18:2164-2166).

Although the rice germplasm with different amylose contents is created at present, the rice germplasm is obtained by screening different genotypes of Wx or editing Wx genes, so that the identification and cloning of new amylose content genes enriches rice gene resources and lays a foundation for cultivating or creating new high-quality rice germplasm.

The high amylose starch is a main source of natural resistant starch food, and can effectively improve intestinal health of human body and prevent diabetes and cardiovascular diseases. Rice is the main grain crop in China, but the resistant starch content in the conventional rice endosperm starch is lower. Inhibition of starch branching enzyme gene Sbel/IIb expression by antisense RNA technology has been reported to significantly increase amylose content (Qinfeng Ling, university of Yangzhou Shuiche paper 2011, doi:10.7666/d.y 2049980). Cloning new amylose content genes will also lay the foundation for creating new high-amylose resistant starch rice germplasm by biotechnology methods.

Disclosure of Invention

Aiming at the defects of the current rice in the aspect of amylose content control gene resource, the invention obtains the amylose content control gene OsACF1 and the application method for changing the amylose content by using the same through a large number of experiments. The rice amylose content is controlled by utilizing the characteristics of the OsACF1 gene and the protein thereof, and the rice amylose content is controlled by utilizing a transgenic technology, so that novel amylose content character rice is generated by mutating the protein sequence, inhibiting or excessively expressing the protein, and the rice amylose content character rice has very important application in agricultural production.

According to one aspect of the present invention, there is provided an amylose content controlling gene OsACF1, wherein the amino acid sequence encoded by the amylose content gene OsACF1 is shown as SEQ ID NO.1 or the amino acid sequence encoded by the amylose content gene OsACF1 is at least 90% homologous to the sequence shown as SEQ ID NO. 1.

Preferably, the nucleotide sequence of the amylose content gene OsACF1 is shown as SEQ ID No.2, or the nucleotide sequence of the amylose content gene OsACF1 is at least 90% homologous with the sequence of SEQ ID No. 2.

According to a second aspect of the present invention, there is provided an application of the above amylose content controlling gene OsACF1, characterized in that the application comprises: editing, knocking out, changing, inhibiting or overexpressing the rice gene of the amylose content regulating gene OsACF1 to change the gene expression level of the amylose content regulating gene OsACF1 in the target rice variety, thereby obtaining the rice varieties with different amylose content traits

According to a third aspect of the present invention, there is provided a method of breeding rice plant lines of different amylose content, comprising the steps of: selecting conventional rice varieties, treating and cultivating to obtain rice with different amylose contents, wherein the conventional method is adopted to ensure that the nucleotide sequence for coding the amino acid shown as SEQ ID NO.1 in the rice is deleted, mutated or inhibited, so that the expression level of the polypeptide corresponding to the amino acid sequence is reduced, increased or activity is changed.

Preferably, the rice variety is japonica rice variety "Yinxiang 38" or "Nippon", or indica rice variety "9311".

Preferably, the method further comprises: and carrying out gene editing on the target rice variety, so that the nucleotide sequence shown as SEQ ID NO.2 contained in the target rice seed is mutated into the nucleotide sequence shown as SEQ ID NO.3, or the amino acid sequence shown as SEQ ID NO.1 in the target rice seed is mutated into the amino acid sequence shown as SEQ ID NO.4, and obtaining the rice strain with low amylose content.

More specifically, the invention adopts a conventional method to mutate the nucleotide sequence shown as SEQ ID NO.2 in a conventional rice variety into SEQ ID NO.3, so as to obtain a rice strain with low amylose content, namely 'Yindaf 38' (osdaf 1-1) natural mutant.

The invention adopts a conventional method to mutate the amino acid sequence shown as SEQ ID NO.1 in a conventional rice variety into SEQ ID NO.4, so as to obtain a rice strain with low amylose content, namely a natural mutant of Yindaf 38 (osdaf 1-1).

Preferably, the method further comprises the steps of: the CRISPR-CAS9 gene editing technology is adopted to change the nucleotide sequence SEQ ID NO.2 of the amino acid sequence shown as SEQ ID NO.1 so that the activity of the polypeptide corresponding to the amino acid sequence is lost or reduced.

Preferably, the construction method of the CRISPR-CAS9 gene editing vector in the CRISPR-CAS9 gene editing technology comprises the following steps:

(a) Selecting a specific fragment of 20bp total from 1 st to 20 th of a nucleotide sequence shown in SEQ ID NO.2 of an OsACF1 gene coding region sequence as a target site;

(b) The method in reference (Zhang et al, the CRISPR/Cas9system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J.12,797-807 (2014)) combines a pair of primers as in SEQ ID No.5 and SEQ ID No.6 in a PCR apparatus at 95℃to 20℃with a 1sec drop of 0.1 ℃. Restriction endonuclease Bsa1 cleaves vector RCKO and T4 ligase is used to ligate the vector to the target sequence. After sequencing verification, the RCKO-OsDAF1 plasmid is successfully constructed, and agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 is transformed.

(c) Transferring agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 containing OsACF1 CRISPR-CAS9 knockout RCKO-OsDAF1 into japonica rice 'Nippon' and culturing to obtain;

the invention adopts a CRISPR-CAS9 method to mutate the nucleotide sequence shown as SEQ ID NO.2 in the conventional rice variety into SEQ ID NO.7, thereby obtaining a rice strain with low amylose content, namely osdaf1-2 mutant.

The invention adopts a CRISPR-CAS9 method to lead the amino acid sequence shown as SEQ ID NO.1 in the conventional rice variety to move and terminate in advance, mutate into SEQ ID NO.8, and further obtain the rice strain with low amylose content, namely the osdaf1-2 mutant.

In a fourth aspect, the present invention provides a use of low amylose rice in rice breeding, wherein the low amylose rice line obtained by the above application is used as a parent, and other parents with advantages of yield and resistance properties are matched to cultivate high yield and high resistance high quality rice, so as to perform conventional rice breeding.

In a fifth aspect, the present invention also relates to a method for restoring the low amylose content trait of rice, comprising the steps of: the OsACF1 gene is transferred into rice with low amylose content obtained by the application by adopting a conventional genetic means, so that the amylose content of the silver aroma 38 (osdaf 1-1) rice mutated by the OsACF1 gene is restored to normal japonica rice.

Preferably, the method comprises the steps of: transferring agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 complementarily constructed by OsACF1 into the rice with low amylose content, and culturing to obtain the rice; wherein the OsACF1 complementation structure contains a nucleotide sequence shown in SEQ ID NO. 9.

Preferably, the method specifically comprises the following steps:

(a) Synthesizing a promoter sequence fragment shown in SEQ ID NO.9 of the OsACF1 gene and comprising 2673bp and a 2172bp gene coding sequence fragment by using a Japanese sunny genome as a reference sequence by using a chemical synthesis method;

(b) Providing agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 carrying a complementarily constructed vector expressing osacp 1;

(c) Transferring agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 complementarily constructed by OsACF1 into the rice with low amylose content, and culturing to obtain the rice; wherein the OsACF1 complementation construction contains a nucleotide with a sequence shown as SEQ ID NO. 9.

Preferably, the step (c) is specifically:

contacting a rice cell or tissue or organ with the agrobacterium tumefaciens of step (b) such that the nucleotide sequence encoding the amino acid set forth in SEQ ID No.9 is transferred into the rice cell and integrated into the chromosome of the rice cell;

selecting rice cells or tissues transferred with the nucleotide for regeneration to obtain rice plants.

The beneficial effects are that:

the inventor of the invention discovers a novel gene resource capable of effectively controlling the amylose content, obtains rice variant strains with different amylose contents by controlling rice OsACF1 genes and encoding proteins thereof, realizes the purpose of controlling the amylose content of rice seed endosperm, and current experimental results show that the range of adjusting the amylose content is 7% -16%. The rice mutant obtained by the invention has no obvious difference between the nutrition period and the source parent or the corresponding complementary plant, has the main character of different amylose content of seed endosperm in the mature period, and has very important application in high-quality rice cultivation and special-function rice cultivation.

Drawings

Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:

FIG. 1 shows the amylose content of OsACF1 natural mutant "Yinxiang 38" (osdaf 1-1), transgenic complementary plant (pOsACF 1-OsACF1 CDS/"Yinxiang 38"), "Nippon" and "Nippon" background Gene editing mutant osdaf1-2 brown rice, respectively, wherein: FIG. 1A shows, from left to right, osACF1 natural mutant "Yinxiang 38" (osdaf 1-1) brown rice, transgenic complementary plant (pOsACF 1-OsACF1 CDS/"Yinxiang 38") brown rice, "Nippon brown rice and" Nippon "background Gene editing mutant osdaf1-2 brown rice, respectively. FIG. 1B shows, from left to right, the amylose content of the natural mutant OsACF1, "Yinxiang 38" (osdaf 1-1) and the amylose content of the transgenic complementary plant (pOsACF 1-OsACF1 CDS/"Yinxiang 38"), the amylose content of "Nipponbare" and the amylose content of the gene editing mutant osdaf1-2 in the background of "Nippon".

FIG. 2 is a schematic diagram of OsACF1 gene localization, structure and mutation site; wherein, fig. 2A is a schematic diagram of OsACF1 gene localization, and the numbers marked on the vertical line are the names, recombinants and genetic distances of the used primers; chr.6 indicates that the gene is located on chromosome 6; the red area is the interval position where OsACF1 is positioned; FIG. 2B is a schematic diagram of the gene structure and mutation sites.

Detailed Description

The invention will be further illustrated with reference to specific examples. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental procedure, in which no specific conditions are noted in the examples below, can be generally followed by conventional conditions, such as for example the molecular cloning of Sambrook et al: conditions described in the laboratory Manual (New York: cold Spring Harbor LaboratoryPress, 1989) or as recommended by the manufacturer. The OsACF1 gene is a nucleotide sequence for encoding an amino acid sequence shown as SEQ ID NO. 1.

EXAMPLE 1 cloning of amylose content controlling Gene OsACF1

1.1 "silver aroma 38" (osacf 1-1) Low amylose Rice screening

In the embodiment, natural rice lines are screened in a large scale, and a natural mutant rice with low amylose content character is screened, wherein the content of amylose in the rice with the low amylose content of 'Yinxiang 38', 'Yinxiang 38' is 8.5%. The coding region sequence of the amylose content trait gene OsACF1 is shown as SEQ ID NO.2 by map-based cloning and positioning to the OsACF1 gene.

1.2 genetic mapping of Rice amylose content control Gene

A rice gene targeting clone (map-based cloning or position cloning) population constructed by the inventors and comprising an amylose content controlling protein gene OsACF1 and a mutant gene OsACF1 thereof is used to target 1 small genome fragment, for example, within 100Kb, according to molecular markers. On this basis, genomic DNA clones containing the fragment were isolated by conventional methods. One of the rice amylose content control proteins OsACF1 is determined by sequencing and further hybridization identification.

The analysis result of the full nucleotide sequence shows that: the total length of the rice amylose content regulating gene OsACF1 is 7044bp (SEQ ID NO.10, comprising a regulating region and an intron). The ORF of the cDNA clone is shown as SEQ ID NO.2, the rice amylose content control protein with the total length of 723 amino acids is encoded, and the amino acid sequence of the rice amylose content control protein is shown as SEQ ID NO. 1.

1.3 Point mutation of Rice Low amylose Gene OsACF1

The OsACF1 mutant material is obtained by natural mutation of the conventional japonica rice variety 'Yinxiang 38' through an OsACF1 gene sequence, and the rice amylose content is reduced due to the deletion of key amino acids of the rice amylose content control protein through sequence comparison of an OsACF1 mutant gene OsACF 1; the OsACF1 mutant gene of the embodiment is characterized in that base substitution occurs at 1221 base pairs of a coding region (the sequence of the OsACF1 mutant gene is shown as SEQ ID NO. 3), so that a stop codon is formed in advance, the key amino acid of the rice amylose content control protein OsACF1 is deleted and the function is lost, the silver aroma 38 rice has low amylose content, and the amylose content of the silver aroma 38 rice is 8.5%.

1.4 method for restoring the Low amylose content Properties of the osacf1 mutant

The gene promoter sequence and the gene coding region sequence of the OsACF1 gene are transferred into a silver incense 38 (osdaf 1-1) mutant plant, so that the amylose content of the silver incense 38 (osdaf 1-1) mutant can be restored to the normal japonica rice level. Synthesizing a promoter sequence fragment shown in SEQ ID NO.9 of an OsACF1 gene and comprising 2673bp and a 2172bp gene coding sequence fragment into pCAMBIA1301 by using a Japanese genome as a reference sequence, sequencing and verifying to be correct, introducing the vector into agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 by a heat shock method to obtain an OsDAF1 complementarily constructed agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105, transforming mature embryo callus of "Yindaf 38" (OsDAF 1-1) by using a genetic transformation means, so that a nucleotide coding an amino acid shown in SEQ ID NO.1 is transferred into a rice cell and integrated into a chromosome of the rice cell; regenerating to obtain rice plants; to see if the amylose content of the "silver aroma 38" (osdaf 1-1) seeds would be restored to normal japonica rice levels.

The T0 generation of the complementary plants and the T1 seeds from the T0 generation of the complementary plants were obtained, and fig. 1B shows that the amylose content of the T1 seeds was 15.4%, indicating that the amylose content of the rice of the complementary plants was restored to the normal level of japonica rice (fig. 1B).

Example 2 creation of Rice with Low amylose content Using different methods

1.1 OsACF1 in Rice variety was knocked out by CRISPR-CAS9 means to create Osacf1-2 Rice with Low amylose content

In order to apply the OsACF1 protein, an OsACF1 gene CRISPR-CAS9 knockout vector is constructed in the embodiment, and wild type Japanese sunny plants are transformed so as to knockout the expression of the OsACF1, thereby achieving the purpose of changing the amylose content of rice.

The CRISPR-CAS9 knockout process is: the method in reference (Zhang et al, the CRISPR/Cas9system produces specific and homozygous targeted gene editing in rice in one generation, plant biotechnology j.12,797-807 (2014)) utilizes a pair of primers as shown in SEQ ID No.5 and SEQ ID No.6 that are annealed and bound together in PCR following a procedure of 95 ℃ -20 ℃ for 1sec to 0.1 ℃ to form a target sequence.

OsACF1-F:5’TGTGTGGGGTCGATGACGTCAGCCAT 3’(SEQ ID NO.5)

OsACF1-R:5’AAACATGGCTGACGTCATCGACCCCA 3’(SEQ ID NO.6)

Vector RCKO (existing vector, see Zhang et al, the CRISPR/Cas9system produces specific and homozygous targeted gene editing in rice in one generation, plant Biotechnol J.12,797-807 (2014)) was digested with restriction enzyme Bsa1, and the vector and target sequence were ligated with T4 ligase. After sequencing verification, the RCKO-OsDAF1 plasmid is successfully constructed, and agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 is transformed.

Agrobacterium containing the RCKO-OsDAF1 construct was streaked on YEB plates containing Kan (50 mg/ml) to obtain single colonies. Single colonies were picked and inoculated into 3ml of YEB liquid medium containing (Kan and rif) antibiotics at 28℃overnight with shaking, at 1% on day 2The medium was transferred into 50ml of YEB liquid medium containing antibiotics, and shaking culture was continued at 200rpm until OD was reached ₆₀₀ When the ratio is about 0.3 to 0.6, fresh agrobacterium tumefaciens bacteria liquid is centrifuged at 5000rpm for 5 minutes, collected and resuspended in 1/3 volume of AAM-AS liquid medium, and then the liquid is used for transforming various receptor materials of rice.

The present example uses conventional agrobacterium transformation methods to transform mature seed calli from rice Nipponbare. Mature seeds are taken for induction, callus is induced on an N6D2 culture medium, and the culture is carried out at 26+/-1 ℃ in dark condition for 15 days for subsequent generation, and the culture is carried out for 8 days for transformation. The callus was immersed in fresh AAM agrobacterium solution and shaken from time to time, after 20 minutes the rice material was removed, excess solution was blotted onto sterile filter paper and subsequently transferred to N6D2C medium and co-cultured at 26 ℃ for 3 days. During co-cultivation, acetosyringone was added to the co-cultivation medium at a concentration of 100mM/L. After 3 days, the callus was removed from the co-culture medium, the embryo was excised and transferred to a selection medium containing 50mg/L hygromycin and timentin for selection. After 12 days, the resistant calli were transferred to selection medium containing 50mg/L hygromycin and timentin for further selection. After 12 days, transferring the vigorous growth resistant callus to a differentiation medium for culturing for about two weeks (24 hours of illumination), and changing a new differentiation medium after green buds grow out to continue differentiation culture until buds grow out. The regenerated plantlets are rooted and strengthened on a 1/2M culture medium, and then transferred into a special transgenic experimental field.

Extracting total DNA of leaves from positive plants, sequencing and identifying by an identification primer, and verifying that transgenic plants OsACF1-2 of OsACF1 are knocked out successfully, so that a nucleotide sequence shown as SEQ ID NO.2 in a conventional rice variety is mutated into SEQ ID NO.7, and an amino acid sequence shown as SEQ ID NO.1 in the conventional rice variety is subjected to frame shifting and early termination, and mutated into SEQ ID NO.8, thereby obtaining the rice with low amylose content of OsACF 1-2.

From the phenotype, the transparency of the OsACF1-2 mutant brown rice is slightly lower in the mature period (figure 1A), and the amylose content of the OsACF1-2 brown rice is found to be 7.6 percent (figure 1A) by measuring the amylose content of the OsACF1-2 brown rice, compared with that of control Japanese sunny rice, the amylose content of the OsACF1-2 brown rice is obviously reduced (figure 1A), which shows that the amylose accumulation of the brown rice is inhibited after the OsACF1 gene is knocked out, and new rice with low amylose content can be obtained.

1.2 obtaining Rice with Low amylose content by hybridization means

The osdaf1-1 mutant rice 'Yinxiang 38' is hybridized with japonica rice variety 'Jiahua No. 1' and indica rice variety '9311', rice plants with low amylose content appear in the F2 generation and accord with the 3:1 separation rule, and further, when the nucleotide sequence of the OsACF1 gene changes in other rice varieties, the OsACF1 gene can also generate plants with low amylose content.

Example 3 creation of Rice with high amylose content Using modern biotechnology means

1.1 production of high amylose Rice by over-expressing OsACF1

When the OsACF1 gene is mutated or expressed down, rice with low amylose content is produced, and conversely, rice with high amylose content can be produced by over-expressing the OsACF1 gene. The corn Ubiquitin promoter is utilized to drive the over-expression of the OsACF1 in the conventional rice, so that the rice with high amylose content can be obtained.

In conclusion, the invention realizes the control of the amylose content of rice by controlling the OsACF1 gene encoding the rice zinc finger protein transcription factor and the variant strains with different amylose content of the rice obtained by the encoding protein thereof; the rice variant strain obtained by the invention has no obvious difference with a source parent or a corresponding complementary plant in the nutrition growth period, the amylose content mode of the mature seed brown rice in the later development period is changed, and the rice variant strain has very important application in agricultural production.

While the principles of the invention have been described in detail in connection with the preferred embodiments thereof, it should be understood by those skilled in the art that the foregoing embodiments are merely illustrative of the implementations of the invention and are not intended to limit the scope of the invention. The details of the embodiments are not to be taken as limiting the scope of the invention, and any obvious modifications based on equivalent changes, simple substitutions, etc. of the technical solution of the invention fall within the scope of the invention without departing from the spirit and scope of the invention.

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ccctccctcc cgccgccccc gccgctcggc tcgtcgcgcc ccgagcgcta ccgcgacaac 240

caccaccggg gaggaggcgg tggccggggt gggggtagtt ccagcccccc gccgtatcgg 300

agtggccgcc gccactcccc gtcgaggaga tccccttcgc cgccgttcaa gaggtcgcgg 360

cgggacgacg ggtacgaccg ccgtggcggc cgtgggagcc cgccgccgcg gtacgggtac 420

ggcgacagga ggtatggata tgaccacgag cgtggtggag gcagaggtgg gtatgatgat 480

gaccgatacc atggcaggta tcaaaatcgc gcagcagatt gggccgattc agggtttggg 540

gcatccaatg atggtcctgg aattacccaa agggaaggac tgatgactta caaacagttc 600

atccaagttc ttgaggatga tatttcacct gctgaagctg agaaacggta tcaagaatac 660

aggacagagt acatcactac tcaaaaacgt gcttattttg accttaacaa gaatgatgat 720

cggttgaaag acaagtacca tccgaccaac ttgtcatctg ttattgacag gaggaatgat 780

agttgtaagg caacagcaaa ggatttcttt catgatttgc aaaatggaac tctggacctt 840

ggccctggaa taactgcggc tgcagcaagt ggcagtgatg gaaattctga tgatgatgga 900

gacagtgaca agagaagaaa gcatggcagg ggttcctcaa aagaaacaga ccctctttct 960

ggtgctcccg tggctcatcc agttagctct gaatctcgac gggttcaagt tgacattgaa 1020

caagctctag cccttgtgcg taagcttgac actgagaagg gtattgtggg gaatatccta 1080

tcaagtggcg atcatgacaa atcagatgta gacaagtctc atattggatc tatggggcct 1140

ataattataa tccgaggctt aaccactgtc aaaggccttg aaggtgttga gctcctagat 1200

actcttctta cctatttatg gcgtattcat ggtgttgatt actatggcat gtctgagaca 1260

aatgaagcaa aaggcagtcg ccatgtcaga gcagacaata agacgtctaa tacaaccaat 1320

attaatgccg ctgactggga aaagaaggtg gatactttct ggcaagaaag gctgagaggt 1380

caggacccca tggtaatatt agcagccaag gacaaaatcg atgcagcagc tgtggaagtt 1440

ctggaacctt atgtcaggaa gataagggat gaaaaatatg gttggaaata tggctgtgga 1500

gctaagggtt gtacgaaact tttccatgct cctgagttcg ttcacaagca tttgaggctg 1560

aagcatccag agcttgtgtt agagttgact tccaaagtcc gagaggatct ctatttccaa 1620

aattacatga atgatcctaa tgcacctggt ggaactccag ttatgcaaca gtctgcacca 1680

gacaaatcaa gacagagacc tggtatggat aatcgtctga gatatgaccg tgccaatcgt 1740

agagaatatg atagggcaga gagagatgga agcagatatg gtagaggtga tcgttctcca 1800

agtcttgatg gcgctgatga tcagatgttt gatgctttcc gtgggcgagg tccaaatgct 1860

ccttttgttc ctgaacttcc cgctccgcca attttgatgc ctattcctgg tgctggtcct 1920

ttgggtccat ttgttcctgc acctccagaa atagccatgc atatgctgag agagcaaggg 1980

ccgccacctc catttgaacc aaacggacct cctcatgcca acccaggagt gcttggacca 2040

atgatgggtg gtcctgcgcc aattataacc atgcctccat cttttcgtca agatcctcgc 2100

cgtttgcgaa gttacaatga ccttgatgct ccggacgagg aagttaccgt tcttgactac 2160

agaagtttgt ag 2172

<210> 3

<211> 2172

<212> DNA

<213> Rice (Oryza sativa)

<400> 3

atggctgacg tcatcgaccc cgcctccacc gaggcccccc gcgcgcgccg cccgccgccg 60

cctccgcccg acagcccgga gggccgctcg ccgccgctcc cgcccccgcc ccccggtggc 120

ccgccgcagc cggcggccac ccgcaagcgg agccgctcgc caccgccgcc tcccccgccg 180

ccctccctcc cgccgccccc gccgctcggc tcgtcgcgcc ccgagcgcta ccgcgacaac 240

caccaccggg gaggaggcgg tggccggggt gggggtagtt ccagcccccc gccgtatcgg 300

agtggccgcc gccactcccc gtcgaggaga tccccttcgc cgccgttcaa gaggtcgcgg 360

cgggacgacg ggtacgaccg ccgtggcggc cgtgggagcc cgccgccgcg gtacgggtac 420

ggcgacagga ggtatggata tgaccacgag cgtggtggag gcagaggtgg gtatgatgat 480

gaccgatacc atggcaggta tcaaaatcgc gcagcagatt gggccgattc agggtttggg 540

gcatccaatg atggtcctgg aattacccaa agggaaggac tgatgactta caaacagttc 600

atccaagttc ttgaggatga tatttcacct gctgaagctg agaaacggta tcaagaatac 660

aggacagagt acatcactac tcaaaaacgt gcttattttg accttaacaa gaatgatgat 720

cggttgaaag acaagtacca tccgaccaac ttgtcatctg ttattgacag gaggaatgat 780

agttgtaagg caacagcaaa ggatttcttt catgatttgc aaaatggaac tctggacctt 840

ggccctggaa taactgcggc tgcagcaagt ggcagtgatg gaaattctga tgatgatgga 900

gacagtgaca agagaagaaa gcatggcagg ggttcctcaa aagaaacaga ccctctttct 960

ggtgctcccg tggctcatcc agttagctct gaatctcgac gggttcaagt tgacattgaa 1020

caagctctag cccttgtgcg taagcttgac actgagaagg gtattgtggg gaatatccta 1080

tcaagtggcg atcatgacaa atcagatgta gacaagtctc atattggatc tatggggcct 1140

ataattataa tccgaggctt aaccactgtc aaaggccttg aaggtgttga gctcctagat 1200

actcttctta cctatttatg acgtattcat ggtgttgatt actatggcat gtctgagaca 1260

aatgaagcaa aaggcagtcg ccatgtcaga gcagacaata agacgtctaa tacaaccaat 1320

attaatgccg ctgactggga aaagaaggtg gatactttct ggcaagaaag gctgagaggt 1380

caggacccca tggtaatatt agcagccaag gacaaaatcg atgcagcagc tgtggaagtt 1440

ctggaacctt atgtcaggaa gataagggat gaaaaatatg gttggaaata tggctgtgga 1500

gctaagggtt gtacgaaact tttccatgct cctgagttcg ttcacaagca tttgaggctg 1560

aagcatccag agcttgtgtt agagttgact tccaaagtcc gagaggatct ctatttccaa 1620

aattacatga atgatcctaa tgcacctggt ggaactccag ttatgcaaca gtctgcacca 1680

gacaaatcaa gacagagacc tggtatggat aatcgtctga gatatgaccg tgccaatcgt 1740

agagaatatg atagggcaga gagagatgga agcagatatg gtagaggtga tcgttctcca 1800

agtcttgatg gcgctgatga tcagatgttt gatgctttcc gtgggcgagg tccaaatgct 1860

ccttttgttc ctgaacttcc cgctccgcca attttgatgc ctattcctgg tgctggtcct 1920

ttgggtccat ttgttcctgc acctccagaa atagccatgc atatgctgag agagcaaggg 1980

ccgccacctc catttgaacc aaacggacct cctcatgcca acccaggagt gcttggacca 2040

atgatgggtg gtcctgcgcc aattataacc atgcctccat cttttcgtca agatcctcgc 2100

cgtttgcgaa gttacaatga ccttgatgct ccggacgagg aagttaccgt tcttgactac 2160

agaagtttgt ag 2172

<210> 4

<211> 406

<212> PRT

<213> Rice (Oryza sativa)

<400> 4

Met Ala Asp Val Ile Asp Pro Ala Ser Thr Glu Ala Pro Arg Ala Arg

1 5 10 15

Arg Pro Pro Pro Pro Pro Pro Asp Ser Pro Glu Gly Arg Ser Pro Pro

20 25 30

Leu Pro Pro Pro Pro Pro Gly Gly Pro Pro Gln Pro Ala Ala Thr Arg

35 40 45

Lys Arg Ser Arg Ser Pro Pro Pro Pro Pro Pro Pro Pro Ser Leu Pro

50 55 60

Pro Pro Pro Pro Leu Gly Ser Ser Arg Pro Glu Arg Tyr Arg Asp Asn

65 70 75 80

His His Arg Gly Gly Gly Gly Gly Arg Gly Gly Gly Ser Ser Ser Pro

85 90 95

Pro Pro Tyr Arg Ser Gly Arg Arg His Ser Pro Ser Arg Arg Ser Pro

100 105 110

Ser Pro Pro Phe Lys Arg Ser Arg Arg Asp Asp Gly Tyr Asp Arg Arg

115 120 125

Gly Gly Arg Gly Ser Pro Pro Pro Arg Tyr Gly Tyr Gly Asp Arg Arg

130 135 140

Tyr Gly Tyr Asp His Glu Arg Gly Gly Gly Arg Gly Gly Tyr Asp Asp

145 150 155 160

Asp Arg Tyr His Gly Arg Tyr Gln Asn Arg Ala Ala Asp Trp Ala Asp

165 170 175

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

180 185 190

Gly Leu Met Thr Tyr Lys Gln Phe Ile Gln Val Leu Glu Asp Asp Ile

195 200 205

Ser Pro Ala Glu Ala Glu Lys Arg Tyr Gln Glu Tyr Arg Thr Glu Tyr

210 215 220

Ile Thr Thr Gln Lys Arg Ala Tyr Phe Asp Leu Asn Lys Asn Asp Asp

225 230 235 240

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

245 250 255

Arg Arg Asn Asp Ser Cys Lys Ala Thr Ala Lys Asp Phe Phe His Asp

260 265 270

Leu Gln Asn Gly Thr Leu Asp Leu Gly Pro Gly Ile Thr Ala Ala Ala

275 280 285

Ala Ser Gly Ser Asp Gly Asn Ser Asp Asp Asp Gly Asp Ser Asp Lys

290 295 300

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

305 310 315 320

Gly Ala Pro Val Ala His Pro Val Ser Ser Glu Ser Arg Arg Val Gln

325 330 335

Val Asp Ile Glu Gln Ala Leu Ala Leu Val Arg Lys Leu Asp Thr Glu

340 345 350

Lys Gly Ile Val Gly Asn Ile Leu Ser Ser Gly Asp His Asp Lys Ser

355 360 365

Asp Val Asp Lys Ser His Ile Gly Ser Met Gly Pro Ile Ile Ile Ile

370 375 380

Arg Gly Leu Thr Thr Val Lys Gly Leu Glu Gly Val Glu Leu Leu Asp

385 390 395 400

Thr Leu Leu Thr Tyr Leu

405

<210> 5

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 5

tgtgtggggt cgatgacgtc agccat 16

<210> 6

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 6

aaacatggct gacgtcatcg acccca 16

<210> 7

<211> 2173

<212> DNA

<213> Rice (Oryza sativa)

<400> 7

atgggctgac gtcatcgacc ccgcctccac cgaggccccc cgcgcgcgcc gcccgccgcc 60

gcctccgccc gacagcccgg agggccgctc gccgccgctc ccgcccccgc cccccggtgg 120

cccgccgcag ccggcggcca cccgcaagcg gagccgctcg ccaccgccgc ctcccccgcc 180

gccctccctc ccgccgcccc cgccgctcgg ctcgtcgcgc cccgagcgct accgcgacaa 240

ccaccaccgg ggaggaggcg gtggccgggg tgggggtagt tccagccccc cgccgtatcg 300

gagtggccgc cgccactccc cgtcgaggag atccccttcg ccgccgttca agaggtcgcg 360

gcgggacgac gggtacgacc gccgtggcgg ccgtgggagc ccgccgccgc ggtacgggta 420

cggcgacagg aggtatggat atgaccacga gcgtggtgga ggcagaggtg ggtatgatga 480

tgaccgatac catggcaggt atcaaaatcg cgcagcagat tgggccgatt cagggtttgg 540

ggcatccaat gatggtcctg gaattaccca aagggaagga ctgatgactt acaaacagtt 600

catccaagtt cttgaggatg atatttcacc tgctgaagct gagaaacggt atcaagaata 660

caggacagag tacatcacta ctcaaaaacg tgcttatttt gaccttaaca agaatgatga 720

tcggttgaaa gacaagtacc atccgaccaa cttgtcatct gttattgaca ggaggaatga 780

tagttgtaag gcaacagcaa aggatttctt tcatgatttg caaaatggaa ctctggacct 840

tggccctgga ataactgcgg ctgcagcaag tggcagtgat ggaaattctg atgatgatgg 900

agacagtgac aagagaagaa agcatggcag gggttcctca aaagaaacag accctctttc 960

tggtgctccc gtggctcatc cagttagctc tgaatctcga cgggttcaag ttgacattga 1020

acaagctcta gcccttgtgc gtaagcttga cactgagaag ggtattgtgg ggaatatcct 1080

atcaagtggc gatcatgaca aatcagatgt agacaagtct catattggat ctatggggcc 1140

tataattata atccgaggct taaccactgt caaaggcctt gaaggtgttg agctcctaga 1200

tactcttctt acctatttat ggcgtattca tggtgttgat tactatggca tgtctgagac 1260

aaatgaagca aaaggcagtc gccatgtcag agcagacaat aagacgtcta atacaaccaa 1320

tattaatgcc gctgactggg aaaagaaggt ggatactttc tggcaagaaa ggctgagagg 1380

tcaggacccc atggtaatat tagcagccaa ggacaaaatc gatgcagcag ctgtggaagt 1440

tctggaacct tatgtcagga agataaggga tgaaaaatat ggttggaaat atggctgtgg 1500

agctaagggt tgtacgaaac ttttccatgc tcctgagttc gttcacaagc atttgaggct 1560

gaagcatcca gagcttgtgt tagagttgac ttccaaagtc cgagaggatc tctatttcca 1620

aaattacatg aatgatccta atgcacctgg tggaactcca gttatgcaac agtctgcacc 1680

agacaaatca agacagagac ctggtatgga taatcgtctg agatatgacc gtgccaatcg 1740

tagagaatat gatagggcag agagagatgg aagcagatat ggtagaggtg atcgttctcc 1800

aagtcttgat ggcgctgatg atcagatgtt tgatgctttc cgtgggcgag gtccaaatgc 1860

tccttttgtt cctgaacttc ccgctccgcc aattttgatg cctattcctg gtgctggtcc 1920

tttgggtcca tttgttcctg cacctccaga aatagccatg catatgctga gagagcaagg 1980

gccgccacct ccatttgaac caaacggacc tcctcatgcc aacccaggag tgcttggacc 2040

aatgatgggt ggtcctgcgc caattataac catgcctcca tcttttcgtc aagatcctcg 2100

ccgtttgcga agttacaatg accttgatgc tccggacgag gaagttaccg ttcttgacta 2160

cagaagtttg tag 2173

<210> 8

<211> 2

<212> PRT

<213> Rice (Oryza sativa)

<400> 8

Met Glu

1

<210> 9

<211> 4845

<212> DNA

<213> Artificial Sequence

<400> 9

ggtaaatcaa gttatggtcc attgccaggg gattaaaaaa ctgtggctct agctattcca 60

ttctaaattt gaggatatta gatagtgtga tatactgata ttgtttcctg ttatactggt 120

cactggtgtg actggttgac tttcaatcca tagttgtaaa cttaaaatca tgacacataa 180

ttggtttagc tatgggaaga tttaatgctg acggttgggc atctttgcat gttattttct 240

tctctccttg gtattcaaaa tatgaaagct gtcctataac tgtggctttt gtttactgcc 300

ggttaaatat gaacttgttt tcctttattt gcactggcag cctgaggcta gcttcagcat 360

caatatactg tagtttacat ttgtttctga cggcagtatg gcttataatg atgtggccat 420

ttaagaattt taggggttgt tttgttttga ggaattggat ggccaatgga ccatcagata 480

ctcccaagtc tcactttttt gttcggttcg aggaatggaa tgtgttggtc cataaccacc 540

tcatccctcg atatgtgatt gattcctcga aagaaacagc ctaagttatg gcccatttca 600

ggcatcaagt ataatgtcct aaaagtttct gtatcctgtt acaaccaaca aattctcttt 660

tccggtctct gtagcttctt caatatgcct tgatatttca aacaaatcaa tttttgaagc 720

ttctgctact tttcctccac agaatggaag tgctgcagca cctaccaccg agcaagaaat 780

tcctgcacca gccactgtca atggccacga atcatcaacg tgagtatagc gatgtttgtc 840

ttgtggagga tcgcgaacat catgccggca ttatatccat gtagtttaac tgtctgtact 900

ataacttgat gagaacctgt taggtaccta ggccatttct ttaccatcat ttagggtttg 960

aatgttgttc tgcatatgtt gtaagccatg aaaaatcatg cttccggtca ttatggccgt 1020

agcaatttta tttcttgtta gactcttggg tggaagtgag aacagagaag gtgatgaagt 1080

gaatatttta cttgtgtctt gtagttattt atatttatct caaaattgat ttatcccata 1140

cagctgtttc gtttgttacc gttttgctgg ccaaccgttg gaagtttcca aaaatataaa 1200

gggctccttt ggaacgaaat aattttacac gaaccagttc aattctcgtg aaaaatcgtt 1260

gatcaaatga tagtaaaagt ttaccggaga aaatactcaa atccactttg aagaataccc 1320

cttgaagagg tccccctttg tcgttcaaga tgccgttcgg ttatttggat ataaagatac 1380

ttgcgagaaa caagagcgat cccgggtgtg tttgtttact ttgttgaaga aaaaagaaat 1440

taaaaaaagt tactttctcc attctataat ataagacgca taagtatttt aatatttaac 1500

ttttaaaaca tctaactaat aattagtata atataaatat acattttatt tgttgaaaat 1560

gatattattt gattgtcact caaatttact tttatattgc tatagttttg ttatagcata 1620

cgagaaatta taagccaaac attagtatta aactaataaa gattatgtta agtatgattg 1680

tcttatatta tttatattat gggataaaga gattgctttg ttgaagaaaa aaatatcaaa 1740

aaagcagaag tactactttc tatatcctaa aatatactac tgttatgaga gtaagacata 1800

atcatttttt tcttatatct cataatataa tgtacatgca tacatacatt aactatcact 1860

tcttaattct ttgtatttgg tttattttaa atcttccaca agtcccttaa taatcatata 1920

gtcgagtctt atggtatcga gggagtacta cgagatgaat cttaatttta aaagtaaaac 1980

aatacgagat gaatcttaat ttaaaaagta aaataatacg atgggacgaa ttaagccctg 2040

attagttttt acgtaaaaac ttttcacctt atcatatcga atgtttgaat acatgcatgg 2100

agtattaaat atagaaaaaa aaaataacta cacagattat gtaaatacaa tacgaatctt 2160

ttaagcctaa tcgcgccatg atttgacaat gggtgggttc ggatgagatt gccgtggctt 2220

cagcttcaca gcacaacaac tacagcacta gcagtaaatt ttgtgctgct agtactgttg 2280

atatagccaa cagcagttca acagctgcaa tatgtagctc aagtgaacac ggccaatgtg 2340

gtcatacagt aaacatttgc taatgacgaa ttaattaggc ttaataaaaa cgtctcacag 2400

tttatatgta taatatgatg tttaataatt taaatgtgtg tccgtatatc ctatgatagg 2460

gctacacaca aacagggaac ccgatcagca gccgtgcacg tgtcccatac gccgtttcac 2520

actttttttc tagacgcttc cttcccttgc cgctaccgcc tcgtgcgccg cagcccgcag 2580

cctagcctac cctctctctc tcccccctcc tcaagctgtg cgcgattcgc ttcctcactc 2640

ccaaacccta accccaccgc gacgctcccc cccatggctg acgtcatcga ccccgcctcc 2700

accgaggccc cccgcgcgcg ccgcccgccg ccgcctccgc ccgacagccc ggagggccgc 2760

tcgccgccgc tcccgccccc gccccccggt ggcccgccgc agccggcggc cacccgcaag 2820

cggagccgct cgccaccgcc gcctcccccg ccgccctccc tcccgccgcc cccgccgctc 2880

ggctcgtcgc gccccgagcg ctaccgcgac aaccaccacc ggggaggagg cggtggccgg 2940

ggtgggggta gttccagccc cccgccgtat cggagtggcc gccgccactc cccgtcgagg 3000

agatcccctt cgccgccgtt caagaggtcg cggcgggacg acgggtacga ccgccgtggc 3060

ggccgtggga gcccgccgcc gcggtacggg tacggcgaca ggaggtatgg atatgaccac 3120

gagcgtggtg gaggcagagg tgggtatgat gatgaccgat accatggcag gtatcaaaat 3180

cgcgcagcag attgggccga ttcagggttt ggggcatcca atgatggtcc tggaattacc 3240

caaagggaag gactgatgac ttacaaacag ttcatccaag ttcttgagga tgatatttca 3300

cctgctgaag ctgagaaacg gtatcaagaa tacaggacag agtacatcac tactcaaaaa 3360

cgtgcttatt ttgaccttaa caagaatgat gatcggttga aagacaagta ccatccgacc 3420

aacttgtcat ctgttattga caggaggaat gatagttgta aggcaacagc aaaggatttc 3480

tttcatgatt tgcaaaatgg aactctggac cttggccctg gaataactgc ggctgcagca 3540

agtggcagtg atggaaattc tgatgatgat ggagacagtg acaagagaag aaagcatggc 3600

aggggttcct caaaagaaac agaccctctt tctggtgctc ccgtggctca tccagttagc 3660

tctgaatctc gacgggttca agttgacatt gaacaagctc tagcccttgt gcgtaagctt 3720

gacactgaga agggtattgt ggggaatatc ctatcaagtg gcgatcatga caaatcagat 3780

gtagacaagt ctcatattgg atctatgggg cctataatta taatccgagg cttaaccact 3840

gtcaaaggcc ttgaaggtgt tgagctccta gatactcttc ttacctattt atggcgtatt 3900

catggtgttg attactatgg catgtctgag acaaatgaag caaaaggcag tcgccatgtc 3960

agagcagaca ataagacgtc taatacaacc aatattaatg ccgctgactg ggaaaagaag 4020

gtggatactt tctggcaaga aaggctgaga ggtcaggacc ccatggtaat attagcagcc 4080

aaggacaaaa tcgatgcagc agctgtggaa gttctggaac cttatgtcag gaagataagg 4140

gatgaaaaat atggttggaa atatggctgt ggagctaagg gttgtacgaa acttttccat 4200

gctcctgagt tcgttcacaa gcatttgagg ctgaagcatc cagagcttgt gttagagttg 4260

acttccaaag tccgagagga tctctatttc caaaattaca tgaatgatcc taatgcacct 4320

ggtggaactc cagttatgca acagtctgca ccagacaaat caagacagag acctggtatg 4380

gataatcgtc tgagatatga ccgtgccaat cgtagagaat atgatagggc agagagagat 4440

ggaagcagat atggtagagg tgatcgttct ccaagtcttg atggcgctga tgatcagatg 4500

tttgatgctt tccgtgggcg aggtccaaat gctccttttg ttcctgaact tcccgctccg 4560

ccaattttga tgcctattcc tggtgctggt cctttgggtc catttgttcc tgcacctcca 4620

gaaatagcca tgcatatgct gagagagcaa gggccgccac ctccatttga accaaacgga 4680

cctcctcatg ccaacccagg agtgcttgga ccaatgatgg gtggtcctgc gccaattata 4740

accatgcctc catcttttcg tcaagatcct cgccgtttgc gaagttacaa tgaccttgat 4800

gctccggacg aggaagttac cgttcttgac tacagaagtt tgtag 4845

<210> 10

<211> 7020

<212> DNA

<213> Rice (Oryza sativa)

<400> 10

gtgcgccgca gcccgcagcc tagcctaccc tctctctctc ccccctcctc aagctgtgcg 60

cgattcgctt cctcactccc aaaccctaac cccaccgcga cgctcccccc catggctgac 120

gtcatcgacc ccgcctccac cgaggccccc cgcgcgcgcc gcccgccgcc gcctccgccc 180

gacagcccgg agggccgctc gccgccgctc ccgcccccgc cccccggtgg cccgccgcag 240

ccggcggcca cccgcaagcg gagccgctcg ccaccgccgc ctcccccgcc gccctccctc 300

ccgccgcccc cgccgctcgg ctcgtcgcgc cccgagcgct accgcgacaa ccaccaccgg 360

ggaggaggcg gtggccgggg tgggggtagt tccagccccc cgccgtatcg gagtggccgc 420

cgccactccc cgtcgaggag atccccttcg ccgccgttca agaggtcgcg gcgggacgac 480

gggtacgacc gccgtggcgg ccgtgggagc ccgccgccgc ggtacgggta cggcgacagg 540

aggtgagggg tttcttcttg gtcatttggt cgaaatctgt actggattgg tggttagttc 600

ttcgaggctc tgcggtttca tcgcgtgtgc ttgggtgatg tgttgggtag gtatggatat 660

gaccacgagc gtggtggagg cagaggtggg tatgatgatg accgatacca tggcaggtat 720

caaaatcgcg cagcaggtga ggattcttct cctcgggcaa agtttcgttt cgatctcaga 780

agtaagctgt ttgagtagca cagcatgagc gaaccccaaa tggtatgagg gaaattgatt 840

atttgcctgt agttagttcc ccaccaaatt agattggttt tgatgatcta caacatagtt 900

tagtgaaaac tatcagatcc tttgctccat agtcagtatg agtttactga attcaagtga 960

acagcatggg gctgttctac ttggtaaaca ttagcaacct tgctttgtta gcacttgcta 1020

atgtactccc tgcggtcata aatatttgac gtttagaaca aaattcggtt gaattttcaa 1080

aattccgact gtaatttccc aaatgcttag ttttaaaaca aaataaaatg ttgtatatag 1140

attttccttg aaaagtacta tcataatata aaaagttatt agattttata aacttatttc 1200

tactacaaaa ttgatggttg taattttaaa ttttgaccaa atcttgtcct aaatgttaaa 1260

tatttatggc agggaggttc aggatttcag atctgtagat gttgggtgtt agggctatta 1320

gctctttgat ttgtttggtg tgttatagtt gtagtaagaa ttttaccact atcttctaat 1380

ttgttcctgc ttcgaatcat tctagtcggg atgaagacaa agtatatgca tggctgtttt 1440

tttttacttt agctagcact tacactcctc atttagaaca tagattcttg atacttgtga 1500

atatgttctg acaattcgat ttcaatgtca gattgggccg attcagggtt tggggcatcc 1560

aatgatggtc ctggaattac ccaaaggtac ttattccttt accatgttta ctctttctga 1620

ttgttaggca ctttgatgtt gtctcctggg tactgtaact atgtagcgat cgacaatact 1680

ggagctaatt gccactacta ttgggaaatg caaattaagc cttctatgct agatgctgta 1740

aggcatcgtt aacagataga gaatcttctc agaaaattct ccaaatttga tgtttgaaat 1800

ctgactgatt ctttgagaac tgtgagcctg tagttaagct atcagcttga aatcctttac 1860

tgcagcacta catactccgg aaccttgtta ctgttattag ccaccagcaa gccagctcaa 1920

tttacagtta gtgccaccac aaaaggactc ttcccagtta tatgtaagct ataccaccca 1980

gatattcagc tacaccttga aggcataaga agcatatgct tttggataca atcaaatgga 2040

gaatctgatg ggaatattct ttatctgtta acaacaaaca aaggctcaga ggctttattt 2100

gaatctctag tagtggattt ctttttcttt ctttttttta attctatacc atgttatctt 2160

aaatatacat tgataactcc atggaacaca acacttccaa tgctaatttg cataagtaga 2220

tgttatcatg ttaacactat gcatttttgt cctattgatg gtagactgtt agattgtgtt 2280

aacattgtta acaggaaaaa ggtgagggat tcttgttttc ttctcttgta tatcttcttt 2340

tgccactatt tagttgctga aatatgttca tcaatttatg atacttgttc agaggaacga 2400

aatggatgaa ttgcttccta tgtaacatga aatcattgaa gcatgatcac gcttgttagg 2460

agtagttgat gcttgggctt ttcattagaa aattataatt tagtgttttt agtgttaatt 2520

ctctataata ctgggcttgc taatcaattt tgtattctgt tgaattgtag ccttttattt 2580

tagtgatgct attaataaac ctgagggtct tttcttggat ttggttgctg gcattgagaa 2640

attagattcg ttctccttat ctttgcttcc cgtagttgtc atctgaagga attctagaat 2700

tttcttggac aaaaacaata gcggaaagat agtaattgga atcagctgat ggatgggcag 2760

aagttattgg tataccccat caaccatcac atggttgtaa tgacttcttc aagtttttaa 2820

gaaataacaa ttattctatg ctgataatac ttttggtggc aacgcatgta attagcaaat 2880

gtttattcag ttgtttcctc tagttatgtg tatgtcacat ttacatttat cattgagtgt 2940

tattgtgtgc ttaaggtgtc ttagttagga agaacatgaa atgttagaga aggtagacca 3000

tgttgctaag gaagataata tttagttagt tattggtcag gatttttttt aattaggaga 3060

atcttttgcc cgaccaatgc agttgttcct ggtgaccgat ttgcataaaa cattgtaatg 3120

agtggcagtt catatgaagg ccacatgtta gatgacatct ctgtagggag aggtggattg 3180

ccttcttttt tgttatttgt aaaaaaatat tcaacatgca taccttttaa actgatcttt 3240

gaactgttga aacagattta tgaaaacttc tattgcagat taaagaaatc tgaactgttg 3300

tcacatttaa acttgttttc aaatattgtt ccctccattc attttgacga gctaaccgtt 3360

ctaacatatt gagcagagga ttgggatata gtttcatgag tttctcttag tgtatttctg 3420

ttatcttata tgattatgca tcttttcagg gaaggactga tgacttacaa acagttcatc 3480

caagttcttg aggatgatat ttcacctgct gaagctgaga aacggtaaat gcacaacact 3540

tactgattat atctttgtgc taccttttta gtattgatgg gctatgtctg tttaaaaagg 3600

tatcaagaat acaggacaga gtacatcact actcaaaaac gtgcttattt tgaccttaac 3660

aagaatgatg atcggtaagt caaaatgatt tagctgtaca caactaggaa caaaaatggt 3720

ccacttgctt taactgacat ttcatttgct ttcacttgca ggttgaaaga caagtaccat 3780

ccgaccaact tgtcatctgt tattgacagg tggagttgaa ttccttttta ttgagcctgt 3840

tcccttatgc attcataaac attattatgt tgtggaaaaa attttcttgc agaacaatac 3900

cctttatact gctcatctta actcctttta catttttgtt aagtaaattt cagaaaacta 3960

caggtgcttt gaccaaatta tcacaaaagt atagatttaa ggcgctgtat cacaaaacta 4020

catatttgat ttcgaagtta tcacaaaact gcagatatta caatttaaat ccctagtact 4080

actgttatgt tagagttata aatgttgtag tttcgtctaa ctgcaacttt tccatataat 4140

gcaggaggaa tgatagttgt aaggcaacag caaaggattt ctttcatgat ttgcaaaatg 4200

gaactctgga cctgtgagtt atatctgcac agcttgtgtt atgatgatct tctggacttc 4260

ttgtttatac cttgattttt tactgagcag tggccctgga ataactgcgg ctgcagcaag 4320

tggcagtgat ggaaattctg atgatgatgg agacagtgac aagagaagaa agcatggcag 4380

gggttcctca aaagaaacag accctctttc tggtgctccc gtggctcatc cagttagctc 4440

tgaatctcga cgggttcaag ttgacattga acaagctcta gcccttgtgc gtaagcttga 4500

cactgagaag ggtattgtgg ggaatatcct atcaagtggc gatcatgaca aatcagatgt 4560

agacaagtct catattggat ctatggggcc tataattata atccgaggct taaccactgt 4620

caaaggcctt gaaggtgttg agctcctaga tactcttctt acctatttat ggcgtattca 4680

tggtgttgat tactatggca tgtctgagac aaatgaagca aaaggcagtc gccatgtcag 4740

agcagacaat aagacgtcta atacaaccaa tattaatgcc gctgactggg aaaagaaggt 4800

ggatactttc tggcaagaaa ggctgagagg tcaggacccc atggtaatat tagcagccaa 4860

ggacaaaatc gatgcagcag ctgtggaagt tctggaacct tatgtcagga agataaggga 4920

tgaaaaatat ggttggaaat atggctgtgg agctaagggt tgtacgaaac ttttccatgc 4980

tcctgagttc gttcacaagc atttgaggct gaagcatcca gagcttgtgt tagagttgac 5040

ttccaaagtc cgagaggatc tctatttcca aaattacatg aagtatgtac atatgatttt 5100

ctgcctgtgc tacttttttt taaggaggtg ttactgatct ggatgtttct ttatgaacag 5160

tgatcctaat gcacctggtg gaactccagt tatgcaacag tctgcaccag taagaacctc 5220

atactctatt acttgcttaa ataaaacaga acaattctac aagtgaattc catgcataat 5280

tacataccag tatatcacat atgtgctata cacatgttac attataactt cgaataaaag 5340

ttccctgcaa aaaagaactt caaataaaat ttgcttttgc ttttatccca gctgcttcct 5400

gtaggttgtt tctttttcat ttgtcagtaa accccagctc ccttttaaga ataatttgta 5460

tgcctgtgcc ttttggttac tagtttgtgt acacatggac catataccat tccacccctt 5520

tgttccttct acagattttt ccttttaggt gctaagccta cattagatga actatacggt 5580

atcagtcaga cagtcactta tgtggcctaa ccggtgacgt gagagttaaa ggagggttgg 5640

cttatttgag ggaatgatca ggcctggaca gaatcagtgg aaggaatctg actaaagctt 5700

ttagtgatgg gtcaataccc tactgaagaa tttagctgac acttctctaa gtatcattaa 5760

tggataaata cattgagtgc aggtggaaac tgcagttaca tggattcatt gaaatccttg 5820

acaaatattt atacttctga tttgcaggac aaatcaagac agagacctgg tatggataat 5880

cgtctgagat atgaccgtgc caatcgtaga gaatatgata gggcagagag agatggaagc 5940

agatatggta gaggtgatcg ttctccaagt cttgatggcg ctgatgatca gatgtttgat 6000

gctttccgtg ggcgaggtcc aaatgctcct tttgttcctg aacttcccgc tccgccaatt 6060

ttgatgccta ttcctggtgc tgggtaggtg ctgtgagaag atatgatttc aatttttgtt 6120

ctgatagtat aaaagactgc taatgagcgt ggctggtttt attttcagtc ctttgggtcc 6180

atttgttcct gcacctccag aaatagccat gcatatgctg agagagcaag ggccgccacc 6240

tccatttgaa ccaaacggac ctcctcatgc caacccagga gtgcttggac caatgatggg 6300

tggtcctgcg ccaattataa ccatgcctcc atcttttcgt caagatcctc gccgtttgcg 6360

aaggttagta attattcatt cataccattg aattccatga tgtctattct cctattttgc 6420

ttggattggc ttgattatgc cacattctga ccaacaattt ggccacctag ggcttgccac 6480

caagccttac accttgcttt agtttgtata tgattacttc tactcgaggg cttaaccata 6540

ctgtctttat tatcatagag gcaaaaatag tatgttgatt tactgtgcca ttgtactata 6600

ttttacaacc ggcaacttaa cccatccatg atgacgattc ttctgcagtt acaatgacct 6660

tgatgctccg gacgaggaag ttaccgttct tgactacaga agtttgtaga gcttgccctg 6720

gtgtaattgt aatttgccaa tcacaactct agcatctccg gtctagtcta ggttggtgat 6780

gtattctttt tcagacatag gggatgtcat gaacaataga gcattttttg aggtgtaatg 6840

cgtcagaaac tactgttgta atttcaaatg gcaacatctg ttattgaact gtgcaccacg 6900

tgcacttgta gtcccaagaa gtgttgaacg cagtttgata aaatgtaatt tttgagactt 6960

tatatgacaa cgttttagct gacaacattt tacttctcca ttggaagtaa ttaatttatt 7020

Claims

1. Amylose content regulating geneOsACF1Characterized in that the amylose content geneOsACF1The nucleotide sequence of (2) is shown as SEQ ID NO.3 or 7.

2. A method for cultivating rice plant germplasm with different amylose content, comprising the following steps: and carrying out gene editing on the target rice variety to ensure that the nucleotide sequence shown in SEQ ID NO.3 or 7 contained in the target rice seed is obtained to obtain the rice strain with low amylose content.

3. The method of claim 2, wherein, when the target rice variety is subjected to gene editing, a CRISPR-CAS9 gene editing technology is adopted, and the construction method of the adopted CRISPR-CAS9 gene editing vector comprises the following steps: (a) Selection ofOsACF1The specific fragment of 20bp total from 1 st to 20 th of the nucleotide sequence shown in SEQ ID NO.2 is taken as a target site; (b) Annealing a pair of primers shown as SEQ ID No.5 and SEQ ID No.6 in PCR and binding together to form a target sequence, restriction endonucleaseBsa1 digestion of vector RCKO, ligation of vector and target sequence with T4 ligase, successful construction of RCKO after sequencing validationOsDAF1Plasmid, transformed Agrobacterium tumefaciens @Agrobacterium tumefaciens) EHA105, (c) RCKO-containing reaction chamberOsDAF1The Agrobacterium tumefaciens EHA105 is transferred into seeds of japonica rice "Nipponbare" and the seeds are cultivated.