CN112080481A - Spike-type related gene OsFRS5 and application and phenotype recovery method thereof - Google Patents

Abstract

The invention relates to an application of an ear type gene OsFRS 5; the amino acid sequence of the spike-type related gene OsFRS5 is shown in SEQ ID NO.1, and the application is as follows: knocking out, changing or inhibiting an OsFRS5 gene by a conventional method or based on a CRISPR/Cas9 system, so that the expression level of the OsFRS5 gene in a conventional rice variety is reduced, and a spike variant strain of rice is obtained. The invention can restore the ear type to the wild type phenotype by amplifying the OsFRS5 gene through the primer and using the means of genetic transformation. The rice osfrs5 mutant has no obvious abnormality in the vegetative growth stage, and after the rice osfrs5 mutant enters reproductive growth, the number of secondary branches and the number of spikelets are reduced sharply under the high-temperature condition; however, under the low temperature condition, the number of secondary branches and the number of spikelets are increased, the seeds are enlarged, the thousand seed weight is increased, the potential of increasing the yield is realized, and the method has an important application prospect in agricultural production.

Description

Spike-type related gene OsFRS5 and application and phenotype recovery method thereof

Technical Field

The invention relates to a method for creating a rice strain in the technical field of bioengineering, in particular to a spike-type related gene OsFRS5 and an application and phenotype recovery method thereof.

Background

Rice is one of the main food crops in the world, and more than half of the population in the world is cultivated. Therefore, the improvement of the rice yield is a big matter related to the national civilization. In the process of crop improvement, in order to improve the rice yield, the spike type, the number of grains per spike and the like of rice are focused. The grain number of the spike is determined by the number of the first branch and the number of the second branch in the spike. Therefore, the gene which can affect the number of the primary and secondary branches can be a favorable resource in the improvement of rice crops. In the former work, there have been many reports on genes affecting rice panicle type, but functional studies on these genes have shown that most of them affect meristem size and thus panicle formation via auxin or cytokinin pathways. However, the relationship between the genes is reported less, so that the spike-type related gene regulation network is not known much. On the other hand, there are few reports on how these panicle regulatory genes are affected by the environment and used in production.

For example: chinese patent publication No. CN102703497A, published as 2012, 10 and 3, entitled "method for creating rice short-stalk lines and use thereof, method for recovering short-stalk traits" discloses a method for creating rice short-stalk lines and use thereof, and method for recovering short-stalk traits in the field of bioengineering technology, but the patent document does not disclose panicle type-related gene regulation.

The OsFRS5 gene belongs to a gene for coding transposase derived protein. The research on the genes is mainly carried out in arabidopsis thaliana, and the functions of the genes mainly relate to accumulation of phytochrome in cell nucleus, regulation of biological clock in plants and the like. The function of the gene in rice has not been reported.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides an ear type related gene OsFRS5, application thereof and a method for phenotype recovery, and the invention discovers that the OsFRS5 gene has close relation with the development of rice ear type for the first time. And the phenotype of the mutant shows great difference under the conditions of high temperature and low temperature. The OsFRS5 gene and the characteristic that the protein thereof participates in regulation and control of the rice panicle type, the transgenic technology is utilized to control the growth of the rice panicle type, and a new rice panicle type variant strain is generated by mutating the protein sequence or inhibiting the expression of the protein, so that the method has very important application in agricultural production.

The purpose of the invention is realized by the following technical scheme:

in a first aspect, the invention relates to a rice panicle-associated gene OsFRS5, wherein an amino acid sequence coded by the panicle-associated gene OsFRS5 is shown as SEQ ID No. 1.

Preferably, the nucleotide sequence of the coding spike-type related gene OsFRS5 is shown in SEQ ID No. 2.

In a second aspect, the invention relates to an application of a rice panicle-type related gene OsFRS5, wherein an amino acid sequence coded by the panicle-type related gene OsFRS5 is shown as SEQ ID No.1, and the application specifically comprises knocking out, changing or inhibiting an OsFRS5 gene by a conventional method or based on a CRISPR/Cas9 system, so that the expression level of the OsFRS5 gene in a conventional rice variety is reduced, and a rice panicle-type variant strain is obtained.

In a third aspect, the invention relates to a method for preparing a spike-type mutant strain of rice, which comprises the following steps: selecting a conventional rice variety, processing and cultivating to obtain the spike-type rice variant strain, wherein the processing is that a nucleotide sequence which codes the amino acid shown as SEQ ID No.1 in the rice is deleted, mutated or inhibited by adopting a conventional method or based on a CRISPR/Cas9 system, so that the expression level of the polypeptide corresponding to the amino acid sequence is reduced or the activity is lost;

the rice variety is japonica rice variety 9522, indica rice 9311 or Guangdong short No. 4; more preferably japonica rice cultivar 9522.

Preferably, the nucleotide sequence of the rice coding amino acid shown as SEQ ID No.1 is shown as SEQ ID No. 2.

Preferably, the preparation method of the spike variant strain of rice comprises the following steps: the nucleotide sequence shown as SEQ ID No.2 in the conventional rice variety is mutated into SEQ ID No.8 by adopting a physical mutagenesis method, and then the spike-type rice mutant strain, namely osfrs5 mutant, is obtained.

Preferably, the preparation method of the spike variant strain of rice comprises the following steps: the physical mutagenesis method is adopted to mutate the amino acid sequence shown as SEQ ID No.1 in the conventional rice variety into SEQ ID No.9, and then the spike-type mutant strain of rice, namely osfrs5 mutant, is obtained.

Preferably, the CRISPR/Cas 9-based system specifically comprises: the OsFRS5 gene is knocked out by using a CRISPR/Cas9 system site-directed knockout method, and the expression of a nucleotide sequence encoding an amino acid sequence shown as SEQ ID No.1 is inhibited.

More preferably, the method for site-directed knockout of CRISPR/Cas9 system comprises the following steps:

a) synthesizing a single nucleotide sequence, wherein the primers are shown as SEQ ID No.3 and SEQ ID No. 4;

OsFRS5CRISPR-F(SEQ ID No.3):

TAGGTCTCCTGACAGAAGCCTGTTTTAGAGCTAGAA

OsFRS5CRISPR-R(SEQ ID No.4):

CGGGTCTCAGTCAGAATCGCCTGCACCAGCCGGG

b) forming a dimer structure by the synthesized mononucleotide sequence through annealing reaction, and carrying out connection reaction with pRGEB32 carrier fragment to construct OsFRS5-RGEB32 plasmid containing rice OsFRS5 gene target sequence; the target sequence is shown as SEQ ID No. 11;

c) infecting rice varieties with Agrobacterium tumefaciens containing OsFRS5-RGEB32 plasmid;

d) and (3) amplifying a genome segment by using a specific primer of the OsFRS5 gene for sequencing, and screening mutant plants.

The rice OsFRS5 gene target sequence is shown as follows:

SEQ ID No.11:GGCGATTCTGACAGAAGCCTAGG

the pRGEB32 plasmid has stronger multi-editing capacity and higher efficiency on the genome.

In a fourth aspect, the invention also relates to the application of the rice panicle type mutant line obtained by the preparation method in rice breeding work.

The application is used for improving the yield of the rice osfrs5 strain under the conditions that the temperature is low (for example, 20-24 ℃) and the sunshine length is 10-13 hours, the rice osfrs5 strain is a high-quality germplasm resource, and the number of grains per spike of osfrs5 material is increased, so that the yield is improved.

Furthermore, the use is for planting in the short-day low-temperature conditions of Hainan province, and the osfrs5 spike variant line of rice can be partly fruited for seed propagation.

In a fifth aspect, the present invention also relates to a method for restoring the panicle phenotype of a rice panicle variant line, comprising the steps of: the OsFRS5 gene is transferred into the panicle type mutant line of the rice obtained by the method by adopting a conventional genetic means, so that the mutant recovers the wild type phenotype.

Preferably, the method comprises the steps of: transferring Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 complementarily constructed by containing OsFRS5 into the panicle type variation strain of rice, and culturing to obtain the mutant strain; wherein the OsFRS5 is complemented to construct nucleotide with a sequence shown as SEQ ID No. 5.

More preferably, the method specifically comprises the steps of:

(a) amplifying OsFRS5 gene, such as 8850bp genome sequence fragment containing promoter sequence, from rice 9522 genome by primers with base sequences shown as SEQ ID No.6 and SEQ ID No. 7;

(b) providing an Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 carrying a complementary construction vector for expressing OsFRS 5;

(c) contacting cells or tissues or organs of the panicle type mutant strain of rice with the agrobacterium tumefaciens in the step (b), so that the nucleotide coding for the amino acid shown as SEQ ID No.1 is transferred into the callus of rice and is integrated on the chromosome of the rice cells;

(d) and (3) selecting the rice cells or tissues or organs which are transferred with the nucleotide, and regenerating to obtain rice plants with recovered spike types.

The research of the invention shows that under the condition of high temperature (such as 30 ℃), the number of osfrs5 secondary branches and the number of spikelets are reduced sharply; and under the condition of low temperature (such as 20-24 ℃), the number of secondary branches and the number of spikelets of osfrs5 are increased slightly, which is beneficial to increasing the yield.

The invention has the following beneficial effects:

the invention obtains the variant strain of the development of the rice panicle type by controlling the panicle type related gene OsFRS5 and the coding protein thereof, thereby realizing the control of the rice panicle type; the rice mutant obtained by the invention has no obvious difference with the original parent in the vegetative stage, abnormal inflorescence development after entering the reproductive growth stage, and very important application in improving the rice agronomic characters and agricultural production.

Drawings

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

fig. 1 is the creation of OsFRS5 CRISPR; wherein, FIG. 1A is a schematic diagram of construction of pRGEB32 vector and OsFRS5 CRISPR; FIG. 1B shows OsFRS5 target site location; fig. 1C is spike-type characteristics of OsFRS5CRISPR line at different temperatures;

FIG. 2 is a schematic representation of morphological observations of osfrs5 mutant plants; wherein, FIG. 2A shows the phenotype of the wild type and osfrs5 mutant as a whole under high temperature conditions; FIG. 2B shows panicle types of wild type and osfrs5 mutants under high temperature conditions; FIG. 2C shows floret phenotype of wild type and osfrs5 mutants at elevated temperature; FIG. 2D shows the phenotype of wild type and osfrs5 mutant as a whole under low temperature conditions; FIG. 2E shows panicle types of wild type and osfrs5 mutants at low temperature; FIG. 2F is floret phenotype of wild type and osfrs5 mutant under cold temperature conditions;

FIG. 3 shows the stimulation performance of osfrs 5; wherein, FIG. 3A shows a comparison of WT versus osfrs5 seed morphology; FIG. 3B shows the hundred particle weight of WT and osfrs 5;

FIG. 4 is a view showing an expression pattern of Osfrs5 gene; wherein, FIG. 4A shows the expression of OsFRS5 gene in each organ; FIG. 4B shows that the expression of OsFRS5 gene shows rhythmicity, in which black line is the change of OsFRS5 expression under short-day condition (10h/14h), and gray line is the change of OsFRS5 expression under long-day condition (14h/10 h);

FIG. 5 shows the spike type of OsFRS5 complementation plant under high temperature condition.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

The OsFRS5 gene is a nucleotide sequence which codes for an amino acid sequence shown as SEQ ID No. 1.

Example 1 method for creating spike variant lines of Rice

1.1 creating osfrs5 Rice panicle type mutant lines by genetic engineering means

The coding region sequence of the OsFRS5 gene in this example is shown in SEQ ID No. 2. The OsFRS5 mutant material of the embodiment is obtained by carrying out sequence variation on an OsFRS5 gene by a conventional genetic engineering method from a conventional japonica rice variety Wuyujing No.7 (also known as 9522). One skilled in the art will appreciate that other means, such as gene editing, can also be used to mutate conventional varieties of rice.

1.2 cloning of the control Gene OsFRS5 for panicle type of Rice

A map-based cloning or position cloning population of rice genes, which is composed of a spike-type related gene OsFRS5 (the nucleotide sequence of which is shown in SEQ ID No. 2) and a mutant gene Osfrs5 (the nucleotide sequence of which is shown in SEQ ID No. 8) and is clear to a person skilled in the art, is positioned in a 1-small genome segment according to molecular markers. On this basis, genomic DNA clones containing this fragment were isolated by conventional methods. One of the two proteins is determined to contain the complete rice panicle development control protein OsFRS5 through sequencing and further hybridization identification.

The whole nucleotide sequence analysis result shows that the whole length of the spike-type related gene OsFRS5 is 3146bp (SEQ ID No.10, including intron). Through software analysis and cDNA cloning, the ORF is shown as SEQ ID No.2, the coded full-length protein OsFRS5 with 988 amino acids is shown as SEQ ID No. 1.

1.3 Point mutation of spike-type control protein Gene of Rice

The Osfrs5 mutant material of the embodiment is obtained by sequence variation of an OsFRS5 gene of Wuyujing No.7 (also known as 9522) of a conventional japonica rice variety, and the spike-type variation of a plant is caused by early termination of spike-type development control protein of rice by comparing the sequences of an Osfrs5 mutant gene and the OsFRS5 gene; in the embodiment, the mutant gene of osfrs5 is inserted into a single base in a coding region (the sequence of the mutant gene is shown as SEQ ID No. 8), so that the amino acid sequence shown as SEQ ID No.1 is mutated into SEQ ID No.9, and the function of the rice panicle development control protein is changed.

1.4 mutation of OsFRS5 Gene in Rice variety by CRISPR means

In order to apply the OsFRS5 protein, a carrier (OsFRS5-RGEB32) of OsFRS5 gene CRISPR is constructed, and a wild 9522 plant is transformed to block the complete expression of OsFRS5, so that the aim of changing the rice spike type is fulfilled. The construction scheme of pRGEB32 vector and OsFRS5CRISPR is shown in FIG. 1. The method comprises the following specific steps:

1.4.1 Synthesis of mononucleotide sequence primers

OsFRS5CRISPR-F (the sequence is shown as SEQ ID No. 3):

TAGGTCTCCTGACAGAAGCCTGTTTTAGAGCTAGAA

OsFRS5CRISPR-R (the sequence is shown as SEQ ID No. 4):

CGGGTCTCAGTCAGAATCGCCTGCACCAGCCGGG

1.4.2 annealing the synthesized mononucleotide sequence to form a dimer structure, and connecting the dimer structure with pRGEB32 vector fragment (as donated by professor Schkan & Chuffi of Huazhong university of agriculture) to construct OsFRS5-RGEB32 plasmid containing rice OsFRS5 gene target sequence (shown as SEQ ID No. 11);

1.4.3 transformation of OsFRS5-RGEB32 plasmid into Agrobacterium tumefaciens to construct Agrobacterium tumefaciens containing OsFRS5-RGEB32 plasmid, streaking and culturing the Agrobacterium tumefaciens on YEB plate containing Kan (50. mu.g/. mu.l) to obtain a single colony. Selecting single colony, inoculating into 3ml YEB liquid culture medium containing antibiotic, shaking and culturing at 28 deg.C overnight, inoculating into 50ml YEB liquid culture medium containing antibiotic at 2 days according to 1% inoculum size, continuing shaking and culturing at 200rpm to OD₆₀₀When the speed is about 0.6 to 0.8, centrifuging fresh agrobacterium tumefaciens bacterial liquid for 5 minutes at 5000rpm, collecting and suspending the liquid in 1/3 volumes of AAM liquid culture medium, wherein the formed AAM agrobacterium tumefaciens bacterial liquid can be used for transforming various receptor materials of rice.

1.4.4 this example describes transformation of young embryogenic calli of rice 9522 using conventional Agrobacterium tumefaciens transformation. Soaking 9522 immature seeds 12-15 days after pollination in 70% ethanol for 1 min, sterilizing in NaClO solution (mixed with water 1:3, adding 2-3 drops of Tween 20) for more than 90 min, washing with sterile water for 4-5 times, picking out young embryos with scalpels and tweezers, inoculating on N6D2 culture medium to induce callus, culturing at 26 + -1 deg.C in dark, and allowing for transformation after 4 days. Soaking the young embryo callus in fresh AAM Agrobacterium tumefaciens bacterial liquid, shaking, removing the young embryo callus after 20 minutes, sucking excessive bacterial liquid on sterile filter paper, transferring to N6D2C culture medium, and culturing at 26 deg.C for 3 days. In the co-culture, acetosyringone was added to the co-culture medium at a concentration of 100. mu.M. After 3 days, the calli were removed from the co-culture medium, the embryos excised and transferred to a selection medium containing 25mg/L Hyg for selective culture. After 7-12 days, the resistant calli were transferred to selection medium containing 50mg/L Hyg for further selection. After 10-12 days, the vigorous resistant callus is transferred to a pre-differentiation culture medium to be cultured for about one week, and then transferred to a differentiation culture medium to be differentiated (12 hours of light/day). Regenerated plantlets were rooted and strong on 1/2MS medium and subsequently transferred to phytotron nutrient solution for cultivation.

1.4.5 extracting total DNA of leaves from the obtained positive plants, and further identifying transformed plants by PCR. Sequencing and detecting the gene sequence of the target site, and if homozygous mutation occurs, the target site is an effective gene knockout plant, namely an OsFRS5CRISPR line plant (namely an Osfrs5 mutant).

1.5 loss or reduction of OsFRS5 protein Activity

Morphological observation of osfrs5 mutant plants grown in Shanghai under long-light high-temperature conditions (average temperature 32 ℃ C., average illumination time 13.5 hours). As shown in FIG. 2, the Osfrs5 mutant has no abnormal plant type compared with the wild type (FIG. 2A), while the wild type 9522 develops normally in the spike type, while the secondary branch of the Osfrs5 mutant decreases sharply (FIG. 2B), and the development of floral organs is arrested; under the short-day and low-temperature conditions (average temperature 23 ℃ C., illumination time period 11.5 hours) of Hainan Mitsui, osfrs5 mutant showed no abnormal plant type as a whole (FIG. 2D), while in spike type, wild type 9522 developed normally, while osfrs5 mutant showed an increased number of secondary branches (FIG. 2E), and no abnormal flower organ development. As can be seen from FIG. 3, the Osfrs5 mutant has increased seeds (FIG. 3A) and thousand kernel weight (FIG. 3B), and compared with the wild type, the Osfrs5 mutant has higher yield and is very important to be applied to agricultural production.

1.6OsFRS5 expression characteristics

The method comprises the steps of extracting RNA from each organ tissue of a parent 9522 from which an OsFRS5 mutant strain is derived, carrying out reverse transcription to obtain a first cDNA chain, determining an expression mode of an OsFRS5 gene (as shown in figure 4A) by a fluorescent quantitative PCR method, and particularly finding that the OsFRS5 gene has wide expression range but high-level expression in the reproductive development period of rice from figure 4A; in addition, the expression of OsFRS5 gene also showed rhythmicity, and the peak of its expression appeared in the evening (see FIG. 4B).

Example 2 method for restoring spike type variation trait of Osfrs5 mutant

The introduction of the genomic nucleotide sequence encoding the OsFRS5 gene into mutant OsFRS5 plants enabled the mutant to revert to the wild type phenotype. Specifically, Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 complementarily constructed by containing OsFRS5 is transferred into the rice panicle type mutant strain OsFRS5 and cultivated to obtain the rice panicle type mutant strain EHA; wherein the OsFRS5 is complemented to construct nucleotide with a sequence shown as SEQ ID No. 5. The method comprises the following specific steps:

3.1 expression of a DNA fragment from the rice 9522 genome with primers:

OsFRS5FL-1301-F (the sequence is shown in SEQ ID NO. 6):

ACATGATTACGAATTCGCCTGATGAAAACAATAACTGCGTTA

OsFRS5FL-1301-R (the sequence is shown in SEQ ID NO. 7):

ATTCGAGCTGGTCACCTCTCCCTATTTACCCATAAACAG

amplifying an 8850bp OsFRS5gDNA fragment (containing a promoter), and connecting a binary vector pCAMBIA1301 of the rice after enzyme digestion by using endonucleases EcoRI and BestEII; after the sequencing is correct, the pCAMBIA1301-OsFRS5 vector is obtained.

3.2 introducing the vector constructed in the previous step into Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 by chemical transformation to obtain OsFRS5 complementary Agrobacterium tumefaciens EHA105, transforming the mature embryo callus of Osfrs5 mutant by genetic transformation, thereby transferring the nucleotide encoding the amino acid shown in SEQ ID NO.1 into rice cells and integrating the nucleotide into the chromosome of the rice cells; regenerating to obtain rice plant; to see if the mutant reverted to the wild type phenotype. Obtaining T₀Generation complementation plants (i.e., Osfrs5 mutant restorer lines), FIG. 5 shows T₀The generation complementary plant can normally develop secondary branches at high temperature, and the wild type phenotype is shown.

In conclusion, the mutant strain with abnormal development of the rice panicle type is obtained by controlling the gene OsFRS5 related to the development of the rice panicle type and the protein coded by the gene OsFRS5, so that the development of the rice panicle type is controlled; the rice mutant obtained by the invention has no obvious difference with the original parent in the vegetative growth period, and after entering the reproductive growth stage, the spike dysplasia and the floret dysplasia cause plant sterility.

It should be noted that the prior art in the protection scope of the present invention is not limited to the examples given in the present application, and all the prior art which is not inconsistent with the technical scheme of the present invention, including but not limited to the prior patent documents, the prior publications and the like, can be included in the protection scope of the present invention.

In addition, the combination of the features in the present application is not limited to the combination described in the claims of the present application or the combination described in the embodiments, and all the features described in the present application may be freely combined or combined in any manner unless contradictory to each other.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.

Sequence listing

<110> Shanghai university of transportation

<120> spike-type associated gene OsFRS5, application thereof and method for phenotype recovery

<130> 2020.0816

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 988

<212> PRT

<213> primer

<400> 1

Met Asp Gln Ser Leu Gln His Thr Asp Gly Gln Asn Gln Ser His Asp

1 5 10 15

Asn Met Glu Ser Asn Glu Val Ser Ser Asp Asp Arg Asn Ser Asp Gly

20 25 30

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

35 40 45

His Tyr Ser Pro Ser Leu Met Glu Leu Asp Asn Ala Arg Pro Pro Glu

50 55 60

Leu Gly Met Lys Phe Ala Thr Leu Glu Asp Ala Gln Arg Phe Tyr Glu

65 70 75 80

Thr His Ala Leu Lys Thr Gly Phe Val Ala Lys Arg Gly Thr Asn Tyr

85 90 95

Arg Arg Lys Lys Phe Thr Ile Glu Cys His Lys Thr Gly Thr Ser Lys

100 105 110

Leu Thr Pro Asn Pro Gln Arg Lys Arg Lys Lys Asn Ile Ile Glu Arg

115 120 125

Thr Gln Cys Gln Ala Lys Val Val Val Lys Leu Asn Lys Gly Gln Trp

130 135 140

Glu Tyr Ala Ala Val Arg Asn Glu His Asn His Pro Leu Cys Pro Ser

145 150 155 160

Tyr Ser Leu Arg Phe Ser Lys Arg Lys Arg Arg Arg Asn Pro Pro Ser

165 170 175

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

180 185 190

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

195 200 205

Asn Glu Val Asn Asn Lys Arg Met Pro Arg His Ala Glu Ile Ser Ile

210 215 220

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

225 230 235 240

Tyr Gln Glu Gln Ser Ser Asn Gly Ile Asp Lys Asn Thr Glu Arg Pro

245 250 255

Ile Val Asp Lys Leu Gly Asp Gln Thr Ser Ser Ile Asp His Ser Leu

260 265 270

Gln His Thr Glu Glu His Asn Arg Ser His Asp Asn Val Glu Ser Ser

275 280 285

Glu Ala Pro Ser Glu Asp Thr Ser Ser Glu Ser Asn Ser Asp Ser Ser

290 295 300

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

305 310 315 320

Phe Asp Glu Leu Lys Asn Ser Arg Pro Pro Glu Pro Gly Met Lys Phe

325 330 335

Pro Ser Leu Gln Ala Ala Gln Arg Phe Tyr Tyr Ala His Ala Leu Leu

340 345 350

Thr Gly Phe Val Gly Lys Arg Gly Thr Asn Tyr Lys Arg Lys Lys Phe

355 360 365

His Leu Glu Cys Asn Lys Ser Gly Lys Met Lys Leu Thr Lys Ser Ser

370 375 380

Glu Asn Pro Met Lys Lys Arg Arg Ser Asn Leu Val Glu Lys Thr Gln

385 390 395 400

Cys Lys Ala Arg Val Ile Val Lys Leu Asp Lys Gly Glu Trp Glu Phe

405 410 415

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

420 425 430

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

435 440 445

Ser Phe Leu Arg Val Leu Gln Gln Asn Arg Val Pro Pro Lys Lys Ile

450 455 460

Met Lys Ile Phe Arg Lys Leu Arg Val Cys Phe Gly Asp Ile Pro Phe

465 470 475 480

Glu Asn Lys Asp Glu His Asn Ile Ala Gln Thr Glu His Arg Lys Ala

485 490 495

Asn Ser Asp Val Glu Ser Ala Leu Lys His Phe Thr Glu Leu Gln Ile

500 505 510

Gln Asn Pro Glu Phe Leu Tyr Val Met Gln Lys Asp Glu Asp Asn Thr

515 520 525

Val Thr Ser Ile Phe Trp Thr Asp Ala Arg Leu Arg Ile Glu Tyr Asp

530 535 540

Ile Phe Gly Asp Leu Ile Met Phe Asp Ala Ala Tyr Ser Thr Asp Met

545 550 555 560

Tyr Asn Met Pro Phe Val Pro Ile Ile Gly Ile Asn Ser His Ala Thr

565 570 575

Pro Phe Leu Leu Gly Cys Ala Leu Leu Lys Asp Glu Lys Val Glu Thr

580 585 590

Phe Glu Trp Met Leu Arg Thr Phe Leu Gln Val Met Gly Gly Lys Met

595 600 605

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

610 615 620

Ala Glu Leu Met Pro His Val Arg Leu Arg Phe Cys Lys Arg His Val

625 630 635 640

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

645 650 655

Asn Ile Asn Thr Asp Leu His Asn Leu Val Gly Asn Ser Leu Val Glu

660 665 670

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

675 680 685

Ser Glu Asn Gln His Leu Gln Leu Met Trp Gln Thr Arg Lys Ser Trp

690 695 700

Ala Pro Val Tyr Phe Arg Glu Asp Leu Tyr Pro Phe Ile Asp Ser Val

705 710 715 720

Gly Ser Asn Glu Gly Ile Asn Ser Leu Phe Lys Gly Asn Met Leu Pro

725 730 735

Lys Asp Thr Ile Asp Lys Phe Ile Gly Gln Phe Lys Glu Ile Gln Glu

740 745 750

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

755 760 765

Leu Lys Tyr Ile Ser Met Gln Pro Ile Glu Gln His Ala Ala His Ile

770 775 780

Tyr Thr Arg Glu Ile Phe Leu Lys Val Gln Glu Glu Leu Leu His Ser

785 790 795 800

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

805 810 815

Glu Lys Val Phe Asn Tyr Glu Asn Pro Glu Phe Asp Arg Asn Tyr Phe

820 825 830

Glu Val Leu Ala Glu Pro Gly Ile Asn Ala Phe Lys Cys Gln Cys Ala

835 840 845

Lys Phe Thr Arg Asp Gly Ile Pro Cys Cys His Ile Phe Arg Leu Phe

850 855 860

Thr Gln Phe Gly Ile Asn Glu Ile Pro Glu Gln Tyr Ile Met Pro Arg

865 870 875 880

Trp Thr Lys Lys Phe Arg Glu Glu Gln Leu Lys Gln Asn Lys Glu Lys

885 890 895

Leu Phe Asp Lys His Gly Ile Lys Asp Ser Glu Asn Thr Leu Arg Tyr

900 905 910

Ala Met Leu Met Ser Lys Ala Ala Glu Ile Cys Pro Lys Ile Cys His

915 920 925

Asp Glu Ala Lys Cys Ser Ile Phe Met Gln Glu Leu Asp Lys Ile Gln

930 935 940

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

945 950 955 960

Cys Arg Glu Gln Gly Ala Leu Gln Gln Glu His Arg Gly Ser Ser Ser

965 970 975

Asn Thr Glu Gln Glu Gln Gln His Thr Gly Gly Leu

980 985

<210> 2

<211> 2967

<212> DNA

<213> primer

<400> 2

atggaccaat cactgcagca tactgatgga caaaaccaat cacatgataa tatggaaagc 60

aatgaagtgt caagcgatga tagaaacagt gatggcgatt ctgacagaag cctaggaaat 120

gaatcagata aagggcttgg agcacactat agtcctagtt taatggaatt ggataatgca 180

agaccaccag aacttggaat gaaatttgca acacttgaag atgcacagag attctatgag 240

acacatgctc ttaaaactgg ctttgttgca aagcggggga cgaactacag gagaaaaaag 300

tttaccatag agtgccacaa gactggcaca tcaaagctaa ctccgaaccc acagaggaag 360

aggaagaaaa atattataga gaggactcaa tgccaggcaa aggtggtagt gaagctcaat 420

aagggacaat gggagtatgc agcagtccgg aatgaacaca accatccatt gtgtcctagc 480

tattcgctta gattctcaaa gcgcaaacgc aggcgaaatc ctccaagcca gaaacagctg 540

gatgttcaga gaaatagtga ccaactgaca caggcagata atcttgagga acggttgtcg 600

caacctctta tttcagctga ttcaaatgaa gtaaacaaca aaaggatgcc tagacatgca 660

gagattagta tatctactgt agagaaggaa cctgttgaca aactacagcg agctggtgat 720

tatcaggagc aatcttcaaa tggcatagac aaaaatacag aacgaccaat tgtagataaa 780

ttgggagacc aaacaagctc gatagaccat tcacttcagc atactgaaga gcataacaga 840

tcacatgata atgtggaaag cagtgaagcg ccaagcgagg atacaagcag tgaaagcaat 900

tcagacagca gctcaggtga tgagtcggat aaggagcttg ggaagtactt ctatcctagt 960

ttcgacgaac tgaagaattc aagaccacca gaacccggaa tgaaatttcc atcccttcaa 1020

gccgcacaaa gattctatta cgcacatgct ctccttactg gttttgtagg aaagagggga 1080

accaactaca agagaaagaa gtttcactta gaatgcaaca agagtggcaa aatgaagtta 1140

acgaagtcat ctgaaaaccc aatgaagaag aggagaagca atcttgtaga gaagacacaa 1200

tgcaaggcaa gggtgatagt gaaactcgat aagggagaat gggagttcac agcagttcgc 1260

catgagcaca atcatccgtt atgcccaagc cctttgcttg caagattcat agtagaccac 1320

aaacaaatgt caactggaga gaagtcattt ttaagagttc tgcaacaaaa tagggtacct 1380

cctaagaaaa ttatgaagat tttcaggaaa ctaagagttt gtttcggaga cataccattt 1440

gaaaataaag atgagcacaa catagcacag acagaacata gaaaagcaaa ctcagatgtt 1500

gaaagcgcat tgaagcactt cacagaattg cagattcaaa acccagaatt tctgtacgta 1560

atgcaaaaag atgaggacaa cacagttaca agcatcttct ggactgatgc aagattgagg 1620

atcgagtatg atattttcgg agatctcatt atgtttgatg ctgcctatag cactgatatg 1680

tataacatgc cttttgtgcc tattattgga ataaatagcc acgcgacacc cttcctgtta 1740

ggatgtgctt tgttaaaaga tgagaaagta gaaaccttcg aatggatgtt gcgtacattt 1800

ttgcaagtga tgggaggaaa aatgccaaga gcggtcataa caaaccagga cacatcaatg 1860

gagaaggcat ttgcagaact catgccgcat gtaaggctta ggttttgcaa gcggcatgtt 1920

atgagcaaag ctcaggaaaa gcttggagac ttcatgacag caagaggtaa cataaataca 1980

gatctacata acttagtagg caactcgctg gtagaaacag aatttgaaga aggatgggtt 2040

gagcttattg agagatacaa tgcaagtgaa aaccaacacc tgcaactcat gtggcaaaca 2100

agaaaaagtt gggcacctgt ctattttaga gaagatctct acccatttat tgactcagtt 2160

ggttccaacg agggaataaa ctcattattt aaaggtaata tgcttccaaa ggacacaata 2220

gataagttca ttgggcaatt taaggagata caagagaata tagcaaaagt tcatgaagaa 2280

gatagatttc agtcaggagc tgaccttaaa tacatctcaa tgcaaccaat agaacaacat 2340

gcagcacata tttacacaag ggaaatattt ctgaaagtac aggaagaact actacattct 2400

actgcattca acgtgcagga gatacaaata gggacagtgt acagacttga aaaggtcttc 2460

aactatgaga acccagagtt tgatagaaat tattttgaag tgcttgctga acctggcatc 2520

aatgcattca agtgccaatg tgcaaaattt acaagggatg gaataccttg ctgccacata 2580

ttcagacttt tcactcagtt tggaatcaat gaaataccag agcagtacat aatgcccaga 2640

tggactaaaa aattcagaga ggagcagcta aaacagaaca aggaaaaatt atttgacaag 2700

catggtataa aggattcaga aaatacattg agatatgcaa tgctaatgag taaagcggct 2760

gaaatttgcc cgaaaatttg ccatgatgaa gcaaaatgca gcatattcat gcaggaacta 2820

gacaagattc aagagaagtt gataacggag agcagagaaa atgcacagaa tgataaccca 2880

tgtcgtgaac aaggggcctt gcaacaggag catcgtggga gcagtagcaa tacagagcaa 2940

gagcaacagc atacaggtgg cttgtaa 2967

<210> 3

<211> 36

<212> DNA

<213> primer

<400> 3

taggtctcct gacagaagcc tgttttagag ctagaa 36

<210> 4

<211> 34

<212> DNA

<213> primer

<400> 4

cgggtctcag tcagaatcgc ctgcaccagc cggg 34

<210> 5

<211> 8849

<212> DNA

<213> primer

<400> 5

gcctgatgaa aacaataact gcgttaacct cgcatttctg tactacgccc taagttacat 60

tctctacaat attgttctcc aaaattctaa aagtaggtta cattctccag atcaaactaa 120

actacatctt acaattaaca ctactgtcta tgtctcaatt catacaatta tattctatgc 180

tctggatcta cacatatgtg ctgtgtgcta cagatatgct aaaatatatg gaactaaaac 240

taaaacgcaa catatatact caaacataac atattagtac aaatgcaaaa gatgtatggg 300

agcaaacctg tgatgaagtg agcgaaccag cagggcttcg ccgctcccct tctgctgccc 360

tctcctctct ctctctttcg ttttttttgg atttttagtg aatataatga aatttcagag 420

aggggagact gggctttata gggggaggca aaaatcgccc tcccccaggg cggcaagggg 480

gccgcctgca aaattccatg ccccctcgcc gcccatttgc aggcggcccc ccgcacagta 540

caaaatcgcc ctagcggagg gcggcaaggg ggccgcctgc aaaattccat gccccctcgc 600

cgcccatttg caggcggccc cccgcacagt acaaaatcgc cctagcggag ggcggcaagg 660

gggccgcctg caaatttcgt tgacggccgt cgcccgaaag cgaacggccg tctgccacgt 720

cattttcgcc gccctctggg atggcgattt ttaaaaatcg ccctcccaga gagcggtagg 780

cgactacttc cgtcaatttt caaaatggaa aattattttt gtaaaacttt taataaaaaa 840

aattaaaaat aaaaaaaatt ccagttgctg ctaggtttga tgcggagtgg aaaaatggaa 900

cgacaggatt ggaatcttct ttcattcgcc agctactcac aatgctcttt gcaggcagaa 960

ttggtggcgc tccagttagc tatggaggcg gccaaatttt tgaattttgc aatttgcagg 1020

gattatcttc ctcactgaca acgggacgaa tcactactta aaaaactatt tttccatacg 1080

gtcaaattga tttttccata cggaggtttc ccccgtctag agccatgcat ctggaaaaat 1140

aaatattttc gcgtgcggac gacccaaccg cacgggataa ttatttttct gtgcggtcgg 1200

gttaagtgga ccgcacggga taatcgatta tcacgtgcag tcaaataatg ccgcccgcac 1260

ggaaccaaaa ataccaaaaa taaaaaaaaa atcgaaaatc gccgcccacc cgcatccggc 1320

cgtcgccacc gcaagctcat cggcgacgga tccgcttcta ctacgcccac cgtcaacgga 1380

tccgtctccc ccgagcccgt caacgttgtc gcgagctcgc cgaggtcagc catacgtcga 1440

ggtcgtcgtc atcgaggcgg gcctcccgag gcagcggcga gggcgagcag ccgctgagcc 1500

aagctaccgc cgcgcctctc gaggtagcag cgacgaacgg cggggatggc ttgcggtggt 1560

ggtgggcggt atcgtcgagg ccattgactc tatggatccg cttctttgtc gcctcgcccg 1620

gccgtcgcct cacccggcca ccgcctcgcc cgctcgccgc cgcgcccagc tgtcgccgcg 1680

accagccgcc gcctcgcccg ctcgccgcac cggtctgccg cctagctccg tttgtcgcct 1740

cgccggcctg acagagggga aggggaggag gagaggggga ggaggagtgg aaggtgtgga 1800

gggagttgac tggggaagag ataaggtaga gaaaggaaga gataaagtag agagagaggg 1860

ggtagtttag agataatagg ggataaagta agagagagag gatgtgaaaa ttttgaagtg 1920

gatgggtggg aaggagggag gtattttcct gtgcggtcca cttaaaaagc ttgtacggaa 1980

aaatcgattt tgtcgtgcgg tccacttaaa tcgaccgcat gagataatga tgattatccc 2040

gtgcggtcag attaggaggc ccgcctgaga aaatcgattt tcctgtgcgg gtgacagccc 2100

cgcgccggtc cccttatttt tccgtgctgt tccacttacg aaccgcttgg aaacaaaagg 2160

ggtggcgtac aggaaaatct atcatgtagt agtgaatgca gacacagcca agaagagaaa 2220

ctttggtcct gtttcgttcc caaaactttt ttccaaaaac atcacatcga atctttgaac 2280

acatgcatgg agtattaaat atagataaat ttgaaaacta attacacagt tagtggggaa 2340

atcacgagac gaatcttttg agtctaatta attcatgatt agccgtaagt gctatagtaa 2400

cccacatgta ctaatgacag attaattagg ctcaaaagat tcgtctcgcg gtttccaggc 2460

gagttatgaa attagttttt tcattcatgt ccgaaaatcc cttccgacat ctggtcaaac 2520

gtctgatgtg acacctaaaa attctctttt agcgaactaa acaggcccat ttgatgagga 2580

tccaggccat tggagtcaaa caaatattct agaaggttcc atggaggtca tgtggattcc 2640

aagggagatc aatagggtgg cagacaagct agttaagtaa aatcttatca gccgcccata 2700

cctcttcatg atcgacacaa catatctcac atcacttatc ctaatagaag ttgtcatgca 2760

agaaatctta gatattattt ctctactgca atctatatca tccatcatgt actctgtttc 2820

tgaataacca aagttgttcc tgaatatttt ttttaataaa aaagagctgg atttttaata 2880

cgagcaacaa cctgcacgga tcttgaagcg atcacggccg tccaacctgg cccaagggtt 2940

ggccatcatc attcctaaca ggccgaaata gacccgccgt cagcgaaaac cggagaaaat 3000

atggtttaaa ctattaagca tggtgaacac ttaaggagtt gacatgtctg gtaacgggga 3060

ttaagaacgc catgcatacg gggggtgctg atacgcgatc agccgcgcgc gcggtcagtc 3120

accccccccc cccccagcta ctgtatacat cttatgtata tacgtcttat gtatatacgt 3180

atgtatatac tatatatata tatataatat atatatatat atatatatat atacgaatat 3240

attttaatgc ataaagaata tatacacaca tacacaaagt ttgtatacgt actaaaaaat 3300

aaaaaaaata gaaaatacat aacgtataga aagtttgtat acgcgtatat aaactttgta 3360

tacacgtata aaaagtttgt atacatgtat agaaagtttg tatacgccta tagaaagttt 3420

gtatacacct atagaaagtt tatatgcgtg tataaaaagt ttatatgcgc atatagaaag 3480

tttgtatatg cgtatagaaa gtttgtatgc gcgtatagaa agttctatac gtacgaatac 3540

aaacttggta tatatacgaa tacaaacttt atatttatac atcttaaaaa aagaaaaaac 3600

caaaagaaga taaactggga aaaaacaaac cgaccagaaa aaaccagaga aaaaaatcca 3660

aacagaaaaa taaaaaccgg agaaaaaaac aaaacaaaca gaaacgaaaa aaaaggaaaa 3720

aacgaaaaac tcgctcgcgg cgcgtcgccg ccccctctca tctccagtcg cgcggggagg 3780

gaggcgcgcg actgatcgcc caatagcatt ctcgcatgcg gactcgacgt aaagttgagg 3840

aaaggccggt ggacttattt cggccgcaga tggcaacggc ccacgaagca caagcggccc 3900

ggcccatcca ccgaccaagc cagatgtatg ggcttgtttc tcacgaaaaa aacaaaagaa 3960

gagaaaggaa aaaagaaaaa agaaaaaact ctcctgtccc cccggcagcc gcggccgctg 4020

ttcccttctc gatgccctag catcctcgcc gccgggaacc gcgaacccta gccctctcct 4080

ccgcccttct ccgggaaggg aatcgattca tctccgccct ccctacccgc tgctgcctcc 4140

tcacgcggcg gcgcaatctc cgtcaccggc ggcggcggcg gcggcgggag gtggggtgtc 4200

cacgaacgaa cgccgtgcgg gctatggagt gatgatgcct tcttctcctc ccgacatgta 4260

tgaggtacgg tcgaagacgc gcgcaactcg cgatttccac tctgttctcc gcaacttccc 4320

gtgcgaggta gtagatgctg tgctgtgagg ggggtttttg attgatttcg tctcctccca 4380

aatccaatcc agaaacaggc acttgttcat ggtggagatg atgagctgct ggcacgggcc 4440

ggccctcctc ccatggagga cgtgctgatg cacatccttc cagttgattc cgaacaggtg 4500

agatcgctat cacatatata tagccaatgt atgtaccctc gccattgcat cttttgcaga 4560

gaattgcaga gaattggaat cggttaagtg ctttcggtga aattgacctt tttaagagat 4620

ctgttctcca ggtttttgct cgtgtaaaca atctggggaa tgaaatttct cctgtacaag 4680

cctggtgcca accagagctg cgcggcgaaa aggtttaact tgaacattat ctgtttacct 4740

accgcgttca tccatcagac aatgacatac ataaatttgg caaaatgcag ctataggtag 4800

taacaaagac ttggctaatt gctttgtctc acatacatgt attacagacc agtaacaatg 4860

atgggcaaac ggtgataact ccagtcgagt tgatcaaggt atttagaatc actttgactt 4920

tttagaatca ctttgtgcct ggcattaact gcgaaacgaa atgcagaaac ctgttgagaa 4980

gcaagtgata acagaaggaa gactgcagct cgctggtgat ggctatcagg tatgaaaacc 5040

agacaatgct tatggagagg tttaatatgg acatttgaat atggcagtga atagctacct 5100

taacaagaaa tattcaattg catgcgtgca gctgctatct tcaaatggca tagacaaaac 5160

tgcagaacaa ctatacgtgg atcaattagg gagacaaacc aactcaatgg accaatcact 5220

gcagcatact gatggacaaa accaatcaca tgataatatg gaaagcaatg aagtgtcaag 5280

cgatgataga aacagtgatg gcgattctga cagaagccta ggaaatgaat cagataaagg 5340

gcttggagca cactatagtc ctagtttaat ggaattggat aatgcaagac caccagaact 5400

tggaatgaaa tttgcaacac ttgaagatgc acagagattc tatgagacac atgctcttaa 5460

aactggcttt gttgcaaagc gggggacgaa ctacaggaga aaaaagttta ccatagagtg 5520

ccacaagact ggcacatcaa agctaactcc gaacccacag aggaagagga agaaaaatat 5580

tatagagagg actcaatgcc aggcaaaggt ggtagtgaag ctcaataagg gacaatggga 5640

gtatgcagca gtccggaatg aacacaacca tccattgtgt cctagctatt cgcttagatt 5700

ctcaaagcgc aaacgcaggc gaaatcctcc aagccagaaa cagctggatg ttcagagaaa 5760

tagtgaccaa ctgacacagg cagataatct tgaggaacgg ttgtcgcaac ctcttatttc 5820

agctgattca aatgaagtaa gcttgctaac aaaggttccc cagatatttt tgttttgtat 5880

acatgtgatg gaagtgattt tttaatgatc tgttatcctg gttgtacctc aggtaaacaa 5940

caaaaggatg cctagacatg cagagattag tatatctact gtagagaagg aacctgttga 6000

caaactacag cgagctggtg attatcaggt atgaaaaggt gaaatctaac aagagtacca 6060

ataataaggt atcaaatagt agcttaacgt aagttattct cttgcatgca ggagcaatct 6120

tcaaatggca tagacaaaaa tacagaacga ccaattgtag ataaattggg agaccaaaca 6180

agctcgatag accattcact tcagcatact gaagagcata acagatcaca tgataatgtg 6240

gaaagcagtg aagcgccaag cgaggataca agcagtgaaa gcaattcaga cagcagctca 6300

ggtgatgagt cggataagga gcttgggaag tacttctatc ctagtttcga cgaactgaag 6360

aattcaagac caccagaacc cggaatgaaa tttccatccc ttcaagccgc acaaagattc 6420

tattacgcac atgctctcct tactggtttt gtaggaaaga ggggaaccaa ctacaagaga 6480

aagaagtttc acttagaatg caacaagagt ggcaaaatga agttaacgaa gtcatctgaa 6540

aacccaatga agaagaggag aagcaatctt gtagagaaga cacaatgcaa ggcaagggtg 6600

atagtgaaac tcgataaggg agaatgggag ttcacagcag ttcgccatga gcacaatcat 6660

ccgttatgcc caagcccttt gcttgcaaga ttcatagtag accacaaaca aatgtcaact 6720

ggagagaagt catttttaag agttctgcaa caaaataggg tacctcctaa gaaaattatg 6780

aagattttca ggaaactaag agtttgtttc ggagacatac catttgaaaa taaagatgag 6840

cacaacatag cacagacaga acatagaaaa gcaaactcag atgttgaaag cgcattgaag 6900

cacttcacag aattgcagat tcaaaaccca gaatttctgt acgtaatgca aaaagatgag 6960

gacaacacag ttacaagcat cttctggact gatgcaagat tgaggatcga gtatgatatt 7020

ttcggagatc tcattatgtt tgatgctgcc tatagcactg atatgtataa catgcctttt 7080

gtgcctatta ttggaataaa tagccacgcg acacccttcc tgttaggatg tgctttgtta 7140

aaagatgaga aagtagaaac cttcgaatgg atgttgcgta catttttgca agtgatggga 7200

ggaaaaatgc caagagcggt cataacaaac caggacacat caatggagaa ggcatttgca 7260

gaactcatgc cgcatgtaag gcttaggttt tgcaagcggc atgttatgag caaagctcag 7320

gaaaagcttg gagacttcat gacagcaaga ggtaacataa atacagatct acataactta 7380

gtaggcaact cgctggtaga aacagaattt gaagaaggat gggttgagct tattgagaga 7440

tacaatgcaa gtgaaaacca acacctgcaa ctcatgtggc aaacaagaaa aagttgggca 7500

cctgtctatt ttagagaaga tctctaccca tttattgact cagttggttc caacgaggga 7560

ataaactcat tatttaaagg taatatgctt ccaaaggaca caatagataa gttcattggg 7620

caatttaagg agatacaaga gaatatagca aaagttcatg aagaagatag atttcagtca 7680

ggagctgacc ttaaatacat ctcaatgcaa ccaatagaac aacatgcagc acatatttac 7740

acaagggaaa tatttctgaa agtacaggaa gaactactac attctactgc attcaacgtg 7800

caggagatac aaatagggac agtgtacaga cttgaaaagg tcttcaacta tgagaaccca 7860

gagtttgata gaaattattt tgaagtgctt gctgaacctg gcatcaatgc attcaagtgc 7920

caatgtgcaa aatttacaag ggatggaata ccttgctgcc acatattcag acttttcact 7980

cagtttggaa tcaatgaaat accagagcag tacataatgc ccagatggac taaaaaattc 8040

agagaggagc agctaaaaca gaacaaggaa aaattatttg acaagcatgg tataaaggat 8100

tcagaaaata cattgagata tgcaatgcta atgagtaaag cggctgaaat ttgcccgaaa 8160

atttgccatg atgaagcaaa atgcagcata ttcatgcagg aactagacaa gattcaagag 8220

aagttgataa cggagagcag agaaaatgca cagaatgata acccatgtcg tgaacaaggg 8280

gccttgcaac aggagcatcg tgggagcagt agcaatacag agcaagagca acagcataca 8340

ggtggcttgt aaggcacagt gatgcttggt ccaaaatgtg cagtgggtca gtagagcatt 8400

acgggagtga gccaattgat ctctttgtgc gggttctggt cttctggagg agactatttt 8460

gtccaggaag tagaatggtt agtatagtta gtttagctga agttagttgt ggtaaacttg 8520

tctgttagga aagtgttggt cgagagcagg aagacgtgta aagatgcatt gtatctatta 8580

gctctcctgt ctaaacatgc atcttggttc gcggcagtat gggaagctga tagttaattg 8640

acatctgtgt tcgccaattt tgcaggtatg ccttcttttt ttgtctgcct ttgttggttt 8700

caaaatgatt tagtgtgtat ttgttgcaat gccatttcgt tgaatcgcct gtaatatgtt 8760

tcataggttt tgatgtagta tgttccgtgc gctttggtct tgattgtatg atctttgttt 8820

cttatactgt ttatgggtaa atagggaga 8849

<210> 6

<211> 42

<212> DNA

<213> primer

<400> 6

acatgattac gaattcgcct gatgaaaaca ataactgcgt ta 42

<210> 7

<211> 39

<212> DNA

<213> primer

<400> 7

attcgagctg gtcacctctc cctatttacc cataaacag 39

<210> 8

<211> 2968

<212> DNA

<213> primer

<400> 8

atggaccaat cactgcagca tactgatgga caaaaccaat cacatgataa tatggaaagc 60

aatgaagtgt caagcgatga tagaaacagt gatggcgatt ctgacagaag cctaggaaat 120

gaatcagata aagggcttgg agcacactat agtcctagtt taatggaatt ggataatgca 180

agaccaccag aacttggaat gaaatttgca acacttgaag atgcacagag attctatgag 240

acacatgctc ttaaaactgg ctttgttgca aagcggggga cgaactacag gagaaaaaag 300

tttaccatag agtgccacaa gactggcaca tcaaagctaa ctccgaaccc acagaggaag 360

aggaagaaaa atattataga gaggactcaa tgccaggcaa aggtggtagt gaagctcaat 420

aagggacaat gggagtatgc agcagtccgg aatgaacaca accatccatt gtgtcctagc 480

tattcgctta gattctcaaa gcgcaaacgc aggcgaaatc ctccaagcca gaaacagctg 540

gatgttcaga gaaatagtga ccaactgaca caggcagata atcttgagga acggttgtcg 600

caacctctta tttcagctga ttcaaatgaa gtaaacaaca aaaggatgcc tagacatgca 660

gagattagta tatctactgt agagaaggaa cctgttgaca aactacagcg agctggtgat 720

tatcaggagc aatcttcaaa tggcatagac aaaaatacag aacgaccaat tgtagataaa 780

ttgggagacc aaacaagctc gatagaccat tcacttcagc atactgaaga gcataacaga 840

tcacatgata atgtggaaag cagtgaagcg ccaagcgagg atacaagcag tgaaagcaat 900

tcagacagca gctcaggtga tgagtcggat aaggagcttg ggaagtactt ctatcctagt 960

ttcgacgaac tgaagaattc aagaccacca gaacccggaa tgaaatttcc atcccttcaa 1020

gccgcacaaa gattctatta cgcacatgct ctccttactg gttttgtagg aaagagggga 1080

accaactaca agagaaagaa gtttcactta gaatgcaaca agagtggcaa aatgaagtta 1140

acgaagtcat ctgaaaaccc aatgaagaag aggagaagca atcttgtaga gaagacacaa 1200

tgcaaggcaa gggtgatagt gaaactcgat aagggagaat gggagttcac agcagttcgc 1260

catgagcaca atcatccgtt atgcccaagc cctttgcttg caagattcat agtagaccac 1320

aaacaaatgt caactggaga gaagtcattt ttaagagttc tgcaacaaaa tagggtacct 1380

cctaagaaaa ttatgaagat tttcaggaaa ctaagagttt gtttcggaga cataccattt 1440

gaaaataaag atgagcacaa catagcacag acagaacata gaaaagcaaa ctcagatgtt 1500

gaaagcgcat tgaagcactt cacagaattg cagattcaaa acccagaatt tctgtacgta 1560

atgcaaaaag atgaggacaa cacagttaca agcatcttct ggactgatgc aagattgagg 1620

atcgagtatg atattttcgg agatctcatt atgtttgatg ctgcctatag cactgatatg 1680

tataacatgc cttttgtgcc tattattgga ataaatagcc acgcgacacc cttcctgtta 1740

ggatgtgctt tgttaaaaga tgagaaagta gaaaccttcg aatggatgtt gcgtacattt 1800

ttgcaagtga tgggaggaaa aatgccaaga gcggtcataa caaaccagga cacatcaatg 1860

gagaaggcat ttgcagaact catgccgcat gtaaggctta ggttttgcaa gcggcatgtt 1920

atgagcaaag ctcaggaaaa gcttggagac ttcatgacag caagaggtaa cataaataca 1980

gatctacata acttagtagg caactcgctg gtagaaacag aatttgaaga aggatgggtt 2040

gagcttattg agagatacaa tgcaagtgaa aaccaacacc tgcaactcat gtggcaaaca 2100

agaaaaagtt gggcacctgt ctattttaga gaagatctct acccatttat tgactcagtt 2160

ggttccaacg agggaataaa ctcattattt aaaggtaata tgcttccaaa ggacacaata 2220

gataagttca ttgggcaatt taaggagata caagagaata tagcaaaagt tcatgaagaa 2280

gatagatttc agtcaggagc tgaccttaaa tacatctcaa tgcaaccaat agaacaacat 2340

gcagcacata tttacacaag ggaaatattt ctgaaagtac aggaagaact actacattct 2400

actgcattca acgtgcagga gatacaaata gggacagtgt acagacttga aaaggtcttc 2460

aactatgaga acccagagtt tgatagaaat tattttgaag tgcttgctga acctggcatc 2520

aatgcattca agtgccaatg tgcaaaattt acaagggatg gaataccttg ctgccacata 2580

ttcagacttt tcactcagtt tggaatcaat gaaataccag agcagtacat aatgcccaga 2640

tggactaaaa aaattcagag aggagcagct aaaacagaac aaggaaaaat tatttgacaa 2700

gcatggtata aaggattcag aaaatacatt gagatatgca atgctaatga gtaaagcggc 2760

tgaaatttgc ccgaaaattt gccatgatga agcaaaatgc agcatattca tgcaggaact 2820

agacaagatt caagagaagt tgataacgga gagcagagaa aatgcacaga atgataaccc 2880

atgtcgtgaa caaggggcct tgcaacagga gcatcgtggg agcagtagca atacagagca 2940

agagcaacag catacaggtg gcttgtaa 2968

<210> 9

<211> 898

<212> PRT

<213> primer

<400> 9

Met Asp Gln Ser Leu Gln His Thr Asp Gly Gln Asn Gln Ser His Asp

1 5 10 15

Asn Met Glu Ser Asn Glu Val Ser Ser Asp Asp Arg Asn Ser Asp Gly

20 25 30

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

35 40 45

His Tyr Ser Pro Ser Leu Met Glu Leu Asp Asn Ala Arg Pro Pro Glu

50 55 60

Leu Gly Met Lys Phe Ala Thr Leu Glu Asp Ala Gln Arg Phe Tyr Glu

65 70 75 80

Thr His Ala Leu Lys Thr Gly Phe Val Ala Lys Arg Gly Thr Asn Tyr

85 90 95

Arg Arg Lys Lys Phe Thr Ile Glu Cys His Lys Thr Gly Thr Ser Lys

100 105 110

Leu Thr Pro Asn Pro Gln Arg Lys Arg Lys Lys Asn Ile Ile Glu Arg

115 120 125

Thr Gln Cys Gln Ala Lys Val Val Val Lys Leu Asn Lys Gly Gln Trp

130 135 140

Glu Tyr Ala Ala Val Arg Asn Glu His Asn His Pro Leu Cys Pro Ser

145 150 155 160

Tyr Ser Leu Arg Phe Ser Lys Arg Lys Arg Arg Arg Asn Pro Pro Ser

165 170 175

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

180 185 190

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

195 200 205

Asn Glu Val Asn Asn Lys Arg Met Pro Arg His Ala Glu Ile Ser Ile

210 215 220

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

225 230 235 240

Tyr Gln Glu Gln Ser Ser Asn Gly Ile Asp Lys Asn Thr Glu Arg Pro

245 250 255

Ile Val Asp Lys Leu Gly Asp Gln Thr Ser Ser Ile Asp His Ser Leu

260 265 270

Gln His Thr Glu Glu His Asn Arg Ser His Asp Asn Val Glu Ser Ser

275 280 285

Glu Ala Pro Ser Glu Asp Thr Ser Ser Glu Ser Asn Ser Asp Ser Ser

290 295 300

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

305 310 315 320

Phe Asp Glu Leu Lys Asn Ser Arg Pro Pro Glu Pro Gly Met Lys Phe

325 330 335

Pro Ser Leu Gln Ala Ala Gln Arg Phe Tyr Tyr Ala His Ala Leu Leu

340 345 350

Thr Gly Phe Val Gly Lys Arg Gly Thr Asn Tyr Lys Arg Lys Lys Phe

355 360 365

His Leu Glu Cys Asn Lys Ser Gly Lys Met Lys Leu Thr Lys Ser Ser

370 375 380

Glu Asn Pro Met Lys Lys Arg Arg Ser Asn Leu Val Glu Lys Thr Gln

385 390 395 400

Cys Lys Ala Arg Val Ile Val Lys Leu Asp Lys Gly Glu Trp Glu Phe

405 410 415

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

420 425 430

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

435 440 445

Ser Phe Leu Arg Val Leu Gln Gln Asn Arg Val Pro Pro Lys Lys Ile

450 455 460

Met Lys Ile Phe Arg Lys Leu Arg Val Cys Phe Gly Asp Ile Pro Phe

465 470 475 480

Glu Asn Lys Asp Glu His Asn Ile Ala Gln Thr Glu His Arg Lys Ala

485 490 495

Asn Ser Asp Val Glu Ser Ala Leu Lys His Phe Thr Glu Leu Gln Ile

500 505 510

Gln Asn Pro Glu Phe Leu Tyr Val Met Gln Lys Asp Glu Asp Asn Thr

515 520 525

Val Thr Ser Ile Phe Trp Thr Asp Ala Arg Leu Arg Ile Glu Tyr Asp

530 535 540

Ile Phe Gly Asp Leu Ile Met Phe Asp Ala Ala Tyr Ser Thr Asp Met

545 550 555 560

Tyr Asn Met Pro Phe Val Pro Ile Ile Gly Ile Asn Ser His Ala Thr

565 570 575

Pro Phe Leu Leu Gly Cys Ala Leu Leu Lys Asp Glu Lys Val Glu Thr

580 585 590

Phe Glu Trp Met Leu Arg Thr Phe Leu Gln Val Met Gly Gly Lys Met

595 600 605

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

610 615 620

Ala Glu Leu Met Pro His Val Arg Leu Arg Phe Cys Lys Arg His Val

625 630 635 640

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

645 650 655

Asn Ile Asn Thr Asp Leu His Asn Leu Val Gly Asn Ser Leu Val Glu

660 665 670

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

675 680 685

Ser Glu Asn Gln His Leu Gln Leu Met Trp Gln Thr Arg Lys Ser Trp

690 695 700

Ala Pro Val Tyr Phe Arg Glu Asp Leu Tyr Pro Phe Ile Asp Ser Val

705 710 715 720

Gly Ser Asn Glu Gly Ile Asn Ser Leu Phe Lys Gly Asn Met Leu Pro

725 730 735

Lys Asp Thr Ile Asp Lys Phe Ile Gly Gln Phe Lys Glu Ile Gln Glu

740 745 750

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

755 760 765

Leu Lys Tyr Ile Ser Met Gln Pro Ile Glu Gln His Ala Ala His Ile

770 775 780

Tyr Thr Arg Glu Ile Phe Leu Lys Val Gln Glu Glu Leu Leu His Ser

785 790 795 800

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

805 810 815

Glu Lys Val Phe Asn Tyr Glu Asn Pro Glu Phe Asp Arg Asn Tyr Phe

820 825 830

Glu Val Leu Ala Glu Pro Gly Ile Asn Ala Phe Lys Cys Gln Cys Ala

835 840 845

Lys Phe Thr Arg Asp Gly Ile Pro Cys Cys His Ile Phe Arg Leu Phe

850 855 860

Thr Gln Phe Gly Ile Asn Glu Ile Pro Glu Gln Tyr Ile Met Pro Arg

865 870 875 880

Trp Thr Lys Lys Ile Gln Arg Gly Ala Ala Lys Thr Glu Gln Gly Lys

885 890 895

Ile Ile

<210> 10

<211> 3146

<212> DNA

<213> primer

<400> 10

atggaccaat cactgcagca tactgatgga caaaaccaat cacatgataa tatggaaagc 60

aatgaagtgt caagcgatga tagaaacagt gatggcgatt ctgacagaag cctaggaaat 120

gaatcagata aagggcttgg agcacactat agtcctagtt taatggaatt ggataatgca 180

agaccaccag aacttggaat gaaatttgca acacttgaag atgcacagag attctatgag 240

acacatgctc ttaaaactgg ctttgttgca aagcggggga cgaactacag gagaaaaaag 300

tttaccatag agtgccacaa gactggcaca tcaaagctaa ctccgaaccc acagaggaag 360

aggaagaaaa atattataga gaggactcaa tgccaggcaa aggtggtagt gaagctcaat 420

aagggacaat gggagtatgc agcagtccgg aatgaacaca accatccatt gtgtcctagc 480

tattcgctta gattctcaaa gcgcaaacgc aggcgaaatc ctccaagcca gaaacagctg 540

gatgttcaga gaaatagtga ccaactgaca caggcagata atcttgagga acggttgtcg 600

caacctctta tttcagctga ttcaaatgaa gtaagcttgc taacaaaggt tccccagata 660

tttttgtttt gtatacatgt gatggaagtg attttttaat gatctgttat cctggttgta 720

cctcaggtaa acaacaaaag gatgcctaga catgcagaga ttagtatatc tactgtagag 780

aaggaacctg ttgacaaact acagcgagct ggtgattatc aggtatgaaa aggtgaaatc 840

taacaagagt accaataata aggtatcaaa tagtagctta acgtaagtta ttctcttgca 900

tgcaggagca atcttcaaat ggcatagaca aaaatacaga acgaccaatt gtagataaat 960

tgggagacca aacaagctcg atagaccatt cacttcagca tactgaagag cataacagat 1020

cacatgataa tgtggaaagc agtgaagcgc caagcgagga tacaagcagt gaaagcaatt 1080

cagacagcag ctcaggtgat gagtcggata aggagcttgg gaagtacttc tatcctagtt 1140

tcgacgaact gaagaattca agaccaccag aacccggaat gaaatttcca tcccttcaag 1200

ccgcacaaag attctattac gcacatgctc tccttactgg ttttgtagga aagaggggaa 1260

ccaactacaa gagaaagaag tttcacttag aatgcaacaa gagtggcaaa atgaagttaa 1320

cgaagtcatc tgaaaaccca atgaagaaga ggagaagcaa tcttgtagag aagacacaat 1380

gcaaggcaag ggtgatagtg aaactcgata agggagaatg ggagttcaca gcagttcgcc 1440

atgagcacaa tcatccgtta tgcccaagcc ctttgcttgc aagattcata gtagaccaca 1500

aacaaatgtc aactggagag aagtcatttt taagagttct gcaacaaaat agggtacctc 1560

ctaagaaaat tatgaagatt ttcaggaaac taagagtttg tttcggagac ataccatttg 1620

aaaataaaga tgagcacaac atagcacaga cagaacatag aaaagcaaac tcagatgttg 1680

aaagcgcatt gaagcacttc acagaattgc agattcaaaa cccagaattt ctgtacgtaa 1740

tgcaaaaaga tgaggacaac acagttacaa gcatcttctg gactgatgca agattgagga 1800

tcgagtatga tattttcgga gatctcatta tgtttgatgc tgcctatagc actgatatgt 1860

ataacatgcc ttttgtgcct attattggaa taaatagcca cgcgacaccc ttcctgttag 1920

gatgtgcttt gttaaaagat gagaaagtag aaaccttcga atggatgttg cgtacatttt 1980

tgcaagtgat gggaggaaaa atgccaagag cggtcataac aaaccaggac acatcaatgg 2040

agaaggcatt tgcagaactc atgccgcatg taaggcttag gttttgcaag cggcatgtta 2100

tgagcaaagc tcaggaaaag cttggagact tcatgacagc aagaggtaac ataaatacag 2160

atctacataa cttagtaggc aactcgctgg tagaaacaga atttgaagaa ggatgggttg 2220

agcttattga gagatacaat gcaagtgaaa accaacacct gcaactcatg tggcaaacaa 2280

gaaaaagttg ggcacctgtc tattttagag aagatctcta cccatttatt gactcagttg 2340

gttccaacga gggaataaac tcattattta aaggtaatat gcttccaaag gacacaatag 2400

ataagttcat tgggcaattt aaggagatac aagagaatat agcaaaagtt catgaagaag 2460

atagatttca gtcaggagct gaccttaaat acatctcaat gcaaccaata gaacaacatg 2520

cagcacatat ttacacaagg gaaatatttc tgaaagtaca ggaagaacta ctacattcta 2580

ctgcattcaa cgtgcaggag atacaaatag ggacagtgta cagacttgaa aaggtcttca 2640

actatgagaa cccagagttt gatagaaatt attttgaagt gcttgctgaa cctggcatca 2700

atgcattcaa gtgccaatgt gcaaaattta caagggatgg aataccttgc tgccacatat 2760

tcagactttt cactcagttt ggaatcaatg aaataccaga gcagtacata atgcccagat 2820

ggactaaaaa attcagagag gagcagctaa aacagaacaa ggaaaaatta tttgacaagc 2880

atggtataaa ggattcagaa aatacattga gatatgcaat gctaatgagt aaagcggctg 2940

aaatttgccc gaaaatttgc catgatgaag caaaatgcag catattcatg caggaactag 3000

acaagattca agagaagttg ataacggaga gcagagaaaa tgcacagaat gataacccat 3060

gtcgtgaaca aggggccttg caacaggagc atcgtgggag cagtagcaat acagagcaag 3120

agcaacagca tacaggtggc ttgtaa 3146

<210> 11

<211> 23

<212> DNA

<213> primer

<400> 11

ggcgattctg acagaagcct agg 23

Claims (10)

1. The panicle-related gene OsFRS5 is characterized in that the amino acid sequence coded by the panicle-related gene OsFRS5 is shown as SEQ ID No. 1.

2. The rice panicle-associated gene OsFRS5 as claimed in claim 1, wherein the nucleotide sequence encoding the panicle-associated gene OsFRS5 is shown in SEQ ID No. 2.

3. The use of the panicle associated gene OsFRS5 as claimed in claim 1 or 2, wherein the amino acid sequence encoded by the panicle associated gene OsFRS5 is shown as SEQ ID No. 1;

the application comprises the following steps: knocking out, changing or inhibiting an OsFRS5 gene by a conventional method or based on a CRISPR/Cas9 system, so that the expression level of the OsFRS5 gene in a conventional rice variety is reduced, and a spike variant strain of rice is obtained.

4. A preparation method of a rice panicle type mutant line is characterized by comprising the following steps: selecting a conventional rice variety, processing and cultivating to obtain the rice panicle type mutant strain;

wherein the processing comprises: by adopting a conventional method or based on a CRISPR/Cas9 system, a nucleotide sequence which codes the amino acid shown as SEQ ID No.1 in rice is deleted, mutated or inhibited, so that the expression level of the polypeptide corresponding to the amino acid sequence is reduced or the activity is lost;

the rice variety is japonica rice variety 9522, indica rice 9311 or Guangdong dwarf No. 4.

5. The method for preparing spike-type variant rice strains according to claim 4, wherein the treatment based on the CRISPR/Cas9 system specifically comprises: the OsFRS5 gene is knocked out at fixed points by using a CRISPR/Cas9 system, and the expression of a nucleotide sequence of an amino acid sequence shown as SEQ ID No.1 is inhibited.

6. The method for preparing spike-type variant rice lines as claimed in claim 5, wherein the method for site-directed knockout of OsFRS5 gene by CRISPR/Cas9 system comprises the following steps:

a) synthesizing a single nucleotide sequence, wherein the primers are shown as SEQ ID No.3 and SEQ ID No. 4;

b) forming a dimer structure by the synthesized mononucleotide sequence through annealing reaction, and carrying out connection reaction with a carrier fragment to construct an OsFRS5-RGEB32 plasmid containing a rice OsFRS5 gene target sequence; the target sequence is shown as SEQ ID No. 11;

c) the rice variety is infected by the agrobacterium tumefaciens containing the OsFRS5-RGEB32 plasmid.

7. Use of a panicle variant line of rice obtained by the method of any one of claims 4 to 6 in rice breeding.

8. A method for restoring the panicle phenotype of a rice panicle variant line is characterized by comprising the following steps: transferring the OsFRS5 gene into a rice panicle type variant strain obtained by the preparation method according to any one of claims 4-6 by using a conventional genetic means, and further restoring the wild type phenotype of the mutant.

9. The method for restoring the panicle phenotype of a rice panicle variant line of claim 8, comprising the steps of: transferring Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 complementarily constructed by containing OsFRS5 into the panicle type variation strain of rice, and culturing to obtain the mutant strain; wherein the OsFRS5 is complemented to construct nucleotide with a sequence shown as SEQ ID No. 5.

10. The method for restoring the panicle phenotype of a rice panicle variant line of claim 9, comprising the steps of:

(a) amplifying a genome sequence fragment of the OsFRS5 gene from a rice 9522 genome by using primers with base sequences shown as SEQ ID No.6 and SEQ ID No. 7;

(b) providing an Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 carrying a complementary construction vector for expressing OsFRS 5;

(c) contacting cells, tissues or organs of the panicle type mutant strain of rice with the agrobacterium tumefaciens in the step (b), so that the nucleotide coding the amino acid shown as SEQ ID No.1 is transferred into the callus of the rice and is integrated on the chromosome of the rice cell;

(d) and (3) selecting the rice cells or tissues or organs which are transferred with the nucleotide, and regenerating to obtain rice plants with recovered spike-type characters.

Images