A gene FBX365 related to rice disintegration value and its application

Abstract

The present invention discloses a gene FBX365 related to the disintegration value of rice and its application, and the nucleotide sequence of the coding region of the gene is shown in SEQ ID NO: 3. The gene of the present invention affects the disintegration value in RVA of rice. The gene FBX365 is edited by gene editing technology, and the edited family can significantly improve the disintegration value in RVA of rice. The FBX365 gene can be used as a gene affecting rice quality in rice breeding in the future, and has great development value and application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural bioengineering, and relates to a gene FBX365 related to the disintegration value in rice RVA and its application. The present invention also relates to a polypeptide sequence encoded by the gene, and a method for cultivating a rice variety with a high disintegration value by gene editing technology. Background Art

[0002] Rice is an important food crop in my country and also an important crop to ensure food security. The public's consumption habits have begun to change from eating enough to eating well, and the requirements for rice quality are increasing.

[0003] Improving the quality of rice requires biochemical analysis of the components of rice, and the evaluation method of rice physicochemical indicators is an important method for evaluating the quality of rice. Among them, the RVA spectrum produced by rapid viscosity analysis (RVA) reflects the gelatinization curve with the same characteristics presented by starch homogenate during heating, continuous high temperature and cooling, which can quickly reflect the quality traits of rice. The secondary data breakdown value (Breakdown Value) generated by it is significantly correlated with the taste value, that is, the larger the breakdown value, the better the edible quality and the greater the breeding potential (Zhu et al., 2019; Han and Hamaker, 2001). Therefore, improving the breakdown value of rice is conducive to the cultivation of high-quality rice.

[0004] At present, the research on rice quality focuses on the detection of QTL at the genetic level and the discovery of excellent haplotypes of cloned genes, including the detection of QTL in the offspring population of both parents and the natural variation population, and the discovery of haplotypes of genes such as Wx. Previous researchers detected that the Wx gene had an effect on the RVA spectrum in the QTL of the offspring population of both parents, and different alleles of Wx produced materials with different RVA spectra (Zhang et al., 2019). At the same time, the Wx gene was also edited to obtain materials with different RVA spectra (Zeng et al., 2020). The use of natural variation populations is helpful to analyze different haplotypes of genes, and the phenotypic differences corresponding to haplotypes are used to predict gene functions, such as the OsbZIP18 gene whose promoter variation affects the level of branched-chain amino acids in rice (Sun et al., 2020) and the OsSPL13 whose non-transcribed region variation affects the length of rice grains (Si et al., 2016).

[0005] Although the use of natural variation populations for genetic haplotype analysis has shown obvious advantages in other traits, there are few studies on the disintegration value in the RVA spectrum in natural variation populations. The understanding of genes that affect the disintegration value in natural variation populations is still very limited.

[0006] FBX family genes are a class of genes containing F-box domains, which are mainly involved in the ubiquitin-proteasome pathway, an important biological regulation system. The OsFBK12 gene reported in rice regulates changes in ethylene levels in the body by participating in the ubiquitin-proteasome pathway, regulating leaf senescence and grain size (Chen et al., 2013). OsDRF1 can be a defense-related gene that upregulates expression and significantly enhances plant disease resistance (Cao et al., 2018). However, our understanding of the effects of FBX genes on rice RVA, especially disintegration value, is still very limited. Summary of the invention

[0007] The purpose of the present invention is to provide a rice FBX365 gene related to the rice disintegration value and a protein encoded by the gene and an application of the gene in regulating the rice disintegration value.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] A rice FBX365 gene. The present invention detects the FBX365 gene and promoter variation of a population, finds that the gene is related to the rice disintegration value, and constructs transgenic plants using gene editing, verifying that knocking out the FBX365 gene can increase the rice disintegration value. The gene has the nucleotide sequence described in (1) or (2):

[0010] (1) the coding region nucleotide sequence as shown in SEQ ID NO: 3;

[0011] (2) A nucleotide sequence that has more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 3 and encodes a protein with the same function.

[0012] The gene also has a genomic nucleotide sequence as shown in SEQ ID NO:2.

[0013] The protein encoded by the rice FBX365 gene also falls within the scope of protection of the present invention. The protein has the amino acid residue sequence shown in (a) or (b) below:

[0014] (a) the amino acid sequence shown in SEQ ID NO: 1;

[0015] (b) An amino acid sequence with the same function derived from the amino acid sequence shown in SEQ ID NO: 1 by substitution and / or deletion and / or addition of one or more amino acid residues.

[0016] The expression cassette, recombinant vector, transgenic cell line or transgenic recombinant bacteria containing the above-mentioned rice FBX365 gene, or the substances used for interfering, inhibiting, silencing, targeted knockout or site-directed mutagenesis of the rice FBX365 gene all fall within the protection scope of the present invention.

[0017] Preferably, the substance used to interfere with, inhibit, silence, target knockout or site-directed mutation of the rice FBX365 gene includes at least one of the following (1) to (6):

[0018] (1) Interference sequence targeting rice FBX365 gene;

[0019] (2) Interference vector of rice FBX365 gene used for interference;

[0020] (3) transgenic cell lines containing rice FBX365 gene interference vectors;

[0021] (4) Primer sequences for targeted knockout or site-directed mutation of the rice FBX365 gene based on gene editing technology;

[0022] (5) Vectors for targeted knockout or site-directed mutation of rice FBX365 gene based on gene editing technology;

[0023] (6) Cell lines with targeted knockout or site-directed mutation of the rice FBX365 gene based on gene editing technology.

[0024] The recombinant vector containing the rice FBX365 gene can be a recombinant cloning vector or a recombinant expression vector, and the recombinant expression vector of the gene can be constructed using existing plant expression vectors.

[0025] The plant expression vector includes binary Agrobacterium vector and vectors that can be used for plant microprojectile bombardment. The plant expression vector may also contain the 3' non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as the Agrobacterium crown tumor induction (Ti) plasmid gene (such as rouge synthase N')s gene), the 3' non-translated region of plant genes (such as soybean storage protein gene) has similar functions.

[0026] When the gene is used to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before the transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter and the ubiquitin promoter of corn, which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a plant expression vector, an enhancer can also be used, including a translation enhancer or a transcription enhancer, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive, and can be natural or synthetic. The translation initiation region can come from a transcription initiation region or a structural gene.

[0027] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical agent resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0028] The application of the above rice FBX365 gene, the above protein, or the above expression box, recombinant vector, transgenic cell line or transgenic recombinant bacteria in regulating the disintegration value of rice or cultivating rice with high disintegration value.

[0029] The above-mentioned substances for interfering, inhibiting, silencing, targeted knockout or site-directed mutation of rice FBX365 gene are used in improving the disintegration value of rice or cultivating rice with high disintegration value. For example, the site-directed mutation vector of rice FBX365 gene is used in improving the disintegration value of rice or cultivating rice with high disintegration value.

[0030] The above application is specifically to improve the disintegration value of rice by gene editing, interference, inhibition or silencing the expression of the rice FBX365 gene, and cultivate rice with high disintegration value.

[0031] A method for cultivating rice with a high disintegration value, which includes increasing the disintegration value of rice by gene editing, interference, inhibition or silencing of the rice FBX365 gene, and cultivating rice with a high disintegration value.

[0032] The research results of the present invention show that the gene editing vector is introduced into the recipient rice, so that the rice FBX365 gene is edited to obtain transgenic rice; compared with the recipient rice, the rice disintegration value of the transgenic rice is increased. The recipient rice is the japonica rice variety Zhonghua 11.

[0033] Beneficial effects of the present invention:

[0034] The plant disintegration value gene FBX365 of the present invention affects the disintegration value of rice. Knocking out the gene can increase the disintegration value of rice, thereby cultivating high disintegration value transgenic rice. The protein and its encoding gene can be applied to plant genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The promoter variation of FBX365 gene in different rice varieties.

[0036] Figure 2 Statistical analysis of disintegration values ​​of varieties with different haplotypes.

[0037] Figure 3 Sequencing analysis of the FBX365 gene-edited families showed that CR2 and CR3 were homozygous knockout families.

[0038] Figure 4 Protein translation analysis after FBX365 gene editing in CR2 and CR3 families.

[0039] Figure 5 Statistical analysis of the disintegration values ​​of the recipient rice, CR2, and CR3 lines. DETAILED DESCRIPTION

[0040] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0041] Embodiment 1,

[0042] Through evolutionary analysis of a large number of rice varieties, the results showed that the protein-coding gene FBX365 (Os10g0139300) containing the F-box domain has different haplotypes in different varieties (Figure 1). The results of sequence analysis showed that there were two single nucleotide variations (SNP1, SNP2) and an insertion-deletion variation (INDEL1) in the promoter region (2Kb before ATG) of Os10g0139300, such as Figure 1 shown.

[0043] The present invention detects the FBX365 gene and promoter variation of a population and finds that the gene is related to the rice disintegration value. The gene has a coding region sequence as shown in SEQ ID NO:3, and the genome sequence of the gene is shown in SEQ ID NO:2.

[0044] Example 2: RVA spectrum determination and disintegration value calculation of different varieties

[0045] The seeds of different varieties were harvested after maturity, dried and placed at room temperature for 3 months to balance the moisture. The seeds were husked with a brown rice machine, refined with a rice polisher, and finally ground into rice flour with a sample grinder. The RVA spectrum of rice flour was measured using a RAV-4 rapid viscosity analyzer produced by Newport, Australia, according to its operating procedures. The results showed that there were differences in the disintegration values ​​of varieties with different haplotypes, such as Figure 2 shown.

[0046] Example 3. Obtaining FBX365 gene-edited transgenic strain and determining its disintegration value

[0047] First, find the appropriate spacer sequence (as shown in nucleotides 317-336 in SEQ ID NO: 3) through the website CRISR-P (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR), and synthesize two complementary primers with adapters (front primer: TGTTGGCGGCTCCGACGGCTGTCCC, rear primer: AAACGGGACAGCCGTCGGAGCCGCCA). After slow annealing in the PCR instrument, a double-stranded oligo was obtained, and the double-stranded oligo was connected to the BGK03 vector digested with BsaI. The constructed vector was transformed into recipient rice callus tissue by Agrobacterium EHA105 to obtain T0 generation gene-edited transgenic plants. A total of 10 knockout plants with edited FBX365 genes were identified by first-generation sequencing, of which CR2 and CR3 were homozygous mutant plants ( Figure 3 ). The FBX365 gene of CR2 lacks a base A at the 320th position after ATG, resulting in a frameshift mutation of 107 amino acids and premature termination of translation (184 amino acids shorter than the wild type). The FBX365 gene of CR3 lacks two bases AC at 320-321bp after ATG, resulting in a frameshift mutation of 107 amino acids and premature termination of translation (303 amino acids shorter than the wild type) ( Figure 4 The disintegration values ​​of the above transgenic knockout lines were measured, and the results showed that the rice disintegration values ​​of the knockout plants CR2 and CR3 were significantly higher than those of the recipient rice ( Figure 5 ).

[0048] The above results show that FBX365 is a gene related to the disintegration value of rice. Rice with high disintegration value can be obtained through gene editing.

[0049] SEQ ID NO:1

[0050] MGAAISCCGCPPRRRAGAADDAAAGLTDDILAEIFLRLPPHPACLRRVSLVSRRFRRVVTSRRFLRRFSDLHGGAPGAPL

[0051] VGFFSNHNHGPWADTRFIPVGVDGTGDSRRSRCRSRRATAARNPGGVLALGDDAEWHVIGCRGGRVLLLSPTRLRLLVLE

[0052] PMLGRRQYIPAPPAPEYRPAYFSNAAVVSAAGGHDELRLRPHLFRVVFVSSNAATKRSTAFVYNSATFRWTKAAATEMSS

[0053] VIDGRPSVLIGQTLYWHLISHGLVAFNLETHELHEILVPADAFDDVHDANLSIVVPRSGGGGVGLAAVSGYILQLWTLRD

[0054] YTHGASTWDLRNIVVLDALLPLRNARLPPPPQLPASAKPMPLVWLMALDEDENVGYVWTAAGVFAVQLDTMNYHKVLGPV

[0055] CRGMQFVFPYKSFFLPQGGSAAMIM

[0056] SEQ ID NO:2

[0057] AGTGTGAGCAGCAGCTTCATCACACCGAGCTCGCAAAAAACACAATCAATCGATCCCCAATTCGATCCCCATGGGCGCCG

[0058] CGATCAGCTGCTGTGGCTGTCCTCCTCGCCGCCGTGCAGGCGCGGCCGACGACGCGGCGGCGGGACTCACCGACGACATC

[0059] CTCGCCGAGATCTTCCTCCGGCTGCCGCCGCACCCGGCCTGCCTCCGCCGCGTCTCCCTCGTCAGCCGCCGGTTCCGCCG

[0060] CGTGGTCACCAGCCGCCGCTTCCTCCGCCGGTTCAGCGACCTCCACGGCGGCGCGCCGGGGGCTCCCCTCGTCGGCTTCT

[0061] TCAGCAACCACAACCATGGCCCCTGGGCGGACACCCGCTTCATCCCTGTCGGCGTCGACGGCACCGGGGACAGCCGTCGG

[0062] AGCCGCTGCCGCAGCCGCCGCGCCACCGCGGCGCGTAATCCCGGCGGGGTGCTCGCCCTGGGCGACGACGCCGAGTGGCA

[0063] CGTCATCGGCTGCCGCGGCGGGCGCGTCCTCCTCCTCAGCCCCACGCGGCTCCGCCTCCTGGTGCTGGAGCCCATGCTCG

[0064] GCCGCCGCCAGTACATCCCGGCGCCGCCGGCGCCGGAGTACAGGCCGGCATACTTCAGCAACGCCGCCGTGGTCTCCGCC

[0065] GCGGGGGGCCACGACGAGCTCCGCCTCCGGCCGCACCTGTTCCGCGTGGTGTTCGTCTCGAGCAACGCCGCCACCAAGCG

[0066] CTCCACCGCCTTCGTCTACAACTCGGCGACGTTCCGGTGGACCAAGGCGGCGGCGACAGAGATGTCGTCGGTGATCGACG

[0067] GCCGGCCGAGCGTCCTGATCGGCCAGACCCTCTACTGGCACCTCATCTCGCACGGCCTCGTCGCGTTCAACCTGGAGACG

[0068] CACGAGCTGCACGAGATCCTGGTGCCGGCCGACGCGTTCGACGACGTGCACGATGCCAACCTGAGCATCGTCGTCCCCAG

[0069] GAGCGGCGGCGGCGGAGTCGGCCTCGCCGCGGTGTCCGGGTACATCCTCCAGCTCTGGACGCTGCGCGACTACACCCACG

[0070] GCGCGTCCACCTGGGACCTGCGCAACATCGTGGTGCTGGACGCGCTGCTCCCCCTGCGCAACGCTCGGCTGCCGCCGCCG

[0071] CCGCAGCTGCCGGCGTCGGCGAAGCCGATGCCTCTGGTGTGGTTGATGGCGCTGGACGAGGATGAGAACGTGGGGTACGT

[0072] GTGGACCGCGGCTGGGGTGTTCGCTGTTCAGCTTGACACCATGAATTACCACAAGGTGCTTGGACCTGTTTGTAGGGGGA

[0073] TGCAATTCGTTTTTCCCTACAAAAGCTTCTTTCTCCCTCAAGGTACATAACATATATACTCCTGATCATAGCTTTACTTA

[0074] ATTTTGGGCACTAAATTTTGAAAAGTTCAATGAACCTAAATTATGCTCTTGGTTGAAAATAATTGTTGTTTTGAACTTGG

[0075] CTGATATATACCAAACTTTAAAAAATTTGACTATAAATCAATTAATTATGAGTTACGACACGCTGATAATTCACTAGTCA

[0076] GAATTAATGATCAACCCGTACTACGTATAAACAGGACATACATTGTAACAATTGTTTGTTTTTATAAAAAATCTTCTCCT

[0077] TTTGTAATTGTTAGTTACATATGTGCAAATCTTGGTCATATAAGGACTTATTAGTTGGAATTTGACATGAAAATTTCTGT

[0078] TTGATAAACTCAATGAACCTAAACCATGTGTTCTTATTTGTACCTTATTAAATACGGATGGCGCTGCAAAGTATTTTTGC

[0079] AAATGCTTATCGTTTGTTATTGGAAACAGTAACATTCAATATTAATTCTCACGCATGTGCTTACACTGTTTTTGTTTTTA

[0080] AGCAGACTATATATTGACGAAAAGTTTAATTAACTGTGTGTTTTGTGCTTTTCCACATTTACTTATAGTCTATTATCACT

[0081] TTCTTAACTCTAACTGCAAGTTTTTGTTTTGGTTTGTTATCATGGTTTTCCATTATTATAGGTGTAAATATATATATATT

[0082] AATTAAACATATTTCAGAGCATATCTAAATTTTTCAGGTGGAAGTGCAGCCATGATTATGTAATTCTACTCCTTCCTGGG

[0083] AAAAGTAAGTTGATTTATCCAACTGCATGAACATAAGATCGAGATAACTAATTAGTGTTTAATAATATTTTGACAGAAAA

[0084] TGACAATATATATGCATTTGTATATCTTGCTTGAAAAATAATAAGTTTATAATATTCTAACTTAGTTTCTTTTTCAACTG

[0085] CATTTAGAAAGAACAGTAGGATAGTAGCTCAATATGCATTTTGAAATAGTATATGGTTGTCCATATTCCACACTTCATCT

[0086] TTCTTTTTGTGACTGAAGGGAGTAGCACGTTTTCACCATGCTCTTTGGACAGATATAATTAATTCAAACAGATAAGTACA

[0087] ATATAAATGAAGGCTGTATGTTGTGTTTTTACATATTACAAATTTCTGTAATCCATAGTGCCTATGTTGGATTTATAAAAA

[0088] GGTGAACCACCTGAATCCTAAAGCAATGTTTTGCTAGTAGTAGGTTTTCATGGTTTAAAATTAGGCTCTTGATGCAGTCC

[0089] CAAAATACAATTGTTTATGAAAACAAATGTAAGACTTGGATCGGAAATCTATATCTATGCAAAATTGCATCGGAAA

[0090] TCTGCTATATCTAGCTAGTTTGTTACTATAGGCACAAAAGATATGATATAAAAGTTATACTACTGCGTACATATGATCGA

[0091] TATTGATGGTACACCATACAACCCATTTATATCCACGCCATTGTTTGATTATTTTTTTCCTGTTTATGTTTTGTTCTT

[0092] CATGTCTGTTGTATTTGATCTATATATGTATATATGTATATATATATATGTATATATATATATGTGTGTGTATATATACA

[0093] TATATACATATATGTATATGTATATATGTATATGTATATATTATATTATTTGTGTATATATATT

[0094] ATATATATATATATATATATATATATATATATATATATATATATATATATATACCTTTCACTGTA

[0095] ATGGAGTTGTATGCATATAATGTGTTTATTGTAGCTATCCGTCCATGTAATCTAAAACAAATTAAGCTGATTCTAAGTA

[0096] ACAGTAATGCAGGTGCTATATATGAGTGGCCTTAAAATTTTCATATAAATTAAATAATTAATTAAATTCTTGTTGTTCTAAAGATCAACT

[0097] GATATAGTTGTGTCCAAGTGATACAAACGGCTTATTTGAATGCCATCTTTTTTTTGGCGTTATGATGATCCAC

[0098] AAACGTATGTTTTGCCATTTTGAATCGTTAATATATATATTTTTTTCACGTAGATGAGAGTTTATTATT

[0099] GTCTTACGGATGTGACATATATATATTGATAAGTGCAAGACATTAGTTGTCCCCTCCATTACATAAATTACACTGGATG

[0100] TTCGAGATCTTTTTCTTTCTACATAAGTATAGTTTCATTTTTTTTCGTAAACTCATGATTTCTATACTCCATTTAC

[0101] TTGATGATATATAAGATGTATATTCTTTAGCATATAATCAATCGGAATGGCAACAATGGAGCTATATATACATAAC

[0102] CAAATCCTAGCTATTTCCTTCCTGCTGTGGATAGTTTGTGACAATCATGTTGGAAGGAAGTAATTAGAATCACATATAG

[0103] AAAAAAATATAACTTCTCTAGTATTGTCAGAAATATTGCCTTTTGTTTCCTATACATTTTGTTTTTGT

[0104] CCCTTAATTATGCATTGGTGAAGCTTTTGGATCAGAATCTTAAAACCTGAAAACTAGCAGTACACAGTTATAACTAGA

[0105] ACAGCACGGTGGTCACTTTTAGATATTTTTATTTTAGTCATTCTTTCTCAGTTGTGGTTATGCATTGGCACACATTTGGA

[0106] TTCGTTTAAAGTGCGATAACTCTGTGAACTTATACATTTTAAATATTCTTCTTTCTATAATTTTTTAAATGTTATTATTA

[0107] TTGTTTATTAGCTGTAATTGTTGTCTCAGCTATTATGTTGGTGGAATAATAGTTTTTATTATTATCTCCAAATAATGAGT

[0108] ATGTTACTACTAAGGGTTGACATAACCTCATTGCATGTAGCAACATTGTTCGCTCTATACTATATCCTTATGATTTTGAA

[0109] AATATACATCCATGGCTTAATTCTGATGAATTCTATGTAATAAGTGAACATAAAAGTCATATGTTACATGCATGCTTGCA

[0110] TGATCATAAATACTAATTAATCAAGCCCTTAATCTCTTCATGTTCTGTTAATGACAGGTGGCAATGCAAGGTGATGACG

[0111] TGAGAATAACCGAGATTTTGCTCATTAAGCTAATATATAATAGGCTGGCTGAAAACTCTCGGAATTAGATGATTTATTTA

[0112] AAGTAATTTGTTGTTGTTTGATGAGACCTTAATTAATTATAGTAAGATGCACTTTTCTGTAATTAACATGTGTGCTTCGG

[0113] TATTTGCTTCTGC

[0114] SEQ ID NO:3

[0115] ATGGGCGCCGCGATCAGCTGCTGTGGCTGTCCTCCTCGCCGCCGTGCAGGCGCGGCCGACGACGCGGCGGCGGGACTCAC

[0116] CGACGACATCCTCGCCGAGATCTTCCTCCGGCTGCCGCCGCACCCGGCCTGCCTCCGCCGCGTCTCCCTCGTCAGCCGCC

[0117] GGTTCCGCCGCGTGGTCACCAGCCGCCGCTTCCTCCGCCGGTTCAGCGACCTCCACGGCGGCGCGCCGGGGGCTCCCCTC

[0118] GTCGGCTTCTTCAGCAACCACAACCATGGCCCCTGGGCGGACACCCGCTTCATCCCTGTCGGCGTCGACGGCACCGGGGA

[0119] CAGCCGTCGGAGCCGCTGCCGCAGCCGCCGCGCCACCGCGGCGCGTAATCCCGGCGGGGTGCTCGCCCTGGGCGACGACG

[0120] CCGAGTGGCACGTCATCGGCTGCCGCGGCGGGCGCGTCCTCCTCCTCAGCCCCACGCGGCTCCGCCTCCTGGTGCTGGAG

[0121] CCCATGCTCGGCCGCCGCCAGTACATCCCGGCGCCGCCGGCGCCGGAGTACAGGCCGGCATACTTCAGCAACGCCGCCGT

[0122] GGTCTCCGCCGCGGGGGGCCACGACGAGCTCCGCCTCCGGCCGCACCTGTTCCGCGTGGTGTTCGTCTCGAGCAACGCCG

[0123] CCACCAAGCGCTCCACCGCCTTCGTCTACAACTCGGCGACGTTCCGGTGGACCAAGGCGGCGGCGACAGAGATGTCGTCG

[0124] GTGATCGACGGCCGGCCGAGCGTCCTGATCGGCCAGACCCTCTACTGGCACCTCATCTCGCACGGCCTCGTCGCGTTCAA

[0125] CCTGGAGACGCACGAGCTGCACGAGATCCTGGTGCCGGCCGACGCGTTCGACGACGTGCACGATGCCAACCTGAGCATCG

[0126] TCGTCCCCAGGAGCGGCGGCGGCGGAGTCGGCCTCGCCGCGGTGTCCGGGTACATCCTCCAGCTCTGGACGCTGCGCGAC

[0127] TACACCCACGGCGCGTCCACCTGGGACCTGCGCAACATCGTGGTGCTGGACGCGCTGCTCCCCCTGCGCAACGCTCGGCT

[0128] GCCGCCGCCGCCGCAGCTGCCGGCGTCGGCGAAGCCGATGCCTCTGGTGTGGTTGATGGCGCTGGACGAGGATGAGAACG

[0129] TGGGGTACGTGTGGACCGCGGCTGGGGTGTTCGCTGTTCAGCTTGACACCATGAATTACCACAAGGTGCTTGGACCTGTT

[0130] TGTAGGGGGATGCAATTCGTTTTTCCCTACAAAAGCTTCTTTCTCCCTCAAGGTGGAAGTGCAGCCATGATTATGTAA。 Sequence Listing <110> Jiangsu Academy of Agricultural Sciences <120> Gene FBX365 Related to Rice Disintegration Value and Its Application <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 425 <212> PRT <213> Oryza sativa L. <400> 1 Met Gly Ala Ala Ile Ser Cys Cys Gly Cys Pro Pro Arg Arg Arg Ala 1 5 10 15 Gly Ala Ala Asp Asp Ala Ala Ala Gly Leu Thr Asp Asp Ile Leu Ala 20 25 30 Glu Ile Phe Leu Arg Leu Pro Pro His Pro Ala Cys Leu Arg Arg Val 35 40 45 Ser Leu Val Ser Arg Arg Phe Arg Arg Val Val Thr Ser Arg Arg Phe 50 55 60 Leu Arg Arg Phe Ser Asp Leu His Gly Gly Ala Pro Gly Ala Pro Leu 65 70 75 80 Val Gly Phe Phe Ser Asn His Asn His Gly Pro Trp Ala Asp Thr Arg 85 90 95 Phe Ile Pro Val Gly Val Asp Gly Thr Gly Asp Ser Arg Arg Ser Arg 100 105 110 Cys Arg Ser Arg Arg Ala Thr Ala Ala Arg Asn Pro Gly Gly Val Leu 115 120 125 Ala Leu Gly Asp Asp Ala Glu Trp His Val Ile Gly Cys Arg Gly Gly 130 135 140 Arg Val Leu Leu Leu Ser Pro Thr Arg Leu Arg Leu Leu Val Leu Glu 145 150 155 160 Pro Met Leu Gly Arg Arg Gln Tyr Ile Pro Ala Pro Pro Ala Pro Glu 165 170 175 Tyr Arg Pro Ala Tyr Phe Ser Asn Ala Ala Val Val Ser Ala Ala Gly 180 185 190 Gly His Asp Glu Leu Arg Leu Arg Pro His Leu Phe Arg Val Val Phe 195 200 205 Val Ser Ser Asn Ala Ala Thr Lys Arg Ser Thr Ala Phe Val Tyr Asn 210 215 220 Ser Ala Thr Phe Arg Trp Thr Lys Ala Ala Ala Thr Glu Met Ser Ser 225 230 235 240 Val Ile Asp Gly Arg Pro Ser Val Leu Ile Gly Gln Thr Leu Tyr Trp 245 250 255 His Leu Ile Ser His Gly Leu Val Ala Phe Asn Leu Glu Thr His Glu 260 265 270 Leu His Glu Ile Leu Val Pro Ala Asp Ala Phe Asp Asp Val His Asp 275 280 285 Ala Asn Leu Ser Ile Val Val Pro Arg Ser Gly Gly Gly Gly Val Gly 290 295 300 Leu Ala Ala Val Ser Gly Tyr Ile Leu Gln Leu Trp Thr Leu Arg Asp 305 310 315 320 Tyr Thr His Gly Ala Ser Thr Trp Asp Leu Arg Asn Ile Val Val Leu 325 330 335 Asp Ala Leu Leu Pro Leu Arg Asn Ala Arg Leu Pro Pro Pro Pro Gln 340 345 350 Leu Pro Ala Ser Ala Lys Pro Met Pro Leu Val Trp Leu Met Ala Leu 355 360 365 Asp Glu Asp Glu Asn Val Gly Tyr Val Trp Thr Ala Ala Gly Val Phe 370 375 380 Ala Val Gln Leu Asp Thr Met Asn Tyr His Lys Val Leu Gly Pro Val 385 390 395 400 Cys Arg Gly Met Gln Phe Val Phe Pro Tyr Lys Ser Phe Phe Leu Pro 405 410 415 Gln Gly Gly Ser Ala Ala Met Ile Met 420 425 <210> 2 <211> 4493 <212> DNA <213> Rice (Oryza sativa L.) <400> 2 agtgtgagca gcagcttcat cacaccgagc tcgcaaaaaa cacaatcaat cgatccccaa 60 ttcgatcccc atgggcgccg cgatcagctg ctgtggctgt cctcctcgcc gccgtgcagg 120 cgcggccgac gacgcggcgg cgggactcac cgacgacatc ctcgccgaga tcttcctccg 180 gctgccgccg cacccggcct gcctccgccg cgtctccctc gtcagccgcc ggttccgccg 240 cgtggtcacc agccgccgct tcctccgccg gttcagcgac ctccacggcg gcgcgccggg 300 ggctcccctc gtcggcttct tcagcaacca caaccatggc ccctgggcgg acacccgctt 360 catccctgtc ggcgtcgacg gcaccgggga cagccgtcgg agccgctgcc gcagccgccg 420 cgccaccgcg gcgcgtaatc ccggcggggt gctcgccctg ggcgacgacg ccgagtggca 480 cgtcatcggc tgccgcggcg ggcgcgtcct cctcctcagc cccacgcggc tccgcctcct 540 ggtgctggag cccatgctcg gccgccgcca gtacatcccg gcgccgccgg cgccggagta 600 caggccggca tacttcagca acgccgccgt ggtctccgcc gcggggggcc acgacgagct 660 ccgcctccgg ccgcacctgt tccgcgtggt gttcgtctcg agcaacgccg ccaccaagcg 720 ctccaccgcc ttcgtctaca actcggcgac gttccggtgg accaaggcgg cggcgacaga 780 gatgtcgtcg gtgatcgacg gccggccgag cgtcctgatc ggccagaccc tctactggca 840 cctcatctcg cacggcctcg tcgcgttcaa cctggagacg cacgagctgc acgagatcct 900 ggtgccggcc gacgcgttcg acgacgtgca cgatgccaac ctgagcatcg tcgtccccag 960 gagcggcggc ggcggagtcg gcctcgccgc ggtgtccggg tacatcctcc agctctggac 1020 gctgcgcgac tacacccacg gcgcgtccac ctgggacctg cgcaacatcg tggtgctgga 1080 cgcgctgctc cccctgcgca acgctcggct gccgccgccg ccgcagctgc cggcgtcggc 1140 gaagccgatg cctctggtgt ggtgatggc gctggacgag gatgagaacg tggggtacgt 1200 gtggaccgcg gctggggtgt tcgctgttca gcttgacacc atgaattacc acaaggtgct 1260 tggacctgtt tgtaggggga tgcaattcgt ttttccctac aaaagcttct ttctccctca 1320 aggtacataa katatatact cctgatcata gctttactta attttgggca ctaaattttg 1380 aaaagttcaa tgaacctaaa ttatgctctt ggttgaaaat aattgttgtt ttgaacttgg 1440 ctgatatata ccaaacttta aaaaatttga ctataaatca attaattatg agttacgaca 1500 cgctgataat tcactagtca gaattaatga tcaacccgta ctacgtataa acaggacata 1560 cattgtaaca attgtttgtt tttataaaaa atcttctcct tttgtaattg ttagttacat 1620 atgtgcaaat cttggtcata taaggactta ttagttggaa tttgacatga aaatttctgt 1680 ttgataaact caatgaacct aaaccatgtg ttcttatttg taccttatta aatacggatg 1740 gcgctgcaaa gtatttttgc aaatgcttat cgtttgttat tggaaacagt aacattcaat 1800 attaattctc acgcatgtgc ttacactgtt tttgttttta agcagactat atattgacga 1860 aaagtttaat taactgtgtg ttttgtgctt ttccacattt acttatagtc tattatcact 1920 ttcttaactc taactgcaag tttttgtttt ggtttgttat catggttttc cattattata 1980 ggtgtaaata tatatatatt aatgaaacat atttcagagc atatctaaat ttttcaggtg 2040 gaagtgcagc catgattatg taattctact ccttcctggg aaaagtaagt tgattatcc 2100 aactgcatga acataagatc gagataacta attagtgttt aataatattt tgacagaaaa 2160 tgacaatata tatgcatttg tatatcttgc ttgaaaaata ataagtttat aatattctaa 2220 cttagtttct ttttcaactg catttagaaa gaacagtagg atagtagctc aatatgcatt 2280 ttgaaatagt atatggttgt ccatattcca cactcatct ttctttgt gactgaaggg 2340 agtagcacgt ttcaccatg ctctttggac agataatt attcaaca gataagtaca 2400 atataaatga aggctgtatg ttgtgttttt acatattaca aatttctgta atccatagtg 2460 cctatgttgg atttataaaa ggtgaaccac ctgaatccta aagcaatgtt ttgctagtag 2520 taggttttca tggtttaaaa ttaggcttt gatgcagtcc caaataca ttgtgtttat 2580 gaaaacaat gtaagacttg gatcggaaaa tctatatcta tgcaaattg catcggaaaa 2640 tctgctatat ctagctagtt tgttactata ggcacaaag atatgatata aaagttatac 2700 tactgcgtac atatgatcga tattgatggt acaccataca acccatttat atccacgcca 2760 ttgtgtttga ttatttttt cctgtttatg tttgttctt catgtctgtt gtatttgatc 2820 father father father father father father father father father 2880 tatatacata tatgtatatg tatatatata tatgtgtata tatgtgtata 2940 grandfather grandfather grandfather grandfather great-grandfather great-grandfather 3000 father father father father fathercc ttcactgta atggagttgt atgcatataa 3060 tgtgtttatt gtagctatcc gtccatgtaa tctaaaacaa attaagctgt attctaagta 3120 acagtaatgc aggtgctata tatgaagtgg ccttaaaattt ttcatacaa cattaattct 3180 tgttgttcta aagatcaact gatatagttg tgtccaagtg atacaaac ggcttatttg 3240 aaatgccatc ttttatttt ggcgttatga tatgatccac aaacgtatgt tttgccattt 3300 tgaatatcgt tatatatat tatatatttt ttcacgtaga tgagtagt tattatatatt gtcttacgga tgtgacatat atattattga taagtgcaag acattagttg tcccctccat 3420 tacataaatt acatagtg ttcgagatct ttttctttct acatagtta atagttttcat 3480 cttttctttc gtaaactcat gatttctata ctccatttac ttgatgatat ataaagtatg 3540 tatattcttt agcatatat caatcggaaa tgcacaca atggagctat atacaatac 3600 caaatcctag ctatttcctt cctgctgtgg ataagtttgt gatacatg ttggaaggaa 3660 gtaattagaa tcacatatag aaaaaaataa taatactctct ctagtaaata ttgtcagaaa 3720 tatattgcct tttgtttc ctataacatt ttgtttttgt cccttaatta tgcattggtg 3780 aagcttttgg atcagacaat cttaaaacct gaaaactagc agtatacagt tataactaga 3840 acagcacggt ggtcactttt agatattttt atttagtca ttctttctca gttgtggtta 3900 tgcattggca cacatttgga ttcgtttaaa gtgcgataac tctgtgaact tatacatttt 3960 aatattctt cttctataa ttttttaaat gttattatta ttgtttatta gctgtaattg 4020 ttgtctcagc tattatgttg gtggaataat agtttttatt attatctcca aataatgagt 4080 atgttactac taagggttga cataacctca ttgcatgtag caacattgtt cgctctatac 4140 tatatcctta tgatttgaa aatacatc catggcttaa ttctgatgaa ttctatgtaa 4200 taagtgaaca taaaagtcat atgttacatg catgcttgca tgatcataaa tactaattaa 4260 tcaagccctt aatctcttca tgttctgtta atgacaggtg gcaatggcaa ggtgatgacg 4320 tgagaataac cgagattttg ccattaagc tatatataa taggctggct gaaaactctc 4380 ggaattagat gatttattta aagtaatttg ttgttgtttg atgagacctt aattaattat 4440 agtaagatgc acttttctgt aattaacatg tgtgcttcgg tatttgcttc tgc 4493 <210> 3 <211> 1278 <212> DNA <213> Oryza sativa L. (rice) <400> 3 atgggcgccg cgatcagctg ctgtggctgt cctcctcgcc gccgtgcagg cgcggccgac 60 gacgcggcgg cgggactcac cgacgacatc ctcgccgaga tcttcctccg gctgccgccg 120 cacccggcct gcctccgccg cgtctccctc gtcagccgcc ggttccgccg cgtggtcacc 180 agccgccgct tcctccgccg gttcagcgac ctccacggcg gcgcgccggg ggctcccctc 240 gtcggcttct tcagcaacca caaccatggc ccctgggcgg acacccgctt catccctgtc 300 ggcgtcgacg gcaccgggga cagccgtcgg agccgctgcc gcagccgccg cgccaccgcg 360 gcgcgtaatc ccggcggggt gctcgccctg ggcgacgacg ccgagtggca cgtcatcggc 420 tgccgcggcg ggcgcgtcct cctcctcagc cccacgcggc tccgcctcct ggtgctggag 480 cccatgctcg gccgccgcca gtacatcccg gcgccgccgg cgccggagta caggccggca 540 tacttcagca acgccgccgt ggtctccgcc gcggggggcc acgacgagct ccgcctccgg 600 ccgcacctgt tccgcgtggt gttcgtctcg agcaacgccg ccaccaagcg ctccaccgcc 660 ttcgtctaca actcggcgac gttccggtgg accaaggcgg cggcgacaga gatgtcgtcg 720 gtgatcgacg gccggccgag cgtcctgatc ggccagaccc tctactggca cctcatctcg 780 cacggcctcg tcgcgttcaa cctggagacg cacgagctgc acgagatcct ggtgccggcc 840 gacgcgttcg acgacgtgca cgatgccaac ctgagcatcg tcgtccccag gagcggcggc 900 ggcggagtcg gcctcgccgc ggtgtccggg tacatcctcc agctctggac gctgcgcgac 960 tacacccacg gcgcgtccac ctgggacctg cgcaacatcg tggtgctgga cgcgctgctc 1020 cccctgcgca acgctcggct gccgccgccg ccgcagctgc cggcgtcggc gaagccgatg 1080 cctctggtgt ggttgatggc gctggacgag gatgagaacg tggggtacgt gtggaccgcg 1140 gctggggtgt tcgctgttca gcttgacacc atgaattacc acaaggtgct tggacctgtt 1200 tgtaggggga tgcaattcgt ttttccctac aaaagcttct ttctccctca aggtggaagt 1260 gcagccatga ttatgtaa 1278

Claims

1. Use of the rice FBX365 gene having a coding region nucleotide sequence as shown in SEQ ID NO: 3 and a protein having an amino acid sequence as shown in SEQ ID NO: 1 in cultivating rice with a high disintegration value, characterized in that: The expression of the rice FBX365 gene is targetedly knocked out, interfered with, inhibited or silenced by gene editing to improve the rice disintegration value and cultivate rice with high disintegration value.

2. Use of a substance for interfering with, inhibiting, silencing or targeted knockout of the rice FBX365 gene having a coding region nucleotide sequence as shown in SEQ ID NO: 3 in cultivating rice with high disintegration value, characterized in that: The rice disintegration value is improved by targeted knockout, interference, inhibition or silencing of the expression of the rice FBX365 gene through gene editing, and rice with high disintegration value is cultivated; The substance used to interfere with, inhibit, silence or target knockout the rice FBX365 gene includes at least one of the following (1) to (6): (1) Interference sequence targeting rice FBX365 gene; (2) Interference vector of rice FBX365 gene used for interference; (3) Transgenic cell lines containing rice FBX365 gene interference vectors; (4) Primer sequences for targeted knockout of rice FBX365 gene based on gene editing technology; (5) A vector for targeted knockout of the rice FBX365 gene based on gene editing technology; (6) Cell lines with targeted knockout of the rice FBX365 gene based on gene editing technology.

3. A method for cultivating rice with high disintegration value, characterized in that: The rice disintegration value is improved by gene editing, targeting knockout, interference, inhibition or silencing of the rice FBX365 gene whose coding region nucleotide sequence is shown in SEQ ID NO: 3, and rice with high disintegration value is cultivated.

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