Application of gene LOC-Os07g49460 in increasing rice grain length

Abstract

The invention discloses an application of a gene LOC-Os07g49460 in increasing the grain length of rice. Belongs to the technical field of gene engineering. According to the application, the function of the LOC-Os07g49460 gene in increasing the grain length is disclosed for the first time, a specific gRNA is constructed by taking the gene as an editing target, the gene is directionally edited to form a mutation type of which the grain length is obviously increased, the mutation generates 403bp large fragment deletion at a first exon and a first intron, and the mutation has a large fragment deletion at a second exon and a second intron. A novel protein sequence is formed, phenotypic investigation shows that the novel protein generated by the mutation type can significantly increase the grain length of rice, and reliable technical support is provided for rapid breeding of long-grain rice varieties.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and more particularly to a gene LOC - Application of Os07g49460 in improving rice grain length. BACKGROUND

[0002] Rice is one of the most important food crops in the world and plays an irreplaceable role in food security. With the continuous growth of global population, the gradual reduction of arable land and the gradual improvement of people's living standards, improving rice yield and quality has become a key problem to be solved in the field of agriculture. Rice grain length is one of the key factors affecting rice yield and quality. Generally speaking, in terms of rice quality, longer rice grains are more favored by consumers in appearance and have relatively higher market value.

[0003] With the rapid development of molecular biology technology, the completion of rice whole genome sequencing and the continuous improvement of genetic linkage map, a solid foundation has been provided for the research of rice grain length related genes. Researchers can locate QTL (quantitative trait locus) of rice grain length with the help of these achievements, and then clone and analyze the function of related important genes.

[0004] However, the genetic mechanism of rice grain length is extremely complex, which is improved by multiple gene loci in coordination, and is also easily affected by environmental factors. At present, although some genes related to rice grain length have been identified, the understanding of the whole genetic improvement network is still very limited, and the genetic control mechanism of grain length traits is not completely clear. There are still a large number of potential key genes to be excavated and researched in the regulation of rice grain length, and the development of more genes regulating rice grain length is of great significance for promoting the process of rice molecular breeding and cultivating new rice varieties with high yield and quality.

[0005] Therefore, how to further develop new rice grain length related regulatory genes is a technical problem to be solved by those skilled in the art. SUMMARY

[0006] Therefore, the present application provides a gene LOC - Application of Os07g49460 in improving rice grain length, the present application first discovers that the gene LOC - Os07g49460 has an important relevance with rice grain length traits, and a specific gRNA is constructed by taking the gene as an editing target, the gene is edited in a directional manner, a mutation type with significantly increased grain length is formed, the mutation produces a large fragment deletion of 403bp at the first exon and the first intron, forming a new type of protein sequence, and the new protein produced by the mutation type can significantly improve the rice grain length through phenotype investigation.

[0007] To solve the above technical problems, the application adopts the following technical solutions:

[0008] Gene LOC - The application of Os07g49460 in improving rice grain length, the gene LOC - The nucleotide sequence of Os07g49460 is any one of the following:

[0009] 1) as shown in SEQ ID NO. 3;

[0010] 2) or a nucleotide sequence shown by the sequence shown in SEQ ID NO. 3 after substitution, deletion and / or addition of one or more bases, which still encodes the same functional protein.

[0011] As a preferred technical solution, the gene LOC - The amino acid sequence encoded by Os07g49460 is any one of the following:

[0012] 1) as shown in SEQ ID NO. 4;

[0013] 2) or an amino acid sequence shown by the amino acid sequence shown in SEQ ID NO. 4 after substitution, deletion and / or addition of one or more amino acids, which still has the same enzyme function.

[0014] Another purpose of the application is to provide: targeting LOC - gRNA1 and gRNA2 of Os07g49460 gene, the sequences of the gRNA1 and gRNA2 are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

[0015] Another purpose of the application is to provide: a DNA molecule encoding the gRNA1 and gRNA2.

[0016] Another purpose of the application is to provide: a biological material, which is any one of the following:

[0017] 1) an expression cassette containing the DNA molecule;

[0018] 2) a recombinant vector containing the DNA molecule, or a recombinant vector containing the expression cassette of 1);

[0019] 3) a transgenic cell line containing the DNA molecule, or a transgenic cell line containing the expression cassette of 1), or a transgenic cell line containing the recombinant vector of 2);

[0020] 4) a recombinant bacteria containing the DNA molecule, or a recombinant bacteria containing the expression cassette of 1), or a recombinant bacteria containing the recombinant vector of 2);

[0021] 5) A transgenic plant containing the DNA molecule, or a transgenic plant containing the expression cassette of 1), or a transgenic plant containing the recombinant vector of 2), or a transgenic plant containing the transgenic cell line of 3).

[0022] Another object of this application is to provide the use of the said gRNA1 and gRNA2, the said DNA molecule, or the said biological material, wherein the use is any one of the following:

[0023] 1) Application in improving rice grain length;

[0024] 2) In gene LOC - Applications in editing Os07g49460;

[0025] 3) Application in the breeding of long-grain rice varieties.

[0026] Another object of this application is to provide: a method for increasing the grain length of rice, comprising the following steps: using LOC - Os07g49460 serves as a gene editing target in targeting LOC. - Guided by gRNA1 and gRNA2 of the Os07g49460 gene, LOC - The Os07g49460 gene was targeted to produce a gain-of-function mutation, forming a novel protein that increases rice grain length.

[0027] As a preferred technical solution, the gene LOC - The nucleotide sequence of Os07g49460 is any one of the following:

[0028] 1) As shown in SEQ ID NO.3;

[0029] 2) Or a nucleotide sequence that, after substitution, deletion and / or addition of one or more bases, still encodes the same functional protein as the sequence shown in SEQ ID NO.3;

[0030] The gene LOC - The amino acid sequence of the protein encoded by Os07g49460 is any one of the following:

[0031] 1) As shown in SEQ ID NO.4;

[0032] 2) Or an amino acid sequence that still has the same enzyme function after the amino acid sequence shown in SEQ ID NO.4 has been replaced, deleted and / or added to one or more amino acids.

[0033] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0034] This application reveals LOC for the first time. - The function of the Os07g49460 gene in increasing grain length was investigated, and specific gRNA1 and gRNA2 were constructed using this gene as editing targets. Targeted editing of this gene resulted in a mutant type with significantly increased grain length. This mutation produced a large 403bp deletion at the first exon and the first intron, forming a new type of protein sequence. Phenotypic analysis showed that the new protein produced by this mutant type can significantly increase rice grain length, increasing the length of mutant rice grains by 7.6%, providing reliable technical support for the rapid breeding of long-grain rice varieties. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0036] Figure 1 For: Sequence analysis of the rice LOC-Os07g49460 gene and design of gene editing targets; black boxes indicate exons, black lines indicate introns and untranslated regions; Cas9 recognition sequences are marked with arrows.

[0037] Figure 2 The gene is: LOC-Os07g49460 mutant; where WT is the wild-type sequence, cas9-1-cas9-4 are mutant plants; Arabic numerals indicate the number of deleted bases (a "-" before the number indicates a base deletion).

[0038] Figure 3 The image shows a cas9-4 mutant plant with a missing 403 bp sequence and a sequence diagram.

[0039] Figure 4 The study investigated the grain size of the LOC-Os07g49460 gene mutant and its wild type; WT was the wild type, cas9-1 and cas9-2 were small fragment deletion mutants, and cas9-4 was a large fragment deletion mutant with a deletion of 403bp.

[0040] Figure 5The analysis included grain length, grain weight, and thousand-grain weight of the LOC-Os07g49460 gene mutant and its wild type. WT represented the wild-type control plant, cas9-1 and cas9-2 represented small deletion mutant plants, and cas9-4 represented a large deletion mutant plant with a deletion of 403bp. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1

[0043] Cloning of the rice LOC-Os07g49460 gene

[0044] Gene information was obtained based on the LOC-Os07g49460 accession number recorded in the Rice Data Center, and full-length primer amplification sequences were designed:

[0045] F: 5'-CAACCTCTTCGAGTCAGGCTG- 3', SEQ ID NO.1;

[0046] R: 5'-TCATCTGTCCCGCTGCCGCTTCGC-3', SEQ ID NO. 2.

[0047] Using the genomic DNA of high-quality conventional japonica rice Xiushui 134 as a template, the LOC-Os07g49460 genome sequence was amplified. The specific amplification conditions are as follows:

[0048] Amplification reaction system: total volume 25 μL, 5 μL FLU-ARMS5×PCR Mix, 2 μL cDNA, 1.5 μL Primermix, 16.5 μL ddH2O;

[0049] Amplification reaction program: 94 °C pre-denaturation for 4 min, 31 cycles (95 °C denaturation for 30 s; 58 °C annealing for 45 s; 72 °C extension for 2 min), followed by an extension at 72 °C for 7 min outside of the cycles.

[0050] After PCR amplification, the gene LOC-Os07g49460 was cloned, and its nucleotide sequence is as follows:

[0051] LOC-Os07g49460 nucleotide sequence:

[0052]

[0053]

[0054]

[0055] Amino acid sequence corresponding to LOC-Os07g49460:

[0056] MMGTAHHNQTAGSALGVGVGDANDAVPGAGGGGYSDPDGGPISGVQRPPQVCWERFIQKKTIKVLLVDSDDSTRQVVSALLRHCMYEVIPAENGQQAWTYLEDMQNSIDLVLTEVVMPGVSGISLLSRIMNHNICKNIPVIMMSSNDAMGTVFKCLSKGAVDFLVKPIRKNELKNLWQHVWRRCHSSSGSGSESGIQTQKCAKSKSGDESNNNNGSNDDDDDDGVIMGLNARDGSDNGSGTQAQSSWTKRAVEIDSPQAMSPDQLADPPDSTCAQVIHLKSDICSNRWLPCTSNKNSKKQKETNDDFKGKDLEIGSPRNLNTAYQSSPNERSIKPTDRRNEYPLQNNSKEAAMENLEESSVRAADLIGSMAKNMDAQQAARAANAPNCSSKVPEGKDKNRDNIMPSLELSLKRSRSTGDGANAIQEEQRNVLRRSDLSAFTRYHTPVASNQGGTGFMGSCSLHDNSSEAMKTDSAYNMKSNSDAAPIKQGSNGSSNNNDMGSTTKNVVTKPSTNKERVMSPSAVKANGHTSAFHPAQHWTSPANTTGKEKTDEVANNAAKRAQPGEVQSNLVQHPRPILHYVHFDVSRENGGSGAPQCGSSNVFDPPVEGHAANYGVNGSNSGSNNGSNGQNGSTTAVNAERPNMEIANGTINKSGPGGGNGSGSGSGNDMYLKRFTQREHRVAAVIKFRQKRKERNFGKKVRYQSRKRLAEQRPRVRGQFVRQAVQDQQQQGGGREAAADR*, SEQ ID NO. 4

[0057] Example 2

[0058] Construction of gene knockout vector targeting rice LOC-Os07g49460

[0059] (1)Design of specific gRNA

[0060] This invention employs the CRISPR / Cas9 gene editing technology system to conduct related experiments, designing a Cas9 recognition site at a 400bp interval between the first exon and the first intron of the LOC-Os07g49460 gene. Figure 1 , Figure 1 Both gRNA1 and gRNA2 in the sequence have a 3bp PAM sequence at their 3' end, based on SEQ ID NO.5 and SEQ ID NO.6, and specific gRNAs have been configured based on this site. Their nucleotide sequences are as follows:

[0061] gRNA1: 5'-TGCTTTACAATTTGCTGTAG-3', SEQ ID NO.5;

[0062] gRNA2: 5'-GCTCATCACAACCAAACCGC-3', SEQ ID NO.6;

[0063] The selection of these two targets takes into account a variety of factors, including gene function and structure, editing efficiency, and off-target risk, with the aim of achieving precise editing of large segments of the gene.

[0064] (2) Construction of a vector targeting the rice LOC-Os07g49460 gene knockout

[0065] The recombinant plasmid pCXUN-Cas9-gRNA was constructed using the pCXUN-Cas9 vector (purchased from Beijing Weishang Lide Biotechnology Co., Ltd.) as the backbone vector. In this vector, the full-length coding sequence of the Cas9 protein is 4200 bp, encoding 1400 amino acids, and its expression is driven by the Ubiquitin promoter; while the expression of the gRNA is regulated by the U6 promoter. During the construction of the targeting vector, the gRNA forward and reverse strand fragments obtained in step (1) were synthesized first. 1 μL of the forward and reverse strand fragments were mixed with 8 μM Anneal Buffer (TE + 50 mM NaCl), incubated at 95℃ for 10 min, and then cooled to 20℃ at a rate of 0.1℃ / s to allow the gRNA forward and reverse strands to fully anneal and form double-stranded DNA. Subsequently, the double-stranded DNA was directionally ligated to the downstream region of the U6 promoter in the pCXUN-Cas9 vector, which had been linearized by BsaI restriction enzyme digestion. Sequencing confirmed that the gRNA fragment was correctly inserted and without mutation, thus completing the construction of the recombinant plasmid pCXUN-Cas9-gRNA.

[0066] The Bsa I enzyme digestion reaction system and conditions are as follows: 5 μL plasmid (about 1 μg), 10 μL 10× buffer, 1 μL Bsa I enzyme, 84 μL ddH2O, and reacted at 37℃ for 2-3 h.

[0067] Example 3

[0068] Genetic transformation of Agrobacterium-mediated targeting vectors

[0069] Using callus tissue of japonica rice Xiushui 134 as the genetic transformation recipient, the gene editing vector was introduced into the recipient material via Agrobacterium-mediated transformation. The resistant callus was then screened, differentiated, and rooted. The specific operational steps are as follows:

[0070] (1) Culture of callus tissue

[0071] After dehulling and sterilizing mature rice seeds, they were inoculated onto induction medium (NB medium + 2,4-D 2 mg·L⁻¹). -1 (pH 5.8), and cultured in the dark at 28℃ for 7 days; after removing the radicle, cultured in the dark for another 7 days to form embryogenic callus;

[0072] Embryogenic callus was then transferred to fresh induction medium and subcultured every 14-20 days. After three subcultures, naturally dispersed, bright yellow granular callus with a diameter of about 2-3 mm was selected as reserve material for Agrobacterium transformation.

[0073] (2) Preparation of Agrobacterium suspension

[0074] Take a small amount of recombinant Agrobacterium bacterial suspension containing the gene-editing vector, and mix it with kanamycin at a concentration of 50 mg / L. -1 Rifampin 50mg·L -1 Streak the culture on YEB solid medium (pH 5.8) and incubate in the dark at 28°C for 36–72 h to complete activation. Pick a single colony from the activated plate and streak it again. After incubating in the dark at 28°C for 2 days, use a solution containing 100 μM·L⁻¹. -1 The cells of acetylsuccinyl syringone were washed and resuspended in AAM medium (pH 5.2), and the bacterial concentration was adjusted to OD. 600nm =1.5-2.0, after standing for 1 hour, an Agrobacterium suspension suitable for transformation was prepared.

[0075] (3) Transfection

[0076] The embryogenic callus obtained in step (1) was immersed in the Agrobacterium suspension prepared in step (2), and after standing for 30 min, the callus was placed on sterile filter paper to dry, and then inoculated into co-culture medium (NB medium + 2,4-D 2 mg·L⁻¹).-1 + Acetyleugenol 100 μM·L -1 (pH 5.8), and incubated in the dark at 25°C for 3 days;

[0077] (4) Resistant callus culture

[0078] Pick the co-cultured callus and place it in a wide-mouth culture flask. Rinse 3-5 times with sterile water (gently shaking several times each time) until no filamentous bacterial cells remain in the rinse water. For the final rinse, use water containing 250 mg·L⁻¹ sterile water. -1 After sterile water containing carbenicillin was allowed to stand for 1 hour, the callus was placed on sterile filter paper to air dry, and then inoculated into selection medium (NB medium + 2,4-D 2 mg·L⁻¹). -1 + Carbenicillin 250 mg / L -1 + Hygromycin 50mg·L -1 (pH 5.8), cultured in the dark at 28℃, and subcultured every 2 weeks. After 3 weeks, nodular bright yellow resistant callus can be seen growing from the browned and shriveled callus tissue.

[0079] (5) Differentiation and rooting culture

[0080] The resistant callus obtained in step (4) was transferred to differentiation medium (NB medium + 2,4-D 2 mg·L⁻¹). -1 +KT10mg・L -1 +NAA 0.4mg·L -1 After two weeks of culture in a medium (pH 5.8), the callus began to turn green, and after three weeks, buds and roots gradually emerged. The seedlings with buds were then transferred to a rooting medium (1 / 2 MS medium, pH 5.8). Once the seedling roots were fully developed, the residual medium on the roots was washed off, and the seedlings were transplanted to the experimental base for subsequent genotyping and phenotypic evaluation.

[0081] Example 4

[0082] Genotyping and phenotypic analysis of gene-edited organisms

[0083] Using the plants obtained in Example 3 as raw materials, genomic DNA was extracted. The extracted DNA was then used as a template for PCR primer amplification to preliminarily identify the transgenic plants. The specific process is as follows:

[0084] The PCR amplification primers include CZTF-F and CZTF-R, with the following specific sequences:

[0085] CZTF-F: 5'-GGGAGATCCAGCTAGAGGTC-3', SEQ ID NO.7;

[0086] CZTF-R: 5'-GGAAGGAGGAAGACAAGG-3', SEQ ID NO.8;

[0087] Amplification reaction system: total volume 25 μL, 5 μL FLU-ARMS5×PCR Mix, 2 μL cDNA, 1.5 μL Primermix, 16.5 μL ddH2O;

[0088] Amplification reaction program: 94 °C pre-denaturation for 4 min, 31 cycles (95 °C denaturation for 30 s; 58 °C annealing for 45 s; 72 °C extension for 30 s), followed by an extension at 72 °C for 7 min outside of the cycles.

[0089] A total of 15 transgenic positive strains were obtained through identification.

[0090] To further determine the specifics of the gene editing, DNA was extracted from the preliminarily identified transgenic plants. Using the extracted DNA as a template, specific amplification primer sequences were designed targeting the target gene, and PCR amplification was performed to obtain a gene fragment containing the target sequence. The amplified gene fragment was then sent to a professional sequencing company for sequencing analysis.

[0091] The primer sequences for amplifying the target gene are as follows:

[0092] Z25-F: 5'-ACCGCCTGCCTTTCTTCA-3', SEQ ID NO.9;

[0093] Z25-R: 5'-GTTGCTTGCTTATCGTCCTTG-3', SEQ ID NO.10;

[0094] Amplification reaction system: total volume 25 μL, 5 μL FLU-ARMS5×PCR Mix, 2 μL cDNA, 1.5 μL Primermix, 16.5 μL ddH2O;

[0095] Amplification reaction program: 94 °C pre-denaturation for 4 min, 31 cycles (95 °C denaturation for 30 s; 58 °C annealing for 45 s; 72 °C extension for 30 s), followed by an extension at 72 °C for 7 min outside of the cycles.

[0096] Analysis of the sequencing results revealed 10 clones with LOC-Os07g49460 gene editing, most of which had genotypes with deletions of 1-3 bp; cas9-4 had a large deletion mutation, with sequencing analysis confirming a deletion of 403 bp. Figure 2 andFigure 3 This formed a new type of protein sequence, SEQ ID NO.11, as follows:

[0097] , SEQ ID NO.11.

[0098] Further observation of the phenotype of the mutant plants revealed that the new protein produced by the Cas9-4 large fragment deletion modification significantly increased rice grain length, by approximately 7.6% compared to the wild type. Figures 4-5 .

[0099] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0100] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Gene LOC - The application of Os07g49460 in improving rice grain length is characterized by... The gene LOC - The nucleotide sequence of Os07g49460 is any one of the following: 1) As shown in SEQ ID NO.3; 2) Or a nucleotide sequence that, after substitution, deletion and / or addition of one or more bases, still encodes the same functional protein as the sequence shown in SEQ ID NO.

3.

2. The gene LOC according to claim 1 - The application of Os07g49460 in improving rice grain length is characterized by... The gene LOC - The amino acid sequence encoded by Os07g49460 is any one of the following: 1) As shown in SEQ ID NO.4; 2) Or an amino acid sequence that still has the same enzyme function after the amino acid sequence shown in SEQ ID NO.4 has been replaced, deleted and / or added to one or more amino acids.

3. Targeting LOC - The gRNA1 and gRNA2 of the Os07g49460 gene are characterized by, The sequences of gRNA1 and gRNA2 are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

4. A DNA molecule, characterized by, The DNA molecule encodes the gRNA as described in claim 3.

5. A biomaterial, characterized in that, The biomaterial is any one of the following: 1) An expression cassette containing the DNA molecule of claim 4; 2) A recombinant vector containing the DNA molecule of claim 4, or a recombinant vector containing the expression cassette of claim 1; 3) A transgenic cell line containing the DNA molecule of claim 4, or a transgenic cell line containing the expression cassette of claim 1), or a transgenic cell line containing the recombinant vector of claim 2; 4) Recombinant bacteria containing the DNA molecule of claim 4, or recombinant bacteria containing the expression cassette of claim 1), or recombinant bacteria containing the recombinant vector of claim 2; 5) A transgenic plant containing the DNA molecule of claim 4, or a transgenic plant containing the expression cassette of claim 1), or a transgenic plant containing the recombinant vector of claim 2), or a transgenic plant containing the transgenic cell line of claim 3).

6. The application of gRNA1 and gRNA2 as described in claim 3, the DNA molecule as described in claim 4, or the biological material as described in claim 5, characterized in that, The application is any one of the following: 1) Application in improving rice grain length; 2) In gene LOC - Applications in editing Os07g49460; 3) Application in the breeding of long-grain rice varieties.

7. A method for improving the length of rice grains, characterized in that, Includes the following steps: using LOC - Os07g49460 is used as a gene editing target in the targeting LOC described in claim 3. - Guided by gRNA1 and gRNA2 of the Os07g49460 gene, LOC - The Os07g49460 gene was targeted to produce a gain-of-function mutation, forming a novel protein that increases rice grain length.

8. The method according to claim 7, characterized in that, The gene LOC - The nucleotide sequence of Os07g49460 is any one of the following: 1) As shown in SEQ ID NO.3; 2) Or a nucleotide sequence that, after substitution, deletion and / or addition of one or more bases, still encodes the same functional protein as the sequence shown in SEQ ID NO.3; The gene LOC - The amino acid sequence of the protein encoded by Os07g49460 is any one of the following: 1) As shown in SEQ ID NO.4; 2) Or an amino acid sequence that still has the same enzyme function after the amino acid sequence shown in SEQ ID NO.4 has been replaced, deleted and / or added to one or more amino acids.

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