Grain length gene cloned from African wild rice and application thereof

Abstract

The invention discloses a grain length gene cloned from African wild rice and application of the grain length gene. The invention aims to solve the problem of how to regulate and control agronomic traits of plants. The invention provides an application of a protein or a substance for regulating the expression of a coding gene of the protein or a substance for regulating the activity or content of the protein in regulating the agronomic traits of plants, and the protein is an ObGL1 protein, is a protein with an amino acid sequence of SEQ ID NO: 2, and is obtained by translating the grain length gene. The agronomic traits are grain weight and / or grain length and / or plant height and / or grain number per ear and / or single plant yield, and the regulation agronomic traits can be up-regulated, increased or increased grain weight and / or grain length and / or plant height and / or grain number per ear and / or single plant yield of main stem ears. The invention provides a new germplasm resource for genetic breeding work, provides a new material for rice variety breeding, and plays a positive role in accelerating improvement of rice varieties.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a grain length gene cloned from African wild rice and application thereof. Background Art

[0002] Rice is one of China's most important grain crops. Grain size is a key factor in determining rice yield (Xing et al., 2010). Elucidating the complex genetic basis of grain size variation is crucial for improving rice yield. To date, based on information from the China Rice Data Center database (https: / / www.ricedata.cn / gene / ), researchers have cloned the genes qGL3 (Qi et al., 2012), LGY3 (Liu et al., 2018), and GL10 (Zhan et al., 2022) that control grain length; the genes GW5 (Liu et al., 2017), GW2 (Choi et al., 2018), and GS5 (Li et al., 2011) that regulate grain width; and the genes WG1 (Hao et al., 2021), TGW6 (Ishimaru et al., 2013), and GS9 (Zhao et al., 2018) that regulate both grain length and width. These genes were primarily cloned from Asian rice, with only a few cloned from African rice. Therefore, the genetic resources of African rice remain to be explored and utilized. Furthermore, selection for large seeds has been a major goal of plant domestication (Larsen et al., 1995; Fuller et al., 2007). However, unlike the trend toward larger grains in other crops, the seeds of O. glaberrima are typically shorter than those of its ancestor, O. barthii (Wu et al., 2017). Uncovering the molecular genetic basis of selection for small seeds during domestication will help increase yields in cultivated rice in Africa. Previous studies have found that selection for the Ogsh4 allele, which prevents grain shattering, led to smaller grains during domestication, but this is insufficient to explain the small grain size observed in all African cultivated rice species, suggesting that other genes may have been under selection (Wu et al., 2017). Therefore, identifying genes controlling rice grain length using African rice, particularly wild African rice, is crucial for understanding the domestication process of African rice and improving yields in both Asian and African cultivated rice. Summary of the Invention

[0003] The technical problem to be solved by the present invention is how to regulate plant agronomic traits. To this end, the present invention provides the use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity and / or content of the protein. The protein may be ObGL1 protein, specifically any one of the following:

[0004] A1) a protein having an amino acid sequence of SEQ ID NO: 3,

[0005] A2) a protein having the same function as the amino acid sequence shown in SEQ ID NO: 3 after amino acid residue substitution and / or deletion and / or addition,

[0006] A3) a protein having an amino acid sequence identity of at least 80% with that defined in A1) or A2) and having the same function;

[0007] A4) A fusion protein obtained by ligating a tag to the end of any one of the proteins defined in A1) to A3);

[0008] The application may be any of the following:

[0009] B1) regulating plant yield,

[0010] B2) preparing products for regulating plant yield,

[0011] B3) cultivating plants with altered agronomic traits,

[0012] B4) preparing products of cultivating plants with altered agronomic traits,

[0013] B5) Plant breeding,

[0014] B6) Preparation of products for use in plant breeding.

[0015] The indicators of plant breeding include plant agronomic traits, and the purpose of plant breeding includes cultivating plants with changed agronomic traits.

[0016] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0017] Among the above proteins, SEQ ID NO: 2 consists of 340 amino acid residues and is named ObGL1 protein or protein ObGL1. The gene encoding it is ObGL1 gene. SEQ ID NO: 3 is as follows: MADVGMVVVTPAASFHHTHHHHHHHEAAAAAAAAAAAAADPIFPLLSAGPCVLDPDKSAASGSAIQFWQPPPQLPSSAAGGNPNPSSSAFPYLKKPLPMLDTGGGSSGSGGAATCQDCGNQAKKDCGHQRCRTCCKSRGFDCSTHVKSTWVPAARRRERQQLTGSA SSSPATASAAAASKKPRLLTSQTTTSHTSTSNATTPRSFDTTSHQDASFRESLPRQVRAPAVFRCVRVTSIDDGEDEYAYQATVTINGHVFKGFLYDQGVDDGRGLAATSNDDSTAGGVPNISELHLGGASISGNAMREGGSSMVHSDLYGGGGGSGGGPHILGGSSYGNTMN.

[0018] In the above application, the protein may be derived from rice.

[0019] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates the expression of a gene encoding the protein ObGL1.

[0020] The present invention also provides applications of the biomaterial, which may be any of the following:

[0021] C1) regulating plant yield,

[0022] C2) preparing products for regulating plant yield,

[0023] C3) cultivating plants with altered agronomic traits,

[0024] C4) preparing products for cultivating plants with altered agronomic traits,

[0025] C5) Plant breeding,

[0026] C6) preparing products for plant breeding;

[0027] The biological material may be any of the following:

[0028] D1) a nucleic acid molecule encoding said protein,

[0029] D2) an expression cassette containing the nucleic acid molecule described in D1),

[0030] D3) a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2),

[0031] D4) a recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3),

[0032] D5) a transgenic plant cell line containing the nucleic acid molecule described in D1), or a transgenic plant cell line containing the expression cassette described in D2),

[0033] D6) transgenic plant tissue containing the nucleic acid molecule described in D1), or transgenic plant tissue containing the expression cassette described in D2),

[0034] D7) A transgenic plant organ containing the nucleic acid molecule described in D1), or a transgenic plant organ containing the expression cassette described in D2).

[0035] Those skilled in the art can readily mutate the nucleotide sequence of the present invention that inhibits, reduces, or down-regulates the expression of the gene encoding the ObGL1 protein using known methods, such as directed evolution or point mutagenesis. Artificially modified nucleotide sequences that are 80% or more identical to the nucleotide sequence of the present invention that inhibits, reduces, or down-regulates the expression of the gene encoding the ObGL1 protein, and that have the function of inhibiting, reducing, or down-regulating the expression of the gene encoding the ObGL1 protein, are derived from and are equivalent to the nucleotide sequence of the present invention.

[0036] As used herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a search to calculate the identity of the amino acid sequence, the value (%) of identity can then be obtained.

[0037] Herein, the greater than 80% identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0038] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0039] In the above application, the nucleic acid molecule in D1) can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA. Furthermore, the nucleic acid molecule in D1) can be a gRNA targeting the protein-encoding gene.

[0040] Furthermore, in the above application, the recombinant vector in D3) can be a recombinant expression vector containing an expression cassette for the ObGL1 gene encoding the protein ObGL1. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as a pCAMBIA1300 vector, a CRISPR-Cas9 vector, a pCAMBIA1301 vector, pGWB411, pGWB412, pGWB405, DTS9005, pBin438, pBI121, or pCAMBIA1302.

[0041] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as by 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 resistance markers (gentamicin marker, kanamycin marker, etc.), or chemical resistance marker genes (such as herbicide resistance genes).

[0042] Furthermore, in the above application, the recombinant microorganism in D4) can specifically be yeast, bacteria, algae and fungi.

[0043] Furthermore, in the above application, the recombinant microorganism may be Agrobacterium, and the Agrobacterium may be EHA105.

[0044] Furthermore, in the above application, the plant tissue in D6) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and / or anthers.

[0045] Furthermore, in the biological material, the transgenic plant organ in D7) can be the root, stem, leaf, flower, fruit and seed of the transgenic plant.

[0046] The present invention also provides a method for regulating agronomic traits of plants, which includes step M. Step M can be a step of regulating the agronomic traits of plants by regulating the expression of the encoding gene in the target plant.

[0047] The present invention also provides a method for producing plants with altered agronomic traits, the method comprising step M, wherein step M may be a step of obtaining plants with altered agronomic traits by regulating the expression of the encoding gene in the target plant.

[0048] The step M may be to inhibit, reduce or silence the activity and / or content of the protein in the target plant, or / and, inhibit, reduce or silence the expression level of the gene encoding the protein, so as to change the agronomic traits of the plant.

[0049] Inhibiting, reducing or downregulating the expression of the encoding gene can be achieved by gene knockout or gene silencing.

[0050] Gene knockout refers to the inactivation of a specific target gene through gene editing techniques. Gene knockout inactivates a specific target gene by altering its DNA sequence, including but not limited to zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR / Cas system. CRISPR (clustered regularly interspaced short palindromic repeats) is a site in the genome containing multiple short repeats. The Cas9 protein, under RNA guidance, can cleave the target sequence recognized by crRNA–tracrRNA.

[0051] The gene silencing refers to the phenomenon of not expressing or underexpressing a gene without damaging the original DNA. Gene silencing is based on the premise that the DNA sequence is not changed, so that the gene is not expressed or underexpressed. Gene silencing can occur at two levels. One is gene silencing at the transcriptional level due to DNA methylation, heterochromatinization and position effects, and the other is post-transcriptional gene silencing, that is, gene inactivation by specifically inhibiting the target RNA at the level after gene transcription, including antisense RNA, co-suppression, gene repression (quelling), RNA interference (RNAi) and microRNA (miRNA)-mediated translation inhibition, etc.

[0052] In the above method, the gene knockout or gene silencing can be performed by any method in the prior art to cause deletion mutation, insertion mutation or base conversion mutation in the gene, thereby performing gene knockout or gene silencing on the ObGL1 gene.

[0053] In the above method, the ObGL1 gene in the target rice is knocked out or silenced by chemical mutagenesis, physical mutagenesis, RNAi, site-directed gene editing, homologous recombination and the like.

[0054] The above-mentioned regulation can also be achieved through zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology or clustered regularly interspaced short palindromic repeats / CRISPR associated, CRISPR / Cas9 system technology, as well as other technologies that can achieve site-specific genome editing.

[0055] In the above method, the protein may be derived from rice.

[0056] In the above, the regulation can be at least one of the following 6 types of regulation:

[0057] E1) regulation at the transcriptional level of the coding gene,

[0058] E2) regulation after transcription of the coding gene,

[0059] E3) regulation of RNA transport of the coding gene,

[0060] E4) regulation of the translation of the coding gene,

[0061] E5) regulating the degradation of the mRNA encoding the gene,

[0062] E6) Post-translational regulation of the gene.

[0063] In the above applications or methods, the agronomic trait may be grain weight and / or grain length and / or plant height and / or number of grains per ear and / or yield per plant. The change in the agronomic trait may be increased grain weight and / or increased grain length and / or increased plant height and / or increased number of grains per ear per main stem and / or increased yield per plant.

[0064] The regulation may be up-regulation, improvement or increase, and the regulation may also be down-regulation, reduction or decrease.

[0065] The regulation of the expression of the coding gene in the target plant may be achieved by knocking out the coding gene in rice. Specifically, the knocking out of the coding gene in rice may be achieved by inserting a 1 bp thymidine deoxyribonucleotide T between nucleotides 735 and 736 of sequence 2 in the rice genome sequence list.

[0066] The grain weight may be thousand-grain weight.

[0067] In the above application or method, the plant may be any one of the following:

[0068] F1) dicots or monocots, F2) Poaceae, F3) Gramineae, F4) Oryza, F5) Rice.

[0069] The present invention also provides a product, which may be the protein or biological material.

[0070] The present invention identifies the grain length phenotype of an introgression line with an African cultivated rice background infiltrated with African wild rice (constructed through multiple generations of backcrossing using African cultivated rice OP108 as the recurrent parent and African wild rice W1411 as the donor parent) and identifies an introgression line, M8, with significantly longer grain length. To clone the gene that controls the difference in grain length phenotype between M8 and OP108, segregating populations of M8 and OP108 were constructed, and the target gene was located using map-based cloning. Knockout vectors, complementation vectors, and overexpression vectors for the candidate gene were constructed to verify gene function. Experiments have shown that the mutant material obtained in this study can increase rice grain length and improve single-plant yield, providing new germplasm resources for genetic breeding and new materials for rice variety selection, which has a positive effect on accelerating the improvement of rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Phenotypes of the two parents, M8 and OP108, used for cloning the grain length gene.

[0072] Figure 2 The results of map-based cloning, subcellular localization of candidate genes and qRT-PCR analysis are shown.

[0073] Figure 3 These are the corresponding agronomic traits after knockout, complementation, and overexpression of candidate genes in the ZH11 background. DETAILED DESCRIPTION

[0074] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0075] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0076] pYLCRISPR / cas9Pubi-H (Xingliang Ma. et al. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Molecular Plant. Volume 8, Issue 8, 3 August 2015, Pages 1274-1284) was kindly provided by Professor Yaoguang Liu of South China Agricultural University. This biomaterial is available to the public from China Agricultural University for use only in replicating the experiments described in this invention and is not intended for any other purpose.

[0077] The data in the following examples were processed using SPSS 11.5 statistical software. The experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA. P < 0.05 (*) indicated a significant difference, and P < 0.01 (**) indicated a very significant difference.

[0078] Example 1: Location of the ObGL1 gene and construction of related strains

[0079] 1.1 Constructing a group to locate GL1

[0080] The introgression line M8 was constructed by backcrossing multiple generations using the germplasm resource African cultivated rice OP108 (accession No. IRGC 104165) (https: / / www.genesys-pgr.org) as the recurrent parent and the African wild rice OP147 (accession No. IRGC 100119) (https: / / www.genesys-pgr.org) as the donor parent. Figure 1Phenotypic comparison of parents M8 and OP108, where (a) is the comparison of mature grain length of parents (upper: 108, lower: M8, bar = 1 cm); (b) is the comparison of mature grain width of parents (upper: 108, lower: M8, bar = 1 cm); (cd) are the young spikelet shells of parents (bar = 5 mm), the white horizontal line represents the cross section of the paraffin section; (e) is the cross section of the paraffin section of the spikelet shell of the parent (upper: 1 OP108, M8 below, bar = 0.5 mm); (f) 108 above, M8 below; (e) magnified view of the cross-section within the red box (bar = 50 μm); (g) SEM comparison of cells in the outer hull of the parental varieties (108 above, M8 below, bar = 100 μm); (hs) comparison of agronomic traits (n = 15), paraffin sections (n ​​= 3), and SEM data (n = 3) of the parental varieties. Error bars indicate standard deviation. **P < 0.01; *P < 0.05; ns indicates no significant difference (Student's t-test). It can be seen that M8 and OP108 differ significantly in grain length.

[0081] To clone the gene controlling the difference in grain length phenotype between M8 and OP108 (named ObGL1), an F2 segregating population of M8 and OP108 was constructed for preliminary positioning; an F3 segregating population and an F 3:4 Fine mapping of families. Grain length, as a quantitative trait, is regulated by multiple quantitative trait loci and influenced by the environment. Therefore, the mapping strategy involves purifying the population background, expanding the population with heterozygous individuals harboring only the introgressed fragment within the mapped interval, and then screening and exchanging individuals to achieve a population size of at least 2,000. Figure 2 Figure 1 shows the localization and expression pattern of the ObGL1 gene, where (a) shows the map-based cloning of the ObGL1 gene; (b) shows the subcellular localization of the ObGL1 gene in tobacco leaves (bar = 50 μm); and (c) shows the transcription level of the ObGL1 gene in the parental line. Error bars indicate standard deviation.

[0082] Since ObGL1 showed a semi-dominant inheritance pattern, it could only be located using homozygous exchange plants. Therefore, after screening heterozygous exchange plants from the F3 population, the F 3:4 Exchange single-plant families and then perform fine positioning through intra-line phenotypic comparison.

[0083] 2. Verification of Localization Results and Cloning of ObGL1

[0084] 2.1 Construction of knockout vectors, complementation vectors and overexpression vectors of candidate genes.

[0085] 2.1.1 Construction of com-GL1 vector (genome complementation vector)

[0086] Using OP108 genomic DNA as a template, complementary vector primer F: 5'-acagctatgaccatgattacgaattcCATTAATCATGGTTACTGCTG and complementary vector primer R: 5'-gcctgcaggtcgactctagaggatccTGTGCCGCAAAACTACACTT amplified a 7.2 kb fragment including ObGL1, which is sequence 1 in the sequence list (SEQ ID NO: 1, 7265 bp), of which positions 1-3966 are the 5'-regulatory region fragment, positions 5074-7265 are the 3'-regulatory region fragment, positions 3967-4603 and 4691-5073 are exons; the rest are introns.

[0087] Sequence 1 was inserted into the restriction sites EcoRI and BamHI of pCAMBIA1300 vector (Beijing Zhuangmeng International Biogene Technology Co., Ltd., ZK857) by homologous recombination to obtain the ObGL1 gene complementation vector, which was named com-GL1 vector.

[0088] 2.1.2 Construction of Cas9-GL1 vector (CRISPR-Cas9 vector)

[0089] According to the CRISPR-Cas9 knockout vector construction process (https: / / www.sciencedirect.com / science / article / pii / S167420521500204X?via%3Dihub) and knockout target primer design method (http: / / skl.scau.edu.cn / home / ) of Liu Yaoguang's group, the rice snRNA promoter was amplified and the amplified product was ligated into the knockout vector pYLCRISPR / cas9Pubi-H by homologous recombination to construct the CRISPR-Cas9 knockout vector.

[0090] The target sequence T1 selected for knockout of the ObGL1 gene in rice Zhonghua 11 (abbreviated as ZH11) is 5'-TGTTGCAGACGCGTCGTTTAGGG-3' (corresponding to positions 717-739 of SEQ ID 2 in the sequence listing). The corresponding vector's F- and R-directed primers are 5'-TGTTGCAGACGCGTCGTTTAGGGgttttagagctagaaat-3' and 5'-CCCTAAACGACGCGTCTGCAACACggcagccaagccagca-3', respectively. Plant-based testing for gene editing uses the F- and R-directed primers 5'-CCTTCTCACCTCCCAAACCA-3' and 5'-CGTACAAGTCCGAGTGCACC-3', respectively.

[0091]

[0092] The amino acid sequence of the protein ObGL1 encoded by the ObGL1 gene in Sequence 2 is Sequence 3 (SEQ ID NO: 3, 340aa) in the sequence listing, and is as follows:

[0093] MADVGMVVVTPAASFHHTHHHHHHHEAAAAAAAAAAAAADPIFPLLSAGPCVLDPDKSAASGSAIQFWQPPPQL

[0094] PSSAAGGNPNPSSSAFPYLKKPLPMLDTGGGSSGSGGAATCQDCGNQAKKDCGHQRCRTCCKSRGFDCSTHVKS

[0095] TWVPAARRRERQQLTGSASSSPATASAAAASKKPRLLTSQTTTSHTSTSNATTPRSFDTTSHQDASFRESLPR

[0096] QVRAPAVFRCVRVTSIDDGEDEYAYQATVTINGHVFKGFLYDQGVDDGRGLAATSNDDSTAGGVPNISELHLGGASISGNAMREGGSSMVHSDLYGGGGGSGGGPHILGGSSYGNTMN.

[0097] The coding sequence (CDS) of protein ObGL1 is sequence 4 (SEQ ID NO: 4, 1023 bp) in the sequence listing, and is as follows:

[0098]

[0099] 2.1.3 Construction of OE-GL1 vector (overexpression vector)

[0100] Total RNA was extracted from young panicles of rice OP108 and reverse transcribed into cDNA.

[0101] The full-length ObGL1 (i.e., sequence 3) was amplified by PCR using the overexpression vector primer F: 5'-TCGACTCTAGAGGATCCCCGGGTACCATGGCCGACGTCGGGATGGT-3' and the overexpression vector primer R: 5'-TCATGGTCTTTGTAGTCCATGAGCTCGTTCATGGTGTTACCATAGC-3'. The above fragment was inserted into the pCAM BIA1301 vector (Beijing Zhuangmeng International Biogene Technology Co., Ltd., catalog number ZK858) between the restriction sites KpnI and SacI by homologous recombination, and the CaMV 35S promoter was inserted upstream of the fragment to drive gene transcription, thereby obtaining a recombinant expression vector expressing ObGL1, referred to as the OE-GL1 vector.

[0102] 2.2 General process of vector construction

[0103] 1) Amplification of target fragment

[0104] The corresponding vector fragments were amplified using the high-fidelity enzyme TKS (Takara) with the primers mentioned in 2.1. The reaction system and amplification conditions are shown in Tables 1 and 2. The amplified products were detected on 1% agarose gel.

[0105] Table 1 TKS enzyme reaction system

[0106] Volume / μL Template DNA 2 Primer (10 μM) 4 2×Gflex buffer 10 <![CDATA[Tks Gflex TM DNA Polymerase]]> 0.4 ddH20 3.6

[0107] Table 2 TKS enzyme reaction conditions

[0108]

[0109] 2) Vector digestion

[0110] Based on the restriction enzyme sites of different vectors, double enzyme digestion was performed, incubated for 4 h at the optimal reaction temperature for the restriction enzyme, and terminated with 10× loading buffer (Takara). The reaction system is shown in Table 3.

[0111] Table 3 Vector enzyme digestion reaction system

[0112] volume Vector fragment 2-3 μg Restriction endonuclease I 2.5 μL Restriction endonuclease II 2.5 μL 10×buffer 5μL ddH2O Make up to 50 μL

[0113] 3) Purification of target fragment and vector

[0114] The DNA gel recovery kit (GeneSand) was used according to its instructions.

[0115] 4) Carrier connection

[0116] Use the Seamless Assembly Seamless Cloning Kit (CloneSmarter) to incubate at 50°C for 15 minutes and then place on ice for 2-3 minutes (the reaction time can be appropriately extended depending on the length of the ligated fragment). The reaction system is shown in Table 4.

[0117] Table 4 Vector ligation reaction system

[0118] Volume / μL Vector fragment x Amplified fragment y 2×Seamless Assembly 2.5 ddH20 Make up to 5 μL

[0119] Note: The volume to be added should be determined based on the length of the vector and the amplified fragment, ensuring a molar ratio of 1:3.

[0120] 5) E. coli transformation

[0121] Use Qingke Biotechnology Co., Ltd. 5α strain, and specific operations were performed according to its instructions.

[0122] 6) Identification and preservation of positive clones

[0123] Perform PCR amplification using the monoclonal bacterial solution obtained in step 5) as a template, and screen for clones containing the target fragment for sequencing verification. Mix the positive clones with 50% glycerol (v / v) at a 1:1 volume ratio and store at -80°C for long-term storage.

[0124] 7) Plasmid extraction

[0125] The bacterial solution was placed in about 15 mL of LB liquid culture medium and cultured in a shaking incubator at 37° C. overnight. The plasmid was extracted from the obtained bacterial solution using a plasmid extraction kit (TIANGEN) according to the instructions.

[0126] 2.3 Rice genetic transformation

[0127] 2.3.1 Agrobacterium transformation (freeze-thaw method)

[0128] Competent cells of Agrobacterium tumefaciens strain EHA105 used for rice genetic transformation were purchased from Qingke Biotechnology Co., Ltd., and plasmid transformation was performed according to the manufacturer's instructions.

[0129] 2.3.2 Rice callus induction and culture

[0130] 1) Select rice seeds with full and sterile grains and hull them using a thresher.

[0131] 2) Place the seeds in a sterilized Erlenmeyer flask and treat with appropriate amounts of 70% ethanol and 15% NaClO for 2 min and 25 min, respectively.

[0132] 3) Pour off the NaClO solution in a clean bench and rinse the seeds with sterile water until no NaClO remains. Then place them in a petri dish covered with sterile filter paper to dry.

[0133] 4) Place the dried seeds evenly in the NB basic medium, with about 40 seeds per dish.

[0134] 5) Incubate in the dark at 28°C for about 7 days until light yellow callus grows.

[0135] 2.3.3 Agrobacterium infection

[0136] 1) Add the plasmid-transfected Agrobacterium to approximately 10 mL of YEB liquid medium containing antibiotics and culture overnight at 28°C and 220 rpm.

[0137] 2) Pipette 2-3 mL of overnight culture into 60 mL of YEB liquid medium containing antibiotics and adjust the initial OD600 value to between 0.12 and 0.15 using a spectrophotometer.

[0138] 3) Incubate at 28°C, 220 rpm, for 4-5 h until the OD600 value of the bacterial solution reaches 0.5-0.6.

[0139] 4) Centrifuge at 6,000 rpm for 10 min, discard the supernatant, and resuspend the cells in AAM solution containing 20 mg / mL AS.

[0140] 5) Transfer the separated rice callus to a 100 mL Erlenmeyer flask, add AAM resuspension solution, and incubate at 28°C, 220 rpm, for 30 min.

[0141] 6) Remove the liquid and place the callus in a petri dish covered with sterile filter paper to dry.

[0142] 7) Place the dried callus on NB-Ac medium covered with sterile filter paper and culture in the dark at 25°C for about 3 days.

[0143] 8) Wash the cultured calli with sterile water without antibiotics and sterile water containing 200 mg / mL cephalexin and timentin, respectively, dry them, and transfer them to the screening medium.

[0144] 2.3.4 Screening of resistant calli, plant regeneration and testing

[0145] Screening of resistant calli (cultured in the dark at 28°C)

[0146] 1) Add 200 mg / mL cephalexin and timentin and 35 mg / mL hygromycin to the screening medium for the first screening, and the screening time is 21-28 days.

[0147] 2) Add 200 mg / mL cephalexin and timentin and 50 mg / mL hygromycin to the screening medium for a second screening, with the screening time being 21-28 days.

[0148] Plant regeneration

[0149] 1) Predifferentiation: The active calli after screening were transferred to predifferentiation medium and cultured in the dark at 28°C for 7-10 days.

[0150] 2) Differentiation: After pre-differentiation, the callus was transferred to differentiation medium and cultured at 28°C for 21-28 days with a cycle of 16 hours in the light and 8 hours in the dark.

[0151] 3) Rooting: The differentiated bud tissue was transferred to a rooting medium and cultured at 28°C for 14-21 days with a cycle of 16 hours in the light and 8 hours in the dark.

[0152] Positive plant detection

[0153] According to the above method, the ObGL1 gene knockout vector CRISPR-Cas9-ObGL1 was transformed into rice Zhonghua 11 (abbreviated as ZH11) through the mediation of Agrobacterium tumefaciens strain EHA105 to obtain T0 generation ObGL1 gene knockout seedlings. The identification method was to extract DNA from the leaves of the transgenic seedlings and amplify it by PCR using hygromycin primers. The CRISPR-Cas9-ObGL1 vector and sterile water were used as positive and negative controls, respectively. The plant that amplified the same band as the CRISPR-Cas9-ObGL1 vector was the T0 generation com-positive plant.

[0154] Leaves from T0-generation ObGL1 knockout seedlings at the seedling stage were excised and identified for gene knockout using the following method: PCR amplification of sequences near the target site T1 was performed using primers 5'-CCTTCTCACCTCCCAAACCA-3' and 5'-CGTACAAGTCCGAGTGCACC-3'. The resulting PCR products were sequenced to identify the mutations at the target site of the T0-generation ObGL1 knockout strain. The identified T0-generation ObGL1 knockout strain was self-pollinated for two consecutive generations to obtain the T2-generation ObGL1 knockout homozygous mutant gl1. Sequencing confirmed that, compared to the wild-type Zhonghua 11, the genomic DNA of the ObGL1 knockout homozygous mutant gl1 contained the following mutations in the ObGL1 genes on both homologous chromosomes: a 1 bp thymine deoxyribonucleotide T was inserted between nucleotides 735 and 736 of sequence 2 in the rice genome sequence listing, resulting in a frameshift mutation in the protein encoded by the ObGL1 gene and premature termination ( Figure 3 c), thereby knocking out the ObGL1 gene in the rice Zhonghua 11 (ZH11) genome.

[0155] According to the above method, the com-GL1 vector was transformed into the ObGL1 knockout homozygous mutant gl1 via Agrobacterium tumefaciens strain EHA105, resulting in T0-generation seedlings with complemented ObGL1 genes. Transgenic seedlings were identified by extracting DNA from leaves and amplifying it using hygromycin primers. The com-GL1 vector and sterile water were used as positive and negative controls, respectively. Plants that expressed the same band as the com-GL1 vector were considered T0-generation com-positive plants.

[0156] According to the above method, the OE-GL1 vector was transformed into rice Zhonghua 11 (abbreviated as ZH11) via Agrobacterium tumefaciens strain EHA105, resulting in T0-generation seedlings overexpressing the ObGL1 gene. Transgenic seedlings were identified by extracting DNA from leaves and amplifying it using hygromycin primers. The OE-GL1 vector and sterile water were used as positive and negative controls, respectively. Plants that expressed the same band as the OE-GL1 vector were identified as T0-generation OE-positive plants.

[0157] The T0 generation com-positive plants and the T0 generation OE-positive plants were self-pollinated to obtain the T1 generation complementary line com and the overexpression line OE, and the corresponding T2 generation seeds were harvested after maturity.

[0158] Example 2, phenotypic identification

[0159] T2 seeds of the knockout line gl1, the complementation line com, and the overexpression line OE of ZH11 and GL1 were planted in the field at the Shangzhuang Experimental Station of China Agricultural University in Beijing (May to October) or in Sanya, Hainan Province (November to May). A randomized block design was used, with three rows per plot and 10 plants per row. Normal field management was followed.

[0160] Phenotypic findings such as Figure 3 , Figure 3 Transgenic validation of the ObGL1 gene. (a) Plant morphology of the knockout line gl1, complementation line com, and overexpression line OE of ZH11 and GL1 (bar = 10 cm); (b) Comparison of grain length between ZH11 and the three transgenic lines (bar = 5 mm); (c) Sequence comparison of the knockout line gl1 of ZH11 and GL1 at the knockout target site; red boxes indicate positions where a base has been inserted; (d) Comparison of grain length between ZH11 and the three transgenic lines (bar = 1 cm); (e) Comparison of cells visualized by scanning electron microscopy of the outer hulls of ZH11 and gl1 (bar = 100 μm); (fo) Comparison of agronomic traits between ZH11 and the three transgenic lines (n = 15); error bars represent standard deviations. **P < 0.01 (Student's t-test).

[0161] Plant height (cm): Randomly select 15 or more non-side row plants from each line and measure the height from the base of the stem to the highest ear. Figure 3 As shown in (f), the plant height of the mutant gl1 increased by about 11 cm (10%).

[0162] Seed length (mm): Fifteen individual plants were randomly selected, and about 100 seeds of each individual plant were measured using the Wanshen SC-G automatic seed analyzer. Figure 3 As shown in (g), knockout of the candidate gene gl1 in the ZH11 background significantly increased the grain length of the knockout line, while complementation and overexpression of the gene significantly reduced the grain length.

[0163] Seed width (mm): Fifteen individual plants were randomly selected, and about 100 seeds of each individual plant were measured using the Wanshen SC-G automatic seed analyzer. Figure 3 As shown in (h), the grain width of the knockout line gl1 was slightly reduced after knocking out the candidate gene in the ZH11 background, and the grain width was also slightly reduced after complementation and overexpression of the gene.

[0164] Thousand-grain weight (g): Weigh the seeds with measured grain shape and convert them into thousand-grain weight. Figure 3 As shown in (i), after knocking out the candidate gene in the ZH11 background, the thousand-grain weight of the knockout line gl1 was significantly increased, while the thousand-grain weight was significantly reduced by complementation and overexpression of the gene.

[0165] Relative gene expression: Total RNA of 1 cm2 panicles of the wild type Zhonghua 11 (ZH11), T2 generation ObGL1 gene knockout homozygous mutant gl1, com and OE were extracted and reverse transcribed. The cDNA obtained by reverse transcription was used as a template, and the primers were: CACGACGTCGCACACCTCCA and ACGCGCACGCACCTGAACAC for Real Time-PCR identification. The actin gene was used as the internal standard (F primer of the actin gene: GACTCTGGTGATGGTGTCAGC; R primer of the actin gene: GGCTGGAAGAGGACCTCAGG). The expression level of the internal standard gene was set as 1, and the expression level of the ObGL1 gene was determined. The experiment was repeated three times. Figure 3 As shown in (j), the relative expression level of the wild-type ZH11 ObGL1 gene was 1.637, the relative expression level of the knockout line gl1 was 1.031, the relative expression level of the complementation line com was 3.782, and the relative expression level of the overexpression strain OE was 32.067.

[0166] Effective number of ears: Select more than 15 individual plants in non-side rows to investigate the number of normal filling ears of each plant after maturity. Figure 3 As shown in (k), the tiller number of the knockout line gl1 was slightly increased.

[0167] Number of primary branches: Select more than 15 individual plants in non-side rows to investigate the number of primary branches of each main stem ear. Figure 3 As shown in (l), the average number of primary branches in the knockout line gl1 increased by 1.4 (10%).

[0168] Secondary branches: Select 15 or more individual plants from non-side rows and investigate the secondary branches of each individual plant. Figure 3 As shown in the middle (m), the average number of secondary branches in the knockout line gl1 increased by 10 (24%).

[0169] Grain number per ear: Fifteen or more individual plants from non-side rows were selected to measure the number of grains per ear in the main stem of each plant. As shown in the figure (n), the number of grains per ear in the main stem of the knockout line gl1 increased by approximately 32 grains (15%).

[0170] Yield per plant (g): Select 15 or more individual plants not in the side rows and weigh the weight of all the grains per plant. Figure 3 As shown in the middle (o), the yield per plant of the knockout line gl1 increased by about 4.7 g (20.1%).

[0171] The results showed that compared with ZH11 plants, the gl1 knockout line had increased grain length, plant height, number of grains per panicle, and yield per plant. The ObGL1 gene negatively regulates grain length, plant height, number of grains per panicle, and yield in rice.

[0172] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Use of a protein or a substance that regulates the expression of the coding gene of the protein or a substance that regulates the activity or content of the protein, characterized in that, The protein is ObGL1 protein, which is any one of the following: A1) A protein with an amino acid sequence of SEQ ID NO:3, A2) A protein with the amino acid sequence shown in SEQ ID NO:3 through substitution and / or deletion and / or addition of amino acid residues and having the same function, A3) A protein having more than 80% identity with the amino acid sequence defined in A1) or A2) and having the same function; A4) A fusion protein obtained by linking a tag to the end of the protein defined in any one of A1)-A3); The application is any one of the following: B1) Regulating plant yield, B2) Preparing a product for regulating plant yield, B3) Cultivating plants with changed agronomic traits, B4) Preparing a product for cultivating plants with changed agronomic traits, B5) Plant breeding, B6) Preparing a product for plant breeding.

2. The application according to claim 1, wherein The protein is derived from rice.

3. Application of biomaterials, characterized in that, The application is any one of the following: C1) Regulating plant yield, C2) Preparing a product for regulating plant yield, C3) Cultivating plants with changed agronomic traits, C4) Preparing a product for cultivating plants with changed agronomic traits, C5) Plant breeding, C6) Preparing a product for plant breeding; The biological material is any one of the following: D1) A nucleic acid molecule encoding the protein described in claim 1 or 2, D2) An expression cassette containing the nucleic acid molecule described in D1), D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2), D4) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3), D5) A transgenic plant cell line containing the nucleic acid molecule described in D1), or a transgenic plant cell line containing the expression cassette described in D2), D6) A transgenic plant tissue containing the nucleic acid molecule described in D1), or a transgenic plant tissue containing the expression cassette described in D2), D7) A transgenic plant organ containing the nucleic acid molecule described in D1), or a transgenic plant organ containing the expression cassette described in D2).

4. The application according to claim 3, wherein: The nucleic acid molecule of D1) is a gRNA targeting the protein coding gene described in claim 1.

5. A method for regulating plant agronomic traits, characterized in that, The method includes step M, and step M includes the step of regulating the agronomic traits of a plant by regulating the expression of the protein coding gene described in claim 1 or 2 in the plant.

6. A method for producing a plant with altered agronomic traits, characterized in that, The method includes step M, and step M includes the step of obtaining a plant with changed agronomic traits by regulating the expression of the protein coding gene described in claim 1 or 2 in the plant.

7. The method according to claim 5 or 6, characterized in that, The protein is derived from rice.

8. The application according to claim 1 or 3 or the method according to claim 5 or 6, characterized in that The regulation is at least one of the following 6 regulations: E1) Regulation carried out at the transcriptional level of the coding gene, E2) Regulation carried out after transcription of the coding gene, E3) Regulation of the RNA transport of the coding gene, E4) Regulation of the translation of the coding gene, E5) Regulation of the mRNA degradation of the coding gene, E6) Post-translational regulation of the gene.

9. The application according to claims 1-4 or the method according to claims 5-7, characterized in that, The plant is any one of the following: F1) Dicotyledonous plants or monocotyledonous plants; F2) Plants of the order Poales, F3) Gramineous plants, F4) Oryza plants, F5) Rice.

10. A product, characterized in that, The product is the protein described in claim 1 or the biological material described in claim 3.

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