Application of LA6 gene in regulation and control of tillering angle of rice

Abstract

The invention relates to the technical field of biology, and particularly discloses a method for reducing the tillering angle of rice and / or increasing the plant height of the rice and / or increasing the yield of the rice by increasing the expression of an LA6 gene in the rice. The invention clones a new gene LA6 participating in regulation and control of the rice tillering angle, and provides a new molecular theoretical basis for analyzing a rice tillering angle regulation and control network.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology and relates to application of LA6 gene in regulating rice tillering angle. Background Art

[0002] Plant type generally refers to the morphological characteristics of various plant tissues and their three-dimensional structure, that is, the way they are arranged in space. The plant type of the aboveground part of the plant includes branching patterns, the size, shape and location of leaves and flowers, etc.

[0003] Tiller angle, the angle between the lateral tillers and the main stem, reflects the plant's degree of looseness and is a key factor in determining planting density and photosynthetic efficiency. Rice varieties exhibit varying tiller angles. For example, wild rice tends to grow prostrate, while cultivated rice generally tends to grow upright. Indica varieties tend to have larger tiller angles and looser plants, while japonica varieties tend to have smaller tiller angles and more compact plants. Furthermore, within the same rice variety, the tiller angle undergoes dynamic changes throughout its growth cycle, divided into two phases: an early, scattered phase and a later, compact phase. From sowing to 60 days, the tiller angle increases continuously. After 60 days, the tiller angle gradually decreases, reaching its minimum at 100 days. This dynamic change in tiller angle throughout the growth cycle is crucial for improving rice yield. Early tillering reduces shading between leaves, quickly establishing a larger light-intercepting area, creating more space for later tillering growth. This also helps suppress weed growth and improves resistance to weed damage. Later tillering gradually becomes more compact, effectively improving light transmission and ventilation between mature plants, thereby increasing photosynthetic efficiency and reducing disease infection. Furthermore, a more compact plant shape at maturity facilitates mechanized harvesting. Therefore, a proper tillering angle directly affects the photosynthetic efficiency, stress resistance, yield, and quality of a rice population, making it crucial for rice production.

[0004] Therefore, understanding the regulatory mechanism of rice tiller angle has important theoretical and practical significance for breeding high-yield rice with ideal plant morphology. Although multiple genes regulating rice tiller angle have been cloned and identified, the molecular regulatory network of rice tiller angle remains largely unknown. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to regulate the tillering angle of rice to obtain an ideal plant shape, thereby increasing rice yield.

[0006] In order to solve the above technical problems, the present invention provides a method for reducing the tillering angle of rice and / or increasing the plant height of rice and / or increasing the rice yield by increasing the expression of the LA6 gene in rice, wherein the LA6 gene is a gene encoding the LA6 protein, and the LA6 protein is the following A1, A2 or A3 protein:

[0007] A1, the amino acid sequence is the protein of SEQ ID No. 2 in the sequence listing;

[0008] A2, a protein having more than 80% identity with the protein shown in A1 and similar function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2 in the sequence listing;

[0009] A3: A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1 or A2.

[0010] In the above method, SEQ ID No. 2 in the sequence listing consists of 601 amino acid residues.

[0011] In the above methods, identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, the identity of a pair of amino acid sequences can be calculated by searching in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, all filters to OFF, 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. The identity value (%) can then be obtained.

[0012] In the above method, the above 80% or greater identity may be at least 81%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0013] In the above method, the LA6 protein may be derived from rice.

[0014] In the above method, the LA6 gene may specifically be a nucleic acid molecule whose coding sequence of the coding chain is SEQ ID No. 1 in the sequence list.

[0015] The above method includes the steps of introducing the LA6 gene into a recipient rice to obtain rice with a reduced tillering angle and / or rice with an increased plant height and / or rice with an increased yield; the tillering angle of the rice with a reduced tillering angle is smaller than the tillering angle of the recipient rice; the plant height of the rice with an increased plant height is higher than the plant height of the recipient rice; and the yield of the rice with an increased yield is higher than the yield of the recipient rice.

[0016] In the above method, the LA6 gene can be modified as follows before being introduced into the recipient rice to achieve better expression:

[0017] 1) Modifying the gene sequence adjacent to the initiator methionine to allow efficient translation initiation; for example, using sequences known to be effective in plants;

[0018] 2) Connected to various plant-expressed promoters to facilitate their expression in plants; the promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the temporal and spatial requirements of expression, and also depends on the target species; for example, a tissue- or organ-specific expression promoter, depending on the stage of development at which the receptor is required; although many promoters derived from dicots have been shown to be functional in monocots, and vice versa, ideally, dicot promoters are selected for expression in dicots, and monocot promoters are used for expression in monocots; in one embodiment of the present invention, the 35S promoter is used to drive the LA6 gene;

[0019] 3) Linking to a suitable transcription terminator can also improve the expression efficiency of the gene of the present invention; for example, tml from CaMV, E9 from rbcS; any available terminator known to function in plants can be linked to the gene of the present invention;

[0020] 4) Introducing enhancer sequences, such as intron sequences (e.g., from Adhl and bronze) and viral leader sequences (e.g., from TMV, MCMV, and AMV).

[0021] The LA6 gene can be introduced into plant cells using conventional biotechnology methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition).

[0022] In the above method, the rice with reduced tillering angle and / or increased plant height and / or increased yield can be transgenic rice or rice obtained through conventional breeding techniques such as hybridization.

[0023] The present invention also provides a protein, which is the LA6 protein.

[0024] The present invention also provides biological materials related to the LA6 protein, which also fall within the protection scope of the present invention.

[0025] The biological material related to the LA6 protein provided by the present invention is any one of the following B1 to B5:

[0026] B1, a nucleic acid molecule encoding LA6 protein;

[0027] B2, an expression cassette containing the nucleic acid molecule described in B1;

[0028] B3, a recombinant vector containing the nucleic acid molecule described in B1, or a recombinant vector containing the expression cassette described in B2;

[0029] B4, a recombinant microorganism containing the nucleic acid molecule described in B1, or a recombinant microorganism containing the expression cassette described in B2, or a recombinant microorganism containing the recombinant vector described in B3;

[0030] B5. A transgenic plant cell line containing the nucleic acid molecule described in B1, or a transgenic plant cell line containing the expression cassette described in B2, or a transgenic plant cell line containing the recombinant vector described in B3.

[0031] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0032] In the above biological material, the nucleic acid molecule B1 is the LA6 gene, specifically a nucleic acid molecule whose coding sequence of the coding chain is SEQ ID No. 1 in the sequence list.

[0033] In the aforementioned biological material, the expression cassette (LA6 gene expression cassette) described in B2 refers to DNA capable of expressing the LA6 gene in host cells. This DNA may include both a promoter for initiating transcription of the LA6 gene and a terminator for terminating transcription of the LA6 gene. Furthermore, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to, the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP," Chao et al. (1999) Plant Physiology 120:979-992); the chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); the tomato proteinase inhibitor II promoter (PIN2) or the LAP promoter (both inducible by methyl jasmonate); heat shock promoters ( U.S. Pat. No. 5,187,267 ); tetracycline-inducible promoters ( U.S. Pat. No. 5,057,422 ); seed-specific promoters, such as the millet seed-specific promoter pF128 ( CN101063139B ( China Patent No. 2007 06063139B )). 10099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin, and soybean beta-conglycin (Beachy et al. (1985) EMBO J. 4: 3047-3053)). These can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, for example, Odell et al. (1996) EMBO J. 4: 3047-3053). 985 ) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0034] Existing plant expression vectors can be used to construct a recombinant expression vector containing the LA6 gene expression cassette. These include binary Agrobacterium vectors and vectors useful for plant microprojectile bombardment, such as pTCK303, pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb (CAMBIA). These plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3 ' end of the mRNA precursor, such as the non-translated region transcribed at the 3 ' end of Agrobacterium crown gall induction (Ti) plasmid gene (such as nopaline synthase gene Nos) and plant gene (such as soybean storage protein gene) all have similar functions. When using the gene construction plant expression vector of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, but must be identical to the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The source of the translation control signal and the start codon is extensive, and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene. To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be modified to include genes encoding color-changing enzymes or luminescent compounds that can be expressed in plants (e.g., GFP, GUS, luciferase), antibiotic marker genes (e.g., the nptII gene, which confers resistance to kanamycin and related antibiotics; the bar gene, which confers resistance to the herbicide phosphinothricin; the HPT and hph genes, which confer resistance to the antibiotic hygromycin; the dhfr gene, which confer resistance to methatrexate; and the EPSPS gene, which confer resistance to glyphosate), chemical resistance marker genes (e.g., herbicide resistance genes), and mannose-6-phosphate isomerase genes, which provide the ability to metabolize mannose. For the safety of transgenic plants, it is possible to omit any selectable marker genes and directly screen transformed plants using stress.

[0035] In the above-mentioned biological materials, the recombinant microorganisms can specifically be yeast, bacteria, algae and fungi.

[0036] To address the above technical issues, the present invention further provides a botanical agent for regulating rice tillering angle and / or increasing rice yield and / or rice plant height. The active ingredient of the botanical agent is a substance that promotes or increases the expression of the LA6 gene or the abundance of the LA6 protein. The substance may include the LA6 protein and / or the biological material.

[0037] The active ingredients of the above-mentioned botanical agents may also contain other biological components and / or non-biological components. Those skilled in the art can determine the other active ingredients of the above-mentioned botanical agents based on the effects of promoting the regulation of rice tillering angle and / or increasing rice yield and / or increasing rice plant height.

[0038] The present invention also protects the use of the method, the LA6 protein, or the biological material in rice breeding or rice production.

[0039] The present invention also protects the use of the plant reagent in rice production.

[0040] The present invention cloned a new gene LA6 involved in regulating the tillering angle of rice, revealing that LA6 can participate in controlling the formation of the tillering angle of rice by regulating the gravity response of the aboveground part of rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the phenotype of the rice mutant la6 in Example 1 of the present invention, and the control is the wild type Ningjing 3 (labeled as NG3). Figure 1 A shows the phenotypes of the wild type and mutant at the jointing stage, and the scale bar is 20 cm. Figure 1 B shows the phenotypes of the wild type and mutant stem bases at the jointing stage, with a scale of 5 cm. Figure 1 C is the statistical analysis of tiller angles of wild type and mutants at the jointing stage. Figure 1 D is the degree of bending of the aboveground parts of wild-type and la6 mutant seedlings after 72 hours of gravity stimulation. The arrow indicates the direction of gravity, and the scale bar is 2 cm. Figure 1 E shows the dynamic changes in the degree of shoot bending during gravity stimulation in wild type and la6 mutants. The values ​​in the figure are mean ± standard deviation (n = 15 samples), and ** indicates a significant difference at the 0.01 level in the t-test.

[0042] Figure 2 For the cloning and functional complementation verification of the LA6 gene in Example 1 of the present invention, the mutant is la6, the wild type is Ningjing 3 (NG3), and the genetically complemented plant is pLA6C. Figure 2 A is a schematic diagram of the LA6 gene structure, the location of the la6 mutation site, the mutation sequence and the mutated amino acid. The black solid box represents the coding region of LA6, the black line between the two black solid boxes represents the intron, and the black arrow indicates the mutation site. Figure 2B is the expression analysis of LA6 in wild type and mutants. Figure 2 C represents the phenotypes of wild type, mutant and genetically complemented plants at the jointing stage, and the scale bar is 20 cm. Figure 2 D represents the tiller angle statistics of wild-type, mutant, and genetically complemented plants at the jointing stage. The values ​​in the figure are mean ± SD (n = 10 samples). ** indicates a significance level of 0.01 in the t-test. Different lowercase letters in the bar graph indicate significant differences in the analysis of variance and Tukey's test.

[0043] Figure 3 This is the phenotype of the LA6 gene knockout mutant in Example 1 of the present invention. The wild type is Zhonghua 11 (ZH11), and the LA6 knockout mutants obtained using CRISPR-Cas9 technology are CR-la6-1, CR-la6-2, and CR-la6-3. Figure 3 Figure A is a schematic diagram of the CRISPR / Cas9 target site and gene editing results of the LA6 gene. Red font represents the sgRNA targeting sequence, underlined positions indicate the sequence adjacent to the protospacer motif, and dotted lines indicate the deletion sequence. Figure 3 B is the phenotype of the LA6 knockout mutant obtained using CRISPR-Cas9 technology at the heading stage, and the scale bar is 20 cm. Figure 3 C shows the tiller angle statistics of the wild type and LA6 knockout mutant. The values ​​in the figure are mean ± SD (sample number n = 10), ** indicates a significance level of 0.01 in the t-test, and different lowercase letters in the bar graph represent significant differences in the analysis of variance and Tukey's test. Figure 3 D shows the degree of bending of the aboveground part of the wild type and LA6 knockout mutant after 72 hours of gravity stimulation. The arrow indicates the direction of gravity, and the scale bar is 2 cm. Figure 3 Figure E shows the dynamic changes in the degree of shoot bending during gravity stimulation in wild type and LA6 knockout mutants. The values ​​in the figure are mean ± standard deviation (n = 15 samples), and ** indicates a significant level of 0.01 in the t-test. DETAILED DESCRIPTION

[0044] 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.

[0045] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0046] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0047] The field cultivation and phenotypic identification methods of the rice materials in the following embodiments are as follows: select mature and full rice seeds, soak them at room temperature for 3 days, transfer them to a 30°C incubator for germination for 1 day, sow the white seeds on the seedbed for conventional seedling cultivation, and transplant them to the field after about 1 month, with a distance of 30 cm between individual plants. The tillering angle of rice is mainly measured with a protractor during the tillering period or the flowering period to maturity. The leaf angle is the angle between the second leaf of the main ear at maturity. Yield-related traits such as plant height, number of tillers, ear length, number of grains per ear, fruit set rate, single plant yield and 1000-grain weight are mainly counted at maturity, and the experimental data are analyzed using SPSS19.0 and Excel 2010. The experimental fields are located in the Changping Experimental Farm in Beijing and the Nanfan Base in Lingshui County, Hainan Province. Field management such as water, fertilizer, and pest control are carried out according to local production conditions.

[0048] Common molecular cloning experiments involved in the following examples, such as fragment amplification, enzyme digestion, transformation, colony PCR, etc., were completed with reference to the Molecular Cloning Experiment Guide (3rd edition).

[0049] In-fusion (In- HD Cloning Kit, Clontech: 639650) system was used to complete the construction of the vector. For specific operation steps, please refer to the instruction manual of the enzyme.

[0050] In the following examples, PCR (Polymerase Chain Reaction) primer design was primarily performed using PrimerPremier 5 software. qRT-PCR primer design was primarily performed using Beacon Designer 7 software. Sequence review and editing were primarily performed using Vector NTI Advance 10 and BioXML 2.6 software. DNA sequencing results were primarily reviewed and aligned using Lasergene 7 software. All primer synthesis and sequencing (except genome resequencing) described herein were performed by Beijing Ruibiotech Biotechnology Co., Ltd. Genome resequencing was performed by Beijing Berry Genomics Co., Ltd.

[0051] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0052] The rice material Ningjing 3 (Oryza sativa L. subsp. japonica cv. Ninggeng 3, abbreviated as NG3) in the following examples is a variety of the College of Agriculture of Nanjing Agricultural University, which passed the Anhui Provincial Approval in 2009 and is numbered: Wandao 2009005.

[0053] In the following examples, field sampling, phenotypic observation, and photography of all rice materials (including transgenic materials) were completed in Beijing or Hainan.

[0054] Yeast strain Y2Hgold was a product of Clontech.

[0055] In the following examples, the vector pDONR221 is a product of Invitrogen.

[0056] The CRISPR / Cas9 vector system for creating rice gene knockout mutant materials in the following examples was purchased from Beijing Viewsolid Biotechnology Co., Ltd.

[0057] Restriction endonucleases and T4 DNA ligase were products of NEB; rTaq enzyme was a product of TAKARA; high-fidelity enzymes KOD FX (Neo) and KOD Plus (Neo) were products of TOYOBO; DNA recovery kits QIAquick Gel Extraction Kit and QIAEX II Gel Extraction Kit were products of QIAGEN; the recombinase system In- HD Cloning Kit is a product of Clontech; Plasmid Extraction Kit Plus SVMinipreps DNA Purification System is a product of Promega; large-scale plasmid extraction kit Xtra Midi EF is a product of MACHEREY-NAGEL; the RNA extraction reagent Trizol is a product of Invitrogen; the DNA digestion kit TURBO DNA-free TM Kit is a product of Ambion; RNA reverse transcription kit SuperScript TM III First-Strand Synthesis System is a product of Invitrogen; Fluorescent Real-Time Quantitative PCR Kit SsoFast TM Supermix was a product of BIO-RAD; the main ingredient of Murashige and Skoog (MS) nutrient solution, MS, was a product of Dochefa. Other commonly used molecular biology and chemical reagents were imported or domestically produced of analytical grade.

[0058] Example 1 Acquisition of gene LA6

[0059] 1. Cloning of gene LA6

[0060] In order to reveal the molecular regulatory mechanism of rice tiller angle, a series of rice mutants with increased tiller angles were screened in the early stage. The applicant cultivated one of the mutants, la6, in the Ningjing 3 (NG3) background and conducted phenotypic identification of rice materials in the field. Phenotypic observations showed that compared with the wild type Ningjing 3 (NG3), the mutant la6 showed scattered growth characteristics ( Figure 1 A and Figure 1 B), the statistical results showed that the tillering angle of the mutant la6 was significantly increased ( Figure 1 Since the relationship between gravity response and tillering angle of rice is very close, in order to study whether LA6 regulates tillering angle by affecting gravity response, the gravity response of seedlings was tested. The results showed that compared with the wild type, the gravity response of la6 was significantly weakened ( Figure 1 D and Figure 1 Therefore, LA6 regulates rice tiller angle formation by affecting gravity response.

[0061] To clone the gene controlling tiller angle in rice, bulk segregant analysis (BSA) was employed. First, la6 and NG3 were backcrossed. Phenotypic analysis of the F2 segregating population revealed a 3:1 segregation ratio between wild-type and mutant traits, suggesting that the la6 tiller angle phenotype is controlled by a recessive gene. Fifty wild-type and scattered-type plants were selected from the F2 population, and equal leaves from each plant were collected to form two pools. DNA from each pool was extracted using the CTAB method. After quality control, DNA from each pool was sent to Beijing Berry Genomics Co., Ltd. for whole-genome resequencing. MutMap was used to identify target genes. Genomic DNA from NG3 and the la6 mutant was used as templates to amplify the fragments containing the candidate mutation sites. Sequencing and sequence analysis revealed potential mutation sites and candidate genes. Finally, it was found that there was a single base T deletion in an exon (T deletion of SEQ ID No.1 1963) in a region that was well linked to the scattered phenotype. This deletion caused a frameshift and premature termination of protein translation ( Figure 2 A). Based on the mutation site information, a candidate gene was screened and named LA6.

[0062] The full-length sequence of LA6 is shown in SEQ ID No. 1 (wherein positions 1-1770 are the promoter region, positions 1771-2739 are the first exon, positions 2740-3210 are the intron, positions 3211-4047 are the second exon, and positions 4048-4567 are the downstream sequence), the cDNA sequence of LA6 is shown in SEQ ID No. 2, and the amino acid sequence of the encoded LA6 protein is shown in SEQ ID No. 3.

[0063] SEQ ID NO.1

[0064]

[0065] SEQ ID NO.2

[0066]

[0067] SEQ ID NO.3

[0068] MEKIQSDCPYPGCFFCVMKEANPSKRRASVLKFFRELPSQDDDGQVLPISGLWNTAMAHPNDPEFINLGIFECMSALIWKGLKNRRWLSHDQNIYIPYYAAHIIGSYTMNMEEFAERAVRAGVIPPLVELLRGRLTWVEQRVAVRALGHL ATYPSTFPAVADHGEVLELAIQLASSSLEIVYSHFYQFVDRRIGYHCDLLTRGMGGVEMESRKAEEWASQLQCWSLQLINCFAFKSEFLHDICKADFLVKLPGMWGGLVNENSPAGVGLLRTICQSKLGRGHVANIPSVVEALCNIARSS DDWQYMAVDCLLWLVQDSNTCHKVIDRVASTLIDLANISMLGDYKKLGDTIVTVLQECMQQYANSRNSISTHTKEQIDELLSSKQSFKLEKNMPKEDLHIKQAAALVVKLEGNSLFSSGNIAGAAAKYSEALALCPMKSKKERVVLYSNR AQCYLLLQQPLAAISDATRALCLHSPLNRHAKSLWRRAQAYDMLGLAKESLLDAILFINECSQSNDPDLSLKQNKVPDYAERLVKKQMRTAWLFREAALKHGGIHCEGEASDAFGQEADDSEWETASESDAENDATGEADDETEWKNDNQV

[0069] Sequence analysis and alignment revealed that LA6 encodes a conserved protein of unknown function in plants. qRT-PCR was used to detect the expression of LA6 in wild-type NG3 and mutant la6. The primer sequences for the LA6 gene are as follows:

[0070] LA6-qPCR-F:TTGTGGACCGAAGGATTG

[0071] LA6-qPCR-R:GCGAAGCAGTTAATGAGTTG

[0072] Rice UBIQUITIN (LOC_Os03g13170) was used as an internal reference. The primer sequences for the internal reference are as follows:

[0073] Ubi-qPCR-F:AACCAGCTGAGGCCCAAGA

[0074] Ubi-qPCR-R: ACGATTGATTTAACCAGTCCATGA

[0075] Total RNA from wild-type NG3 was extracted using Trizol (Life Technologies: 15596-018). The extracted total RNA was dissolved in 50 μL of nuclease-free water and analyzed for quality by electrophoresis on a 1.2% (w / v) formaldehyde-denaturing agarose gel. The integrity and expression levels of 28S, 18S, and 5S rRNAs were assessed. RNA was quantified by measuring the OD values ​​at 230, 260, and 280 nm using a NanoDrop 2000 UV-Vis spectrophotometer.

[0076] The quantified RNA was analyzed using TURBO DNA-free TM Kit (Life Technologies: AM1907) was used to remove the DNA that may remain during the total RNA extraction process. The first-strand cDNA was prepared using total RNA as a template using the III First-Strand Synthesis System (Life Technologies: 18080-051).

[0077] qRT-PCR reactions were prepared using Bio-RAD's SsoFast EvaGreen supermix, consisting of 5 μL of supermix, 1 μL of Primer-F, 1 μL of Primer-R, 1 μL of cDNA, and 2 μL of H₂O. The quantitative PCR instrument was a Bio-RAD CFX96. The PCR protocol was as follows: denaturation at 95°C for 30 seconds, followed by 40 cycles of melting at 95°C for 5 seconds, annealing and extension at 60°C for 10 seconds, and plate reading for 5 seconds. Melting curve analysis was performed at 65-95°C, 0.5°C / 5 seconds, with a plate reading for 5 seconds.

[0078] The results showed that the expression of LA6 in the mutant was significantly reduced ( Figure 2 B), indicating that the mutation of the LA6 gene may lead to the scattered phenotype of the mutant.

[0079] 2. Construction of transgenic plants

[0080] 2.1 Construction of transgenic vector

[0081] (1) Construction of LA6 gene genetic complementation vector

[0082] The gDNA of Ningjing 3 (NG3) seedlings was extracted and used as a template to amplify a 4567 bp genomic sequence (SEQ ID NO.1), including the promoter sequence, coding region, and 520 bp downstream of the stop codon. The LA6 full-length complementary sequence fragment (SEQ ID NO.1) was recovered and the binary vector pCambia1300 was digested with BamH I-HF and Sal I-HF, and the correctly sequenced LA6 full-length complementary transgenic vector was obtained by in-fusion homologous recombination.

[0083] Full-length complementary construct:

[0084] The forward primer is LA6 ORF-F, and the reverse primer is LA6 ORF-R. The primer sequences are as follows:

[0085] Forward primer: CGGTACCCGGGGATCCGCTATTGTGACTTTGTATGCCAGGT

[0086] Reverse primer: ATGCCTGCAGGTCGACAACTACTCCCCGAATAGAACACATC

[0087] (2) Construction of LA6-CR transgenic vector

[0088] The full-length CDS sequence of LA6 was analyzed using the CRISPRdirect online gene knockout target design tool (http: / / crispr.dbcls.jp / ), and two target sites with low off-target efficiency were finally selected in the coding region, namely positions 2657-2676 of SEQ ID No.1 and positions 2722-2741 of SEQ ID No.1.

[0089] Target sequence:

[0090] Target 1:TTGCCCGTTCTTCAGATGAC

[0091] Target 2: TCAAATACATGTCATAAGGT

[0092] According to the plant Cas9 / gRNA plasmid construction kit (Catalog No. VK005-01) provided by Beijing Viewsolid Biotechnology Co., Ltd., the construction was carried out according to its instructions. The final LA6 knockout transgenic vector expressing the correctly sequenced sgRNA sequences for the two target sites and the Cas9 protein was named vector LA6-CR.

[0093] 2.2 Agrobacterium-mediated genetic transformation of rice

[0094] The Agrobacterium-mediated rice genetic transformation is described in the reference "Hiei, Y., Komari, T., and Kubo, T. (1997). Transformation of rice mediated by Agrobacterium tumefaciens. Plant Mol. Biol. 35, 205-218." The specific process is as follows:

[0095] (1) Callus induction of mature rice embryos: Select mature and plump rice seeds, remove the husks, wash with 75% (v / v) ethanol for 1 minute, then disinfect with 2.5% (v / v) sodium hypochlorite solution for 45 minutes, rinse with sterile water 3-4 times, and then sow on NB medium to induce callus tissue. Subculture once every two weeks. During subculture, select embryonic callus tissue with a smooth, dense surface and light yellow color;

[0096] (2) Agrobacterium transformation and culture: The transgenic plasmid was transformed into Agrobacterium strain EHA105 by electroporation, and then spread on YEP solid medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, and grown at 28°C. After 2-3 days, the positive single colony was picked and inoculated into liquid YEP medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, and cultured at 28°C with shaking until the OD 600 The bacteria were collected by centrifugation at 5,000 rpm for 10 minutes at room temperature and then resuspended in Agrobacterium infection solution to an OD of 0.8-1.0. 600 is about 1, the rice callus to be transformed;

[0097] (3) Co-cultivation of Agrobacterium and Rice Callus: Callus tissue that has been grown for 4 days on fresh subculture medium and is in good condition is co-cultivated with an appropriate amount of Agrobacterium-infused solution for 20 minutes, shaking occasionally. The callus is then removed, and the excess bacterial solution on the surface is removed with sterile filter paper. The callus is then transferred to a culture dish containing multiple layers of sterile filter paper and cultured in the dark at 22°C for 2-3 days. Afterwards, the callus is transferred to NB medium containing 50 mg / L hygromycin and 100 mg / L carbenicillin for selection and culture.

[0098] (4) Screening, differentiation and plant regeneration of resistant callus: The selection medium was changed every 7 days. Each time the medium was changed, resistant callus with a smooth, dense and light yellow surface was selected for further culture. After 4-5 consecutive resistance screenings, the vigorously growing resistant callus was transferred to the differentiation medium. The culture was first cultured in the dark at 28°C for 3-5 days, and then transferred to normal light conditions for culture. After about 4 weeks, seedlings emerged. After cutting off the roots, the seedlings were transferred to the rooting medium for two rooting cultures. After that, the seedlings were moved to the greenhouse and placed in a cool place for a few days before being moved to the field.

[0099] To confirm whether LA6 regulates the tillering angle of rice, the applicant used the Agrobacterium-mediated rice genetic transformation method described above to transform the mutant la6 with the full-length complementation vector. After sequencing, positive genetic complementation plants were obtained. Phenotypes of multiple independent lines of transgenic plants were observed and it was found that the positive lines could well restore the mutant's phenotype of increased tillering angle and reduced plant height ( Figure 2 C and Figure 2 D), indicating that mutations in the LA6 gene are responsible for the la6 phenotype.

[0100] In order to further confirm that the phenotype of the la6 mutant is caused by the mutation of the LA6 gene and to facilitate the subsequent analysis of the genetic relationship of LA6 in the regulation of tillering angle, the applicant used the above-mentioned Agrobacterium-mediated rice genetic transformation method to transform the wild-type rice Zhonghua 11 (ZH11) with the LA6 knockout transgenic vector LA6-CR, and obtained multiple homozygous strains CR-la6 with LA6 gene knockout at different targets (numbered CR-la6-1, CR-la6-2, CR-la6-3, etc., respectively). Figure 3 A is the CRISPR / Cas9 target site of the LA6 gene and the gene editing results of CR-la6-1, CR-la6-2, and CR-la6-3. Phenotypic observation revealed that, consistent with the phenotype of the la6 mutant, the CR-la6 plants showed a larger tillering angle and shorter plant height (see Figure 3 B. Figure 3 C. Figure 3 D and Figure 3 E), thus further confirming that the mutation of the LA6 gene is the cause of the la6 phenotype.

[0101] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A method for reducing the tillering angle of rice and / or increasing the height of rice plants and / or increasing rice yield by increasing the expression of LA6 gene in rice, characterized in that: The LA6 gene is a gene encoding the LA6 protein, and the LA6 protein is the following A1, A2 or A3 protein: A1, the amino acid sequence is the protein of SEQ ID No. 2 in the sequence list; A2, a protein having more than 80% identity with the protein shown in A1 and having similar functions obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2 in the sequence list; A3: A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1 or A2.

2. The method according to claim 1, characterized in that: The coding sequence of the LA6 gene as the coding chain is a DNA molecule of SEQ ID No. 1 in the sequence list.

3. The method according to claim 1 or 2, characterized in that: The method comprises the steps of introducing the LA6 gene into a recipient rice to obtain rice with a reduced tillering angle and / or rice with an increased plant height and / or rice with an increased yield; the tillering angle of the rice with a reduced tillering angle is smaller than the tillering angle of the recipient rice; the plant height of the rice with an increased plant height is higher than the plant height of the recipient rice; and the yield of the rice with an increased yield is higher than the yield of the recipient rice.

4. The LA6 protein of claim 1.

5. The LA6 protein-related biological material according to claim 4, characterized in that: Any one of B1 to B5 below: B1, a nucleic acid molecule encoding LA6 protein; B2, an expression cassette containing the nucleic acid molecule described in B1; B3, a recombinant vector containing the nucleic acid molecule described in B1, or a recombinant vector containing the expression cassette described in B2; B4, a recombinant microorganism containing the nucleic acid molecule described in B1, or a recombinant microorganism containing the expression cassette described in B2, or a recombinant microorganism containing the recombinant vector described in B3; B5. A transgenic plant cell line containing the nucleic acid molecule described in B1, or a transgenic plant cell line containing the expression cassette described in B2, or a transgenic plant cell line containing the recombinant vector described in B3.

6. The biomaterial according to claim 5, characterized in that: The nucleic acid molecule B1 is a nucleic acid molecule whose coding sequence is the coding chain and is SEQ ID No. 1 in the sequence list.

7. A plant agent, characterized in that: The active ingredient of the reagent is a substance that promotes or increases the LA6 gene described in claim 1 or 2, or increases the abundance of the LA6 protein described in claim 1 or 2.

8. The plant agent according to claim 7, characterized in that: The substance comprises the LA6 protein according to claim 4 and / or the biological material according to claim 5 or 6.

9. Use of the method according to any one of claims 1 to 3, or the LA6 protein according to claim 4, or the biological material according to claim 5 or 6 in rice breeding or rice production.

10. Use of the plant agent according to claim 7 or 8 in rice production.

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