Application of CCT5 gene or encoded protein thereof in regulation and control of plant yield

Abstract

The invention discloses an application of a CCT5 gene or an encoded protein thereof in regulation and control of plant yield. Through GWAS analysis, the SiCCT5 gene is found from core germplasm resources of millet, the gene is over-expressed in rice, and it is proved that the SiCCT5 gene of millet has the function of regulating and controlling development of mesocotyl in rice. The OsCCT5 gene is further overexpressed in the rice, the rice with the overexpressed OsCCT5 gene is found to be increased in grain number per ear, grain size and thousand grain weight compared with the wild type, that is, the yield of the rice is obviously improved, and the rice OsCCT5 gene is proved to have the function of positively regulating and controlling the plant yield. The invention has an application prospect in the aspect of regulating and controlling the plant yield.

Description

Technical Field

[0001] The present invention relates to a yield-related gene isolated from a gramineous plant and its use, and in particular to a yield-related gene isolated from a gramineous plant. CCT5 Use of a gene or its encoded protein in regulating plant yield, belonging to CCT5 Genes and their areas of use. Background Art

[0002] Rice ( Oryza sativa ) is one of the world's major food crops. Finding and utilizing key genes that affect rice yield, especially those involved in regulating plant growth, development, and response to environmental stress, is the core direction of modern rice genetic improvement.

[0003] Genetic regulation of rice grain shape is a key area for improving rice quality and yield. In recent years, scientists have revealed a series of genes and their molecular mechanisms that regulate rice grain shape through the study of multiple genes, which has promoted the progress of rice breeding. GS3 The gene is an important regulatory gene for rice grain shape and has been shown to significantly affect grain length. This gene directly affects rice grain shape and yield by regulating cell division. GW2 Genes affect rice grain shape by regulating grain weight and shape. GW2 Mutations in the gene often result in smaller grain weight and may affect the quality of the rice; OsSPL16 The gene is closely related to the width and yield of rice grains. It increases grain width by promoting cell division, thereby improving grain shape and enhancing rice yield and grain quality. GSE5 Genes play a pleiotropic role in controlling rice grain shape, affecting not only rice grain shape but also grain transparency. Analyzing the mechanism of action of genes regulating grain shape not only provides a theoretical basis for improving rice grain shape, but also provides strong support for the development of efficient breeding strategies.

[0004] CCT5 As a newly discovered regulatory protein, the T-complex protein encoded by the gene plays a key role in the embryonic development, cell division and differentiation of plants. T-complex protein promotes the normal growth of rice at different developmental stages by regulating the signal transduction pathway related to plant hormones. Exploring its molecular mechanism for increasing seed yield has great potential for application in increasing crop yield. Summary of the invention

[0005] The main purpose of the present invention is to CCT5 Gene, CCT5 protein, containing CCT5 Gene expression cassette or containing CCT5 Recombinant plant expression vectors of genes are used to regulate plant yield.

[0006] In order to achieve the above object, the main technical solutions adopted by the present invention include: One aspect of the present invention is to CCT5 Gene, CCT5 protein, containing CCT5 Gene expression cassette or containing CCT5 Recombinant plant expression vectors of genes are used to regulate plant yield.

[0007] In a preferred embodiment of the present invention, the plant is a grass plant.

[0008] In a preferred embodiment of the present invention, the regulating plant yield is to increase plant yield; wherein, the increasing plant yield includes increasing the number of grains per ear, grain size or thousand-grain weight.

[0009] For reference, the present invention provides an embodiment, that is, by overexpressing the gene encoding the CCT5 protein related to plant yield in the plant, the expression amount or activity of the CCT5 protein related to plant yield is increased, thereby increasing the plant yield, and then increasing the number of grains per ear, grain size, and thousand-grain weight of the plant.

[0010] A preferred embodiment of the present invention is a method for increasing plant yield, comprising: overexpressing in a plant CCT5 Gene, CCT5 The expression of the gene is increased or the function or activity of the CCT5 protein is enhanced; for example, SiCCT5 Genes or derived from rice OsCCT5 The gene is connected to the expression control element to obtain a recombinant plant expression vector for expressing the gene in plants; the recombinant plant expression vector is transformed into plants, so that CCT5 The gene is overexpressed in plants, and the yield of the resulting transgenic plants is improved.

[0011] For reference, the present invention provides a CCT5 Gene plant recombinant expression vector, including: millet SiCCT5 Genes or derived from rice OsCCT5The gene is connected to the expression control element to obtain a recombinant plant expression vector; the recombinant plant expression vector may be composed of a 5' non-coding region, nucleotides shown in SEQ ID NO.2 or nucleotides shown in SEQ ID NO.4 and a 3' non-coding region; wherein the 5' non-coding region may include a promoter sequence, an enhancer sequence and / or a translation enhancement sequence; the promoter may be a constitutive promoter, an inducible promoter, a tissue or organ specific promoter; the 3' non-coding region may include a terminator sequence, an mRNA cleavage sequence, etc. Suitable terminator sequences may be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase terminator regions.

[0012] The recombinant plant expression vector may also contain a selective marker gene for selecting transformed cells, for selecting transformed cells or tissues. The marker gene includes genes encoding antibiotic resistance and genes conferring resistance to herbicidal compounds. In addition, the marker gene also includes phenotypic markers, such as β-galactosidase and fluorescent protein.

[0013] The transformation protocol and the protocol for introducing the polynucleotide or polypeptide into the plant may vary depending on the type of plant or plant cell being transformed. Suitable methods for introducing the polynucleotide into plant cells include microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-velocity ballistic bombardment. In certain embodiments, various transient transformation methods may be used to transform millet. SiCCT5 Gene or rice OsCCT5 Genes are provided to plants. Transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al. Plant Cell Reports. 1986. 5:81-84).

[0014] In a preferred embodiment of the present invention, the grass plant is rice.

[0015] Another aspect of the present invention is to provide a millet or rice source capable of regulating plant yield. CCT5 Genes and their proteins.

[0016] A preferred specific embodiment of the present invention, the CCT5 The gene is the nucleotide sequence shown in SEQ ID NO.2 OsCCT5 The gene or nucleotide sequence is shown in SEQ ID NO.4 SiCCT5 Gene.

[0017] In addition, those skilled in the art may also optimize the nucleotides shown in SEQ ID NO.2 and SEQ ID NO.4 to enhance the expression efficiency in plants.

[0018] Those skilled in the art can easily adopt known methods, such as directed evolution or point mutation, to CCT5 The nucleotide sequence of the gene is mutated. CCT5 Nucleotides with 75% or higher identity to the nucleotide sequence of a gene are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as the encoded protein has the function of regulating plant yield.

[0019] In addition, the nucleotide sequence described in the present invention can be DNA, such as cDNA, genomic DNA or recombinant DNA; it can also be RNA, such as mRNA or hnRNA.

[0020] In a preferred embodiment of the present invention, the CCT5 protein is derived from a plant of the Poaceae family.

[0021] In a preferred embodiment of the present invention, the CCT5 protein is a protein having 94% or more identity with the amino acid sequence shown in SEQ ID NO.1, and the protein still has the function or activity of regulating plant yield.

[0022] The percentage of sequence identity described in the present invention can be obtained by well-known bioinformatics algorithms, including Myers and Miller algorithm, Needleman-Wunsch global alignment method, Smith-Waterman local alignment method, Pearson and Lipman similarity search method, Karlin and Altschul algorithm, which are well known to those skilled in the art.

[0023] In a preferred embodiment of the present invention, the CCT5 protein is the OsCCT5 protein with the amino acid sequence shown in SEQ ID NO.1 or the SiCCT5 protein with the amino acid sequence shown in SEQ ID NO.3.

[0024] The present invention uses GWAS analysis to analyze genes regulating millet mesocotyl development and clones SiCCT5 Gene, by overexpression in Nipponbare rice SiCCT5 Genes, prove millet SiCCT5 The gene has the function of regulating the development of rice mesocotyls. On this basis, the present invention further conducted sequence alignment and phylogenetic analysis to determine the homologous protein OsCCT5 protein of millet SiCCT5 protein in rice; OsCCT5 Gene and further analysis OsCCT5 The expression pattern of the gene suggests that the gene may also play a role in regulating plant yield. OsCCT5 Rice traits of genes found to be overexpressed OsCCT5 The number of grains per ear, grain size, and 1,000-grain weight of rice with the gene have all increased, indicating that rice yield has been improved. OsCCT5 The gene has the function of positively regulating plant yield.

[0025] Definitions of terms used in this invention Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0026] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs (phosphorothioates, phosphoramidates, etc.) used in antisense technology. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0027] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. That is, descriptions directed to polypeptides apply equally to describing peptides and describing proteins, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked via covalent peptide bonds.

[0028] The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector such as a plasmid or may be integrated into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell, and the host cell may also be a monocotyledonous or dicotyledonous plant cell.

[0029] The term "operably linked" refers to a functional connection between two or more elements. The operably linked elements may be contiguous or non-contiguous.

[0030] The term "recombinant plant expression vector" means one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, together with vectors with helper plasmids, are most commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, selection markers engineered to be expressed in plant cells, heterologous DNA sequences to be transcribed, etc.

[0031] The term "transformation" refers to a process by which a heterologous DNA sequence is introduced into a host cell or organism.

[0032] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a plant cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 For millet SiCCT5 Gene cloning and functional verification; among them, Figure 1 -A is the Manhattan plot of GWAS analysis results; Figure 1 -B is a quantile plot (QQ plot) used to detect whether the p-value observed in the GWAS analysis conforms to the expected distribution; Figure 1 -C is a schematic diagram of the constructed pCam23A-SiCCT5 vector; Figure 1 -D represents wild-type rice WT and overexpression SiCCT5 Rice lines with genes SiCCT5 Gene expression detection results; Figure 1 -E represents wild-type rice WT and overexpression SiCCT5 The results of the mesocotyl length of the rice lines with the gene cultured in continuous darkness for 7 days. The arrows indicate the coleoptile nodes, and the mesocotyl is the part between the arrow and the seed. Figure 1 -F is wild type rice WT and overexpression SiCCT5 Statistical analysis results of mesocotyl length of rice lines with the gene cultured in continuous darkness for 7 days; Figure 1 -G represents wild-type rice WT and overexpression SiCCT5 Longitudinal sections of mesocotyls of genetic rice lines; Figure 1 -H represents wild-type rice WT and overexpression SiCCT5 Statistical analysis data of cell length of mesocotyls in rice lines with the gene; Figure 2 This is a phylogenetic tree constructed based on the foxtail millet SiCCT5 protein sequence; Figure 3 For rice OsCCT5 Gene overexpression and expression pattern analysis; Figure 3 -A is a schematic diagram of the constructed pCam23A-OsCCT5 vector; Figure 3 -B represents wild type rice WT and overexpression OsCCT5 Rice lines with genes OsCCT5 Gene expression detection results; Figure 3 -C is the OsCCT5 Gene expression detection; Figure 3 -D is OsCCT5 The staining results of GUS driven by gene promoter in seeds; Figure 3 -E is OsCCT5 The staining results of GUS driven by gene promoter in mesocytosis; Figure 4 Wild-type rice WT and overexpression OsCCT5 Phenotypic analysis of rice lines expressing genes; Figure 4 -A represents wild-type rice WT and overexpression OsCCT5 Plant type diagram of rice lines with the same gene; Figure 4 -B represents wild type rice WT and overexpression OsCCT5 Grain shape diagram of rice strains with genes, Figure 4 -C represents wild-type rice WT and overexpression OsCCT5 Observation results of the epidermal cell size of the lemma of rice lines with the gene; Figure 4 -D represents wild-type rice WT and overexpression OsCCT5 Statistical analysis results of the number of grains per ear in rice lines with the gene; Figure 4 -E represents wild-type rice WT and overexpression OsCCT5 Statistical analysis results of grain length of rice lines with the gene; Figure 4 -F is wild type rice WT and overexpression OsCCT5 Statistical analysis results of thousand-grain weight of rice lines with the gene; Figure 4 -G represents wild-type rice WT and overexpression OsCCT5 The statistical analysis results of the single-plant yield of rice lines with the same gene, Figure 4 -H wild-type rice WT and overexpression OsCCT5 The statistical analysis results of the width of lemma epidermal cells of rice lines with the gene are shown in Table 1. Figure 4 -I represents wild-type rice WT and overexpression OsCCT5 Statistical analysis results of the lemma epidermal cell length of rice lines with the same gene. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0035] Vectors, strains and experimental materials The pCam23A vector was preserved by the Institute of Biotechnology, Chinese Academy of Agricultural Sciences; Agrobacterium tumefaciens AGL1 was purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd., item number ZK296; the wild type rice was Nipponbare rice ( Oryza sativa L.ssp. Japonica variety Nipponbare ; hereinafter also referred to as wild-type rice WT), is preserved by the Crop High Light Efficiency Functional Genomics Team of the Institute of Biotechnology, Chinese Academy of Agricultural Sciences.

[0036] Test reagents The primers used in PCR were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the sequencing was completed by Beijing Qingke Biotechnology Co., Ltd. Sma I and XB I. Infusion recombinant enzyme and high-fidelity enzyme were purchased from Beijing Liuhetong Economic and Trade Co., Ltd. (TaKaRa); antibiotics were purchased from SIGMA, USA; the remaining reagents were domestic analytical grade.

[0037] The reagent formula used in the experiment is as follows: 2,4-D (2 mg / mL): Dissolve 2,4-D in a microwave oven with 5 mL to 10 mL of 1 N KOH, and add ultrapure water to the volume. Store at room temperature.

[0038] 6-BA (3 mg / mL): Weigh 150 mg of 6-BA, dissolve it in 5 mL of 1 N KOH, and then dilute to 50 mL with sterile water. Sterilize by filtration.

[0039] Tim (200 mg / mL): Dissolve 2 g of Tim in sterile water to make up to 10 mL and filter sterilize.

[0040] G418 (150 mg / mL): Dissolve to volume with sterile water and filter sterilize.

[0041] Rifampicin (25 mg / mL): Dissolve 0.5 g of Rifampicin (Rif) in 1 N NaOH and dilute to 10 mL with methanol; or dilute to 10 mL with DMSO and filter sterilize. Store at -20°C, working concentration 25 μg / mL.

[0042] Kanamycin (50 mg / mL): Weigh 0.5 g of kanamycin sulfate powder, add 10 mL of ultrapure water to dissolve and filter sterilize, the working concentration is 50 μg / mL.

[0043] Data processing GraphPad Prism 8 statistical software was used to process the data. The significance analysis of one set of data was performed using the Student test. P < 0.05 (*) indicated a significant difference, P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****) indicated an extremely significant difference. The significant difference analysis of multiple sets of data was determined by one-way analysis of variance (ANOVA) and Tukey's multiple comparison test. In the results, the letter a was marked on the largest mean. If the second set of data had a significant difference, it was marked with b, and if there was no significant difference, it was marked with a, and so on.

[0044] Test Example 1 Millet SiCCT5 Gene acquisition and functional analysis 1 GWAS analysis of mesocotyl development using foxtail millet core germplasm resources 637 millet germplasm resources were germinated in the dark, and the length of their mesocotyls was counted. Combined with the genomic variation data, GWAS analysis was used to analyze the genes regulating millet mesocotyl development. Figure 1 -A, the blue dashed line in the Manhattan plot represents the genome-wide significance threshold (P = 9.50 × 10 -6 ), the results showed that there was a P value lower than 9.50×10 -6 The main SNP association site (the position indicated by the red triangle) and there is a significant association signal ( Figure 1 -B), cloned to this region through further bioinformatics analysis and verification SiCCT5 gene (Seita.2G010200).

[0045] The amino acid sequence of the SiCCT5 protein is as follows: MALAFDEFGRPFIILREQEQKTRLRGLDAQKANIAAGKAVARILRTSLGPKGMDKMLQSPDGDVTITNDGATILEQMDVDNQIAKLMVELSRSQDYEIGDGTTGVVVMAGALLEQAEKLLERGIHPIRVAEGYEMASRIASEHLERISHKYEFTADNIEPLVQTCMTTLSSKIVNRCKRALAEIAVKAVLAVADLERKDVNLDLIKVEGKVGGKLEDTELIYGIVVDKDMSHPQMPKRIEDAKIAILTCPFEPPKPKTKHKVDIDTVEKFQTLREQEQKYFDEMVQKCKDAGATLVICQWGFDDEANHLLMNRNLPAVRWVGGVELELIAIATGGRIVPRFEELSPEKLGKAGLVREKSFGTTKDRMLYIEQCANSRAVTIFIRGGNKMMIEETKRSLHDALCVARNLIRNNSIVYGGGSAEISCSIAVETAADRHPGVEQYAIRSFADALDAVPLALAENSGLPPIDTLTAVKAQQVKESNPHCGIDCNDVGTNDMKEQNVFETLIGKQQQILLATQVVKMILKIDDVISPSEY (SEQ ID NO.3).

[0046] SiCCT5

[0047] 2 Extraction and reverse transcription of millet RNA RNA extraction: The total RNA of millet was extracted using the RNA prep pureplant kit with the catalog number DP432 from Tiangen Biochemical Technology (Beijing) Co., Ltd. The tissue samples used for RNA extraction were quickly placed in tin foil after field sampling, quickly frozen in liquid nitrogen, and brought back to the Beijing laboratory for RNA extraction. The specific steps for RNA extraction are shown in the instructions.

[0048] RNA reverse transcription: Use the FastKing cDNA First-Strand Synthesis Kit (Genomic Depleted) with the catalog number KR116 from Tiangen Biochemical Technology (Beijing) Co., Ltd. and follow the instructions. The specific method is as follows: Take 50 ng-2 μg of total RNA to establish a 20 μL reaction system. Thaw the template RNA on ice; thaw 5×gDNABuffer, FQ-RT Primer Mix, 10×King RT Bufer, and RNase-Free ddH2O at room temperature and quickly place on ice after thawing. Vortex each solution to mix before use, and briefly centrifuge to collect the liquid remaining on the tube wall. The following steps must be performed on ice.

[0049] The gDNA removal reaction system is 5×gDNA Buffer 2 μL; RNA 50 ng-2 μg; RNase-Free ddH2O is added to 10 μL. Incubate at 42℃ for 3 min and then place on ice.

[0050] The reverse transcription reaction system was 10×King RT Bufer 2 μL; FastKing RT Enzyme Mix 1 μL; FQ-RTPrimer Mix 2 μL; RNase-Free ddH2O was added to make up to 10 μL.

[0051] Add the Mix from the reverse transcription reaction to the reaction solution in the gDNA removal step and mix thoroughly. Incubate at 42°C for 15 minutes. Incubate at 95°C for 3 minutes and place on ice. The obtained cDNA can be used for subsequent experiments or stored at low temperatures.

[0052] 3 SiCCT5 Construction of gene overexpression vector Primers SiCCT5-CDS-F and SiCCT5-CDS-R were designed using primer design software DNAMAN. The specific nucleotide series of SiCCT5-CDS-F / R are as follows: SiCCT5-CDS-F:TTGTAGGTAGAAGAGGTACCCGGGATGGCGCTCGCCTTCGATGA (SEQ IDNO.5); SiCCT5-CDS-R:GCATGCCTGCAGGTCGACTCTAGATCAGTATTCGGATGGTGAGA (SEQ ID NO. 6).

[0053] Using the cDNA obtained above as a template, SiCCT5-CDS-F and SiCCT5-CDS-R as primers, and high-fidelity enzyme PrimeSTAR HS DNA Polymerase with GC Buffer (TaKaRa catalog number: R044A) purchased from Beijing Liuhetong Economic and Trade Co., Ltd., PCR amplification was performed to obtain PCR products.

[0054] The PCR products obtained above were separated by agarose gel electrophoresis, cut and recovered. Sma I and XB The pCam23A vector digested with I was recombined using the recombinase In-Fusion Snap Assembly Master Mix (TaKaRa catalog number: 638948). After sequencing, the recombinant vector pCam23A-SiCCT5 was constructed by transfecting millet SiCCT5 Nucleotide replacement of the CDS sequence of the gene in the pCam23A vector Sma I and XB I site obtained vector ( Figure 1 -C), the arrow in the figure indicates SiCCT5 Gene sequence.

[0055] 4 Overexpression SiCCT5 Preparation of Genetic Nipponbare Rice Lines The recombinant vector pCam23A-SiCCT5 prepared above was introduced into Agrobacterium AGL1 to obtain the recombinant bacteria AGL1 / pCam23A-SiCCT5, and positive recombinant bacteria were obtained after enzyme digestion verification.

[0056] The recombinant bacteria AGL1 / pCam23A-SiCCT5 was transformed into Nipponbare rice using rice genetic transformation to obtain the T0 generation overexpression SiCCT5 Genetic Nipponbare rice strain. The specific steps are as follows: (1) Seed sterilization and callus induction stage: Shell the mature seeds, select 300 plump and complete shelled rice seeds and place them in a 50 mL sterilized centrifuge tube, and wash the seeds three times with sterilized ultrapure water. Then disinfect the surface with 40 mL 75% ethanol for 5 minutes, sterilize with 40 mL 50% sodium hypochlorite for 5 minutes, repeat the sterilization once, and finally rinse with sterile water 10 times until the water is clear. Use sterile filter paper to absorb the sterilized seeds and place them on the induction culture medium, 20 seeds / dish, and culture them in the dark at 28℃ for 28 days until callus tissue the size of a fallen millet grain grows.

[0057] (2) Subculture and pre-culture stage of callus tissue: transfer embryonic callus tissue in good condition to MS medium. About 100 seeds can be placed in each dish. Culture in the dark at 28℃ for 7 days. The picked callus tissue can be put back for further culture. A batch of induced callus can be picked 3-4 times. At the same time, large pieces of callus tissue that have not fallen off can be placed on new MS medium to make them fall off again and picked again.

[0058] (3) Preparation of Agrobacterium: One day in advance, culture Agrobacterium on YEP medium (or LB medium) supplemented with the corresponding resistance screening agent, kanamycin (50 mg / mL) and rifampicin (25 mg / mL). Use a spreading stick to scrape the Agrobacterium colonies on the original culture dish and spread them evenly on the new culture medium. If it is a bacterial liquid, pour a small amount of the bacterial liquid into the culture medium and spread it evenly with a spreading stick. Label the carrier, place it upside down, and incubate it in a 28°C incubator overnight.

[0059] Without activation treatment, the transformed colonies can be directly cultured for 4-5 days. Before shaking the culture, use a sterilized spoon to evenly spread all the colonies, then scrape an appropriate amount of bacteria and shake the culture. Bacteria stored at 4℃ must be activated.

[0060] (4) OD value adjustment: Scrape off the Agrobacterium with a key, place the bacteria in AAM liquid culture medium, and culture at 200 rpm at 28°C for 2 h. Then adjust the OD value of the bacterial solution to 0.12-0.15 using AAM liquid culture medium.

[0061] (5) Infection and co-cultivation: Collect about 100 embryonic calli into a 100 mL Erlenmeyer flask; Pour the adjusted concentration of Agrobacterium into the conical flask for infection, and shake it for 20 min on a shaker at 100 g. After shaking, pour out the infection solution, dry the callus with filter paper, move the infected callus to the co-cultivation medium, and cover it with sterile filter paper to ensure that all callus tissues are in contact with the filter paper surface. Culture in the dark at 22°C for 4 days.

[0062] (6) Resistance screening of transformed callus: Collect the callus tissue that has completed the co-culture stage into a 50 mL sterile centrifuge tube, and rinse the callus tissue 10 times with sterile water until the washing liquid is clear. Then pour into the suspension culture medium, add 1 mL of Tim washing liquid with a concentration of 200 mg / mL, and shake it on a shaker at 100 g for 1 h. After shaking, pour out the filtrate and absorb the water with filter paper. Transfer the callus tissue to the selection culture medium and use tweezers to evenly place the callus particles to prevent contact inhibition and large-area contamination. 2-3 dishes per carrier can be screened. Culture in the dark at 28℃ for 2 weeks. This process is called the first screening. During this period, pay attention to observe whether there is any contamination. After two weeks, subculture once again on the same culture medium, and double the number of screening culture dishes, so that each carrier can have 4-6 dishes. The selection lasts for about 4 weeks in total. This process is called the second screening. When the callus tissue has obvious yellow round particles the size of millet grains falling off, it can proceed to the next stage.

[0063] (7) Differentiation and rooting: Select the white and dense callus and transfer it to the differentiation medium. Before using the differentiation medium, be sure to dry the water vapor. Each dish can hold 20 seeds. Try not to place them on the edge of the culture dish, as they are easy to come into contact with water. Culture under light at 28℃ for 3-4 weeks. Be sure to place the materials in layers. Prevent the lower layer of the material from being burned by high temperature due to light and affecting the differentiation ability. It is best to place it on the bottom layer of the tissue culture room shelf to prevent the generation of water vapor and affect the differentiation of the callus. Then subculture it once on the same culture medium. Be careful to handle it with care to prevent water droplets on the lid from dripping onto the callus. Callus that comes into contact with water will no longer differentiate. Callus that has just been subcultured to the differentiation medium needs to be left for two days or shaded with a black plastic bag to prevent the wound tissue from overheating and browning.

[0064] (8) Strong seedlings: If relatively strong seedlings appear, transfer them to 1 / 2 MS strong seedling medium. Culture under light at 28℃ for 2-3 weeks. Leave the seedlings just transferred to the strong seedling medium for two days before light treatment.

[0065] (9) Transplanting of tissue culture seedlings: Wash off the residual culture medium on the roots and transfer the seedlings with good root systems into the greenhouse, keeping the soil moist in the first few days.

[0066] (10) Overexpression SiCCT5 Creation of T1 transgenic rice lines: SiCCT5 Gene overexpression vector was transferred into wild-type Nipponbare rice through Agrobacterium to obtain multiple millets SiCCT5 The gene was overexpressed in the T0 generation of Nipponbare rice, and the T0 generation plants were self-fertilized to obtain the T1 generation SiCCT5 Among the gene overexpression plants, SiCCT5-OE#1 and SiCCT5-OE#2 were randomly selected and used for subsequent analysis.

[0067] (11) The culture medium formula used is as follows: N6 culture medium: the solutes are potassium nitrate 2830 g / L, ammonium sulfate 463 g / L, calcium chloride (CaCl2·2H2O) 166 g / L, magnesium sulfate (MgSO4·7H2O) 185 g / L, potassium dihydrogen phosphate 400 g / L, ferrous sulfate (FeSO4·7H2O) 27.8 g / L, manganese sulfate (MnSO4·H2O) 4.4 g / L, zinc sulfate (ZnSO4·7H2O) 1.6 g / L, boric acid 0.8 g / L, potassium iodide 1.6 g / L, vitamin B1 (thiamine hydrochloride) 1.0 g / L, vitamin B6 (pyridoxine hydrochloride) 0.5 g / L, niacin 0.5 g / L, and glycine 2.0 g / L, and the solvent is deionized water.

[0068] Induction medium: N6 medium was supplemented with 2,4-D at a final concentration of 2.5 mg / L, 0.8 g / L hydrolyzed casein, 0.3 g / L proline, 30 g / L sucrose and 3 g / L phytagel.

[0069] Suspension medium: N6 medium was supplemented with 2,4-D at a final concentration of 2.5 mg / L, 0.8 g / L hydrolyzed casein, 0.3 g / L proline, 30 g / L sucrose, 10 g / L glucose and 100 μM acetosyringone.

[0070] Co-culture medium: N6 medium was supplemented with 2,4-D at a final concentration of 2.5 mg / L, 0.3 g / L proline, 30 g / L sucrose, 10 g / L glucose, 100 μM acetosyringone and 8 g / L agar powder.

[0071] Selection medium: N6 medium was supplemented with 2.5 mg / L 2,4-D, 0.3 g / L proline, 50 mg / L G418, 200 mg / L timentin, 30 g / L sucrose and 8 g / L agar powder.

[0072] MS medium: solutes are CaCl2·2H2O 440 mg / L, KH2PO4 170 mg / L, MgSO4·7H2O 370 mg / L, NH4NO3 1650 mg / L, KNO3 1900 mg / L, KI 0.83 mg / L, CoCl2·6H2O 0.025 mg / L, H3BO4 6.2 mg / L, Na2MoO4·7H2O 0.25 mg / L, MnSO4·4H2O 22.3 mg / L, CuSO4·5H2O 0.025 mg / L, ZnSO4·7H2O 8.6 mg / L, FeSO4·7H2O 27.8 mg / L, Na2EDTA 37.3 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxine hydrochloride 0.5 mg / L, niacin 0.5 mg / L, inositol 100 mg / L, glycine 2.0 mg / L, sucrose 30000 mg / L, agar powder 7000 mg / L, and the balance is deionized water.

[0073] 1 / 2MS medium: The final concentration of solutes in MS is halved.

[0074] Differentiation medium: MS medium supplemented with 2 mg / L KT, 0.2 mg / L NAA, 2 mg / L 6-BA, 0.2 mg / L IAA, 0.8 g / L hydrolyzed casein, 0.3 g / L proline, 30 g / L sucrose and 3 g / L phytagel, and the balance is sterile water.

[0075] 5 Overexpression SiCCT5 Phenotypic Analysis of Genetic Nipponbare Rice Lines Wild-type rice WT, SiCCT5-OE#1 and SiCCT5-OE#2 were identified and DNAMAN software was used to design SiCCT5 A pair of primers SiCCT5-RT-F and SiCCT5-RT-R for gene quantification, and a rice internal reference gene ACTIN The primers for the gene are RICE-actin-F and RICE-actin-R, and the primer sequences are as follows: SiCCT5-RT-F:GTTCAGACCTGCATGACAACTC (SEQ ID NO.7); SiCCT5-RT-R:TCGAATGGGCATGTTAGGATGG (SEQ ID NO. 8).

[0076] RICE-actin-F: TGCTATGTACGTCGCCATCCAG (SEQ ID NO.9); RICE-actin-R:AATGAGTAACCACGCTCCGTCA (SEQ ID NO. 10).

[0077] RNA from wild-type rice WT, SiCCT5-OE#1, and SiCCT5-OE#2 was extracted and reverse transcribed into cDNA for quantitative PCR experiments. The specific steps were the same as the extraction and reverse transcription of millet RNA. Figure 1 -D shows that SiCCT5 The gene was successfully overexpressed in SiCCT5-OE#1 and SiCCT5-OE#2 strains.

[0078] The mesocotyl development of wild-type rice WT, SiCCT5-OE#1 and SiCCT5-OE#2 seeds was analyzed after 7 days of continuous dark culture. Figure 1 -E and Figure 1 As shown in Figure 5-F, a significant increase in the length of the embryonic axis was observed in both SiCCT5-OE#1 and SiCCT5-OE#2 overexpression lines.

[0079] The longitudinal sections of the mesocotyls of wild-type rice WT, SiCCT5-OE#1 and SiCCT5-OE#2 seeds cultured in continuous darkness for 7 days were analyzed. Figure 1 -G and Figure 1 -H, it can be seen that the cell length of the mesocotyls of the two overexpression lines SiCCT5-OE#1 and SiCCT5-OE#2 increased significantly, that is, the increase in cell length is the main reason for the elongation of the mesocotyls. SiCCT5 The gene has the function of regulating the development of rice mesocotyl.

[0080] Test Example 2 Rice OsCCT5 Gene cloning and functional analysis According to the discovered millet SiCCT5 To investigate the function of the gene in rice mesocotyl development, the homologous sequences of SiCCT5 proteins in different species were retrieved and downloaded from the NCBI database. The sequence alignment and phylogenetic analysis were performed using MEGA11 software, and a phylogenetic evolutionary tree was constructed ( Figure 2 ), the arrows in the evolutionary tree represent millet SiCCT5 Gene( Seita.2G010200 ) and rice OsCCT5 Gene( LOC_Os06g36700 ). Phylogenetic analysis revealed that the millet SiCCT5 protein has only one homologous protein, OsCCT5, in rice. OsCCT5The full length of the cDNA sequence of the gene is 1608 bp, and its nucleotide sequence is shown in SEQ ID NO.2. The amino acid sequence of the OsCCT5 protein is shown in SEQ ID NO.1.

[0081] The amino acid sequence of OsCCT5 protein is as follows: MALAFDEFGRPFIILREQEKKSRLRGLDAQKANIAAGKAVARILRTSLGPKGMDKMLQSPDGDVTITNDGATILEQMDVDNQIAKLMVELSCSQDYEIGDGTTGVVVMAGSLLEQAEKLLERGIHPIRIAEGYELASRIAFDHLEHISHKFEFSATNIEPLVQTCMTTLSSKIVNRCKRTLAEIAVKAVLAVADLERKDVNLDLIKVEGKVGGKLEDTELVYGIIVDKDMSHPQMPKRIEDAKIAILTCPFEPPKPKTK HKVDIDTVEKFQMLREQEQKYFDEMVQKCKDVGATLVICQWGFDDEANHLLMHRNLPAVRWVGGVELELIAIATGGRIVPRFQELSPEKLGKAGIVREKSFGTTKDRMLYIEQCANSRAVTIFIRGGNKMMIEETKRS LHDALCVARNLIRNNSIVYGGGSAEISCSVAVEAAADRYPGVEQYAIRSFADALDAIPLALAENSGLSPIDTLTAVKSQQVKESNPHCGIDCNDVGTNHMKEQNVFETLIGKQQQILLATQVVKMILKIDDVISPSDY (SEQ ID NO. 1).

[0082] OsCCT5

[0083] 1 Overexpression OsCCT5 Preparation of Genetic Nipponbare Rice Lines The same method as in Experimental Example 1 was used to extract RNA from Nipponbare rice and reverse transcribe it into cDNA, which was used as a template for amplification. OsCCT5 CDS sequence of the gene.

[0084] Primers OsCCT5-OE-F and OsCCT5-OE-R were designed using the primer design software DNAMAN. The nucleotide sequences of the primers are as follows: OsCCT5-OE-F:TTGTAGGTAGAAGAGGTACCCGGGATGGCGCTGGCCTTCGAC (SEQ IDNO.11); OsCCT5-OE-R:GCATGCCTGCAGGTCGACTCTAGATCAATAGTCAGAAGGCGAGATAAC (SEQ ID NO. 12).

[0085] The obtained Nipponbare rice cDNA was used as a template, OsCCT5-OE-F and OsCCT5-OE-R were used as primers, and the recombinant vector pCam23A-OsCCT5 was constructed by the same method as in Experimental Example 1. Sma I and XB Insert the nucleotide sequence shown in SEQ ID No.2 between the I sites to obtain Figure 3 -A shows the recombinant vector pCam23A-OsCCT5, and the arrow in the figure indicates OsCCT5 Gene sequence.

[0086] The same method as in Experimental Example 1 was used to construct the rice OsCCT5 The gene overexpression vector pCam23A-OsCCT5 was transformed into wild-type Nipponbare rice by Agrobacterium to obtain multiple rice OsCCT5 The gene was overexpressed in the T0 generation of Nipponbare rice, and the T0 generation plants were self-fertilized to obtain the T1 generation OsCCT5 Among the gene overexpressing plants, OsCCT5-OE#1 and OsCCT5-OE#2 were randomly selected and used for subsequent analysis.

[0087] Wild-type rice WT, OsCCT5-OE#1 and OsCCT5-OE#2 were identified and DNAMAN software was used to design OsCCT5 A pair of primers for gene quantification, OsCCT5-RT-F and OsCCT5-RT-R, and a rice internal reference gene ACTINThe primers for the gene are RICE-actin-F and RICE-actin-R, and the primer sequences are as follows: OsCCT5-RT-F:CCAATATAGAGCCTCTGGTGCA (SEQ ID NO.13); OsCCT5-RT-R: ATGCTTTGTCTTAGGCTTCGGG (SEQ ID NO. 14).

[0088] RICE-actin-F: TGCTATGTACGTCGCCATCCAG (SEQ ID NO. 15); RICE-actin-R:AATGAGTAACCACGCTCCGTCA (SEQ ID NO. 16).

[0089] The same method as in Experimental Example 1 was used to extract RNA from wild-type rice WT, OsCCT5-OE#1, and OsCCT5-OE#2, and reverse transcribed into cDNA for fluorescence quantitative PCR experiments. Figure 3 -B, OsCCT5 The gene was successfully overexpressed in OsCCT5-OE#1 and OsCCT5-OE#2 strains.

[0090] 2 OsCCT5 Gene expression pattern analysis test To clarify OsCCT5 To investigate the tissue expression pattern of the gene, real-time fluorescence quantitative PCR was used to detect the expression level of OsCCT5 gene in rice roots, stems, leaves and panicles of different lengths. Figure 3 -C, OsCCT5 The gene expression level was highest in the root, followed by the ear, especially in the 5 cm ear, which was higher than that in the ears at other stages. GUS staining was performed on the OsCCT5 promoter-driven GUS expression plants, such as Figure 3 -D. Figure 3 -As shown by the arrow in E, it is found OsCCT5 The gene is expressed not only in roots and glumes, but also in coleoptiles. Therefore, it is speculated that the gene can also play a role in regulating crop grain development.

[0091] 3 Overexpression OsCCT5 Experiment on improving rice yield through gene To observe OsCCT5 Traits of gene overexpression lines Wild-type rice WT, OsCCT5-OE#1, and OsCCT5-OE#2 were planted separately, and the relevant traits were statistically analyzed, with 15 rice plants in each line.

[0092] The test results are as follows Figure 4 As shown, compared with wild-type rice WT, OsCCT5 The number of grains per ear in the gene overexpression lines increased significantly ( Figure 4 -A, Figure 4 -D); Grain length increased significantly ( Figure 4 -B, Figure 4 -E); thousand-grain weight increased significantly ( Figure 4 -F); the yield per plant increased significantly ( Figure 4 -G), while the width of the lemma epidermal cells increased significantly ( Figure 4 -C, Figure 4 -H), the length of the lemma epidermal cells increased significantly ( Figure 4 -C, Figure 4 -I). The above data show that overexpression in rice OsCCT5 The gene has the function of increasing the number of grains per rice panicle, grain size or thousand-grain weight, and can significantly improve rice yield.

Claims

1. CCT5 Gene, CCT5 protein, containing CCT5 Gene expression cassette or containing CCT5 The invention discloses the use of a recombinant plant expression vector of a gene in regulating plant yield; the plant is a grass plant.

2. The use according to claim 1, characterized in that The regulating plant yield is to increase rice yield.

3. The use according to claim 2, characterized in that: The method of increasing rice yield includes increasing the number of rice grains per panicle, the size of grains or the thousand-grain weight.

4. The use according to any one of claims 1 to 3, characterized in that: include: Will CCT5 Overexpression of genes in plants increases CCT5 The expression amount or level of the gene, or the function or activity of the CCT5 protein is enhanced.

5. The use according to claim 1, characterized in that: The grass plant is rice.

6. The use according to claim 1, characterized in that: The CCT5 The gene is the nucleotide sequence shown in SEQ ID NO.2 OsCCT5 The gene or nucleotide sequence is shown in SEQ ID NO.4 SiCCT5 Gene.

7. The use according to claim 1, characterized in that The CCT5 protein is derived from Gramineae plants.

8. The use according to claim 7, characterized in that The CCT5 protein is a protein having 94% or more identity with the amino acid sequence shown in SEQ ID NO.1, and the protein still has the function or activity of regulating plant yield.

9. The use according to claim 8, characterized in that The CCT5 protein is the OsCCT5 protein whose amino acid sequence is shown in SEQ ID NO.1 or the SiCCT5 protein whose amino acid sequence is shown in SEQ ID NO.

3.

10. A method for increasing plant yield, characterized in that: include: Build contains CCT5 Gene overexpression recombinant plant expression vector; CCT5 Gene overexpression recombinant plant expression vector transforms plants, CCT5 The gene is overexpressed in plants, and the yield of the resulting transgenic plants is increased; CCT5 The gene is the nucleotide sequence shown in SEQ ID NO.2 OsCCT5 The gene or nucleotide sequence is shown in SEQ ID NO.4 SiCCT5 Gene; the plant is a grass plant.

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