Use of the CCT5 gene or its encoded protein in regulating plant yield

Abstract

The present invention discloses CCT5 the use of a gene or its encoded protein in regulating plant yield. Through GWAS analysis, the present invention finds SiCCT5 the gene from the core germplasm resources of foxtail millet, and overexpresses the gene in rice, proving that the SiCCT5 gene of foxtail millet has the function of regulating the development of rice mesocotyl. The present invention further overexpresses OsCCT5 the gene in rice and finds that the number of grains per panicle, grain size, and 1000-grain weight of the rice overexpressing OsCCT5 the gene are all increased compared with the wild type, that is, the yield of rice is significantly improved, proving that the OsCCT5 gene of rice has the function of positively regulating plant yield. The present invention has an application prospect in regulating plant yield.

Description

Technical Field

[0001] The present invention relates to yield-related genes isolated from Gramineae plants and their uses, in particular to the uses of genes or their encoded proteins isolated from Gramineae plants in regulating plant yield, belonging to CCT5 the field of genes and their uses. CCT5 Background Art

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

[0003] The genetic regulation of rice grain shape is a key area for improving rice quality and yield. In recent years, through the study of various genes, scientists have revealed a series of genes that regulate rice grain shape and their molecular mechanisms, promoting the progress of rice breeding. GS3 Gene is an important regulatory gene for rice grain shape and has been proven to significantly affect grain length. This gene directly affects rice grain shape and yield by regulating cell division; GW2 Gene affects rice grain shape by regulating the weight and shape of grains, GW2 Mutations in Gene usually result in smaller grain weight and may affect the quality of rice; OsSPL16 Gene is closely related to the width and yield of rice grain shape. This gene increases grain width by promoting cell division, thereby improving grain shape, enhancing rice yield and grain quality; GSE5 Gene plays a pleiotropic role in controlling rice grain shape. It not only affects rice grain shape but also involves 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 developing efficient breeding strategies.

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

[0005] The main object of the present invention is to apply CCT5 Gene, CCT5 protein, an expression cassette containing CCT5 Gene or a recombinant plant expression vector containing CCT5 Gene to regulate plant yield.

[0006] To achieve the above object, the main technical solutions adopted by the present invention include:

[0007] One aspect of the present invention is to apply CCT5 genes, CCT5 proteins, expression cassettes containing CCT5 genes or recombinant plant expression vectors containing CCT5 genes to regulate plant yield.

[0008] In a preferred specific embodiment of the present invention, the plant is a gramineous plant.

[0009] In a preferred specific embodiment of the present invention, the regulation of plant yield is to increase plant yield; wherein, the increase in plant yield includes increasing the number of spikelets per ear, grain size or 1000-grain weight of the plant.

[0010] As a reference, the present invention provides an embodiment. That is, by overexpressing the coding gene of the CCT5 protein related to plant yield in the plant, the expression level or activity of the CCT5 protein related to plant yield is increased, thereby increasing plant yield, and further increasing the number of spikelets per ear, grain size, and 1000-grain weight of the plant.

[0011] In a preferred specific embodiment of the present invention, a method for increasing plant yield includes: overexpressing CCT5 genes in the plant to enhance the expression level of CCT5 genes or enhance the function or activity of the CCT5 protein; for example, connecting the SiCCT5 gene derived from foxtail millet or the OsCCT5 gene derived from rice with an expression regulatory element to obtain a recombinant plant expression vector for expressing the gene in the plant; transforming the recombinant plant expression vector into the plant to overexpress CCT5 genes in the plant, and the yield of the obtained transgenic plant is increased.

[0012] As a reference, the present invention provides a CCT5 gene plant recombinant expression vector, including: the SiCCT5 gene of foxtail millet or the OsCCT5A recombinant plant expression vector is obtained by connecting a gene with an expression regulatory element; the recombinant plant expression vector may consist of a 5′-untranslated region, the nucleotide shown in SEQ ID NO.2 or the nucleotide shown in SEQ ID NO.4, and a 3′-untranslated region; wherein, the 5′-untranslated region may include a promoter sequence, an enhancer sequence or / and a translation enhancer sequence; the promoter may be a constitutive promoter, an inducible promoter, a tissue- or organ-specific promoter; the 3′-untranslated region may contain a terminator sequence, an mRNA cleavage sequence, etc. Suitable terminator sequences can be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase terminator regions.

[0013] The recombinant plant expression vector may also contain a selectable marker gene for selecting transformed cells, for selecting transformed cells or tissues. The marker genes include: genes encoding antibiotic resistance and genes conferring resistance to herbicidal compounds, etc. In addition, the marker genes also include phenotypic markers, such as β-galactosidase and fluorescent proteins, etc.

[0014] The transformation protocol and the protocol for introducing the polynucleotide or polypeptide into a plant may vary depending on the type of plant or plant cell to be transformed. Suitable methods for introducing the polynucleotide into a plant cell include: microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-velocity ballistic bombardment, etc. In a specific embodiment, various transient transformation methods can be used to provide the SiCCT5 gene of Setaria italica or the OsCCT5 gene of Oryza sativa to the plant. Stable transformed plants can be regenerated from the transformed cells using conventional methods (McCormick et al. Plant Cell Reports. 1986. 5:81-84).

[0015] In a preferred specific embodiment of the present invention, the gramineous plant is Oryza sativa.

[0016] Another aspect of the present invention is to provide a CCT5 gene and its protein derived from Setaria italica or Oryza sativa that can regulate plant yield.

[0017] In a preferred specific embodiment of the present invention, the CCT5 gene is the OsCCT5 gene with the nucleotide sequence shown in SEQ ID NO.2 or the SiCCT5 gene with the nucleotide sequence shown in SEQ ID NO.4.

[0018] In addition, those skilled in the art can also optimize the nucleotides shown in SEQ ID NO.2 and the nucleotides shown in SEQ ID NO.4 to enhance the expression efficiency in plants.

[0019] Those of ordinary skill in the art can easily adopt known methods, such as directed evolution or site-directed mutagenesis, to mutate CCT5 the nucleotide sequence of a gene. Those nucleotides that have been artificially modified and have 75% or higher identity with CCT5 the nucleotide sequence of the gene, as long as the encoded protein has the function of regulating plant yield, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0020] 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, etc.

[0021] In a preferred specific embodiment of the present invention, the CCT5 protein is derived from a gramineous plant.

[0022] In a preferred specific 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 this protein still has the function or activity of regulating plant yield.

[0023] The percentage of sequence identity described in the present invention can be obtained by well-known bioinformatics algorithms, including the Myers and Miller algorithms, the Needleman-Wunsch global alignment method, the Smith-Waterman local alignment method, the Pearson and Lipman similarity search method, and the algorithm of Karlin and Altschul, which are well-known to those skilled in the art.

[0024] In a preferred specific 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.

[0025] The present invention uses GWAS to analyze genes regulating the development of the mesocotyl in foxtail millet, clones the SiCCT5 gene, and by overexpressing the SiCCT5 gene in Nipponbare rice, it is proved that the foxtail millet SiCCT5 gene has the function of regulating the development of the mesocotyl in rice. On this basis, the present invention further conducts sequence alignment and phylogenetic analysis to determine the homologous protein OsCCT5 protein of the foxtail millet SiCCT5 protein in rice; by overexpressing the OsCCT5 gene in Nipponbare rice and further analyzing the OsCCT5 gene expression pattern, it is speculated that this gene can also play a role in regulating plant yield. Further observing the overexpressionOsCCT5 rice traits of genes, and it was found that overexpression OsCCT5 the number of spikelets per panicle, the grain size, and the 1000-grain weight of rice overexpressing OsCCT5 genes all increased, that is, the yield of rice was improved, proving that rice

[0026] Term definitions involved in the present invention

[0027] Unless otherwise defined, 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.

[0028] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides in single-stranded or double-stranded form, and their polymers. Unless specifically restricted, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically restricted, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs (phosphorothioates, phosphoroamidates, etc.) used in antisense technology. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (including, but not limited to, degenerate codon substitutions) and complementary sequences, as well as the explicitly specified sequences. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue.

[0029] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, a description of a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The term applies to both naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the term encompasses amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are joined by covalent peptide bonds.

[0030] The term "recombinant host cell line" or "host cell" means a cell containing the polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-mating or other methods known in the art. The exogenous polynucleotide can be maintained as a non-integrating vector such as a plasmid or can be integrated into the host genome. The host cell can be a prokaryotic cell or a eukaryotic cell, and the host cell can also be a monocotyledonous or dicotyledonous plant cell.

[0031] The term "operably linked" refers to a functional connection between two or more elements, and the elements that are operably linked may be adjacent or non-adjacent.

[0032] The term "recombinant plant expression vector" means one or more DNA vectors for achieving plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors together with vectors having helper plasmids are most commonly used for Agrobacterium tumefaciens-mediated transformation. Binary vectors generally include: cis-acting sequences required for T-DNA transfer, selectable markers engineered to be able to express in plant cells, heterologous DNA sequences to be transcribed, etc.

[0033] The term "transformation" refers to a method of introducing a heterologous DNA sequence into a host cell or organism.

[0034] 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

[0035] Figure 1 for Setaria italica SiCCT5 cloning and functional verification of genes; wherein, Figure 1 -A is a Manhattan plot of the GWAS analysis results; Figure 1 -B is a quantile plot (QQ plot) for detecting whether the p-values observed in the GWAS analysis conform to the expected distribution; Figure 1 -C is a schematic diagram of the constructed pCam23A-SiCCT5 vector; Figure 1 -D is for wild-type rice WT and overexpression SiCCT5 in rice lines of the gene SiCCT5 gene expression level detection results; Figure 1 -E is for wild-type rice WT and overexpression SiCCT5 in rice lines of the gene, the mesocotyl length results after culturing for 7 days in continuous darkness, the arrow indicates the coleoptile node, and the mesocotyl is the part between the arrow and the seed; Figure 1 -F is the statistical analysis result of the mesocotyl length in wild-type rice WT and rice lines overexpressing SiCCT5 the gene after culturing for 7 days in continuous darkness; Figure 1 -G is the longitudinal section of the mesocotyl in wild-type rice WT and rice lines overexpressing SiCCT5 the gene; Figure 1 -H is the statistical analysis data of the cell length of the mesocotyl in wild-type rice WT and rice lines overexpressing SiCCT5 the gene;

[0036] Figure 2 is a phylogenetic tree constructed based on the Setaria italica SiCCT5 protein sequence;

[0037] Figure 3 for riceOsCCT5 Gene overexpression and expression pattern analysis; among them, Figure 3 -A is a schematic diagram of the constructed pCam23A-OsCCT5 vector; Figure 3 -B is the detection result of the gene expression level in wild-type rice WT and rice lines overexpressing the OsCCT5 gene; OsCCT5 -C is the detection of the gene expression level in various tissues of rice; Figure 3 -C is the detection of the gene expression level in various tissues of rice; OsCCT5 -C is the detection of the gene expression level in various tissues of rice; Figure 3 -D is OsCCT5 the staining result of GUS driven by the gene promoter in seeds; Figure 3 -E is OsCCT5 the staining result of GUS driven by the gene promoter in the mesocotyl;

[0038] Figure 4 is the phenotypic analysis of wild-type rice WT and rice lines overexpressing the OsCCT5 gene; among them, Figure 4 -A is the plant type diagram of wild-type rice WT and rice lines overexpressing the OsCCT5 gene; Figure 4 -B is the grain type diagram of wild-type rice WT and rice lines overexpressing the OsCCT5 gene, Figure 4 -C is the observation result of the size of lemma epidermal cells of wild-type rice WT and rice lines overexpressing the OsCCT5 gene; Figure 4 -D is the statistical analysis result of the number of spikelets per panicle of wild-type rice WT and rice lines overexpressing the OsCCT5 gene; Figure 4 -E is the statistical analysis result of the grain length of wild-type rice WT and rice lines overexpressing the OsCCT5 gene; Figure 4 -F is the statistical analysis result of the 1000-grain weight of wild-type rice WT and rice lines overexpressing the OsCCT5 gene; Figure 4 -G is the statistical analysis result of the yield per plant of wild-type rice WT and rice lines overexpressing the OsCCT5 gene, Figure 4 -H is the statistical analysis result of the width of lemma epidermal cells of wild-type rice WT and rice lines overexpressing the OsCCT5 gene, Figure 4 -I is the statistical analysis result of the length of lemma epidermal cells of wild-type rice WT and rice lines overexpressing the OsCCT5 gene. Specific implementation mode

[0039] 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 described embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements all fall within the protection scope of the present invention.

[0040] Vectors, Strains and Test Materials

[0041] The pCam23A vector is preserved by the Institute of Biotechnology, Chinese Academy of Agricultural Sciences; Agrobacterium tumefaciens AGL1 is purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd., with the product number ZK296; the wild-type rice is Nipponbare rice ( Oryza sativa L.ssp. Japonica variety Nipponbare ; hereinafter also referred to as wild-type rice WT), which is preserved by the Crop High Photosynthetic Efficiency Functional Genomics Team of the Institute of Biotechnology, Chinese Academy of Agricultural Sciences.

[0042] Test Reagents

[0043] The primers used for PCR are synthesized by Beijing Tsingke Biotechnology Co., Ltd., and the sequencing is completed by Beijing Tsingke Biotechnology Co., Ltd. Restriction enzymes Sma I and Xba I, Infusion recombinase, and high-fidelity enzyme are all purchased from Beijing Liuhetong Economic and Trade Co., Ltd. (TaKaRa); antibiotics are purchased from SIGMA Corporation, USA; the rest of the reagents are all domestic analytical pure.

[0044] The reagent formulations used in the test process are as follows:

[0045] 2,4-D (2 mg / mL): First, dissolve 2,4-D in a microwave oven with 5 mL - 10 mL of 1 N KOH, and then add ultrapure water to make up the volume. Store at room temperature.

[0046] 6-BA (3 mg / mL): Weigh 150 mg of 6-BA, first dissolve it in 5 mL of 1 N KOH, and then make up the volume to 50 mL with sterile water. Filter sterilize.

[0047] Timectin (200 mg / mL): Dissolve 2 g of Timectin in sterile water and make up the volume to 10 mL, then filter sterilize.

[0048] G418 (150 mg / mL): Dissolve it in sterile water, make up the volume, and filter sterilize.

[0049] Rifampicin Rif (25 mg / mL): 0.5 g of rifampicin (Rif) was first dissolved in 1 N NaOH and then made up to 10 mL with methanol; or directly made up to 10 mL with DMSO, filtered and sterilized. Store at -20 °C, working concentration 25 μg / mL.

[0050] Kanamycin (50 mg / mL): Weigh 0.5 g of kanamycin sulfate powder, add 10 mL of ultrapure water to dissolve, filter and sterilize, working concentration 50 μg / mL.

[0051] Data processing

[0052] The data was processed using GraphPad Prism 8 statistical software. The significance analysis of a set of data was performed using Student's t-test. P < 0.05 (*) indicates significant difference, P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****) indicates extremely significant difference. The significance difference analysis of multiple groups of data was determined by one-way analysis of variance (ANOVA) and Tukey multiple comparison test. In the results, the letter a was marked on the largest mean value. If there was a significant difference in the second group of data, it was marked b, and if there was no significant difference, it was marked a, and so on.

[0053] Experimental example 1 Foxtail millet SiCCT5 Obtaining and functional analysis of genes

[0054] 1 Performing GWAS analysis on mesocotyl development using foxtail millet core germplasm resources

[0055] 637 foxtail millet germplasm resources were germinated in the dark, and the mesocotyl lengths were respectively counted. Combining with genomic variation data, GWAS was used to analyze the genes regulating foxtail millet mesocotyl development. The results of the GWAS analysis are as Figure 1 shown in -A. The blue dotted line in the Manhattan plot represents the genome-wide significance threshold (P = 9.50×10 -6 ), and the results show that there is a major SNP association locus (the position indicated by the red triangle) on chromosome 2 with a P value lower than 9.50×10 -6 , and there is a significant association signal ( Figure 1 -B). Through further bioinformatics analysis and verification, the gene (Seita.2G010200) located in this interval was cloned. SiCCT5

[0056] ​The amino acid sequence of the SiCCT5 protein is as follows: MALAFDEFGRPFIILREQEQKTRLRGLDAQKANIAAGKAVARILRTSLGPKGMDKMLQSPDGDVTITNDGATILEQMDVDNQIAKLMVELSRSQDYEIGDGTTGVVVMAGALLEQAEKLLERGIHPIRVAEGYEMASRIASEHLERISHKYEFTADNIEPLVQTCMTTLSSKIVNRCKRALAEIAVKAVLAVADLERKDVNLDLIKVEGKVGGKLEDTELIYGIVVDKDMSHPQMPKRIEDAKIAILTCPFEPPKPKTKHKVDIDTVEKFQTLREQEQKYFDEMVQKCKDAGATLVICQWGFDDEANHLLMNRNLPAVRWVGGVELELIAIATGGRIVPRFEELSPEKLGKAGLVREKSFGTTKDRMLYIEQCANSRAVTIFIRGGNKMMIEETKRSLHDALCVARNLIRNNSIVYGGGSAEISCSIAVETAADRHPGVEQYAIRSFADALDAVPLALAENSGLPPIDTLTAVKAQQVKESNPHCGIDCNDVGTNDMKEQNVFETLIGKQQQILLATQVVKMILKIDDVISPSEY (SEQ ID NO.3).

[0057] SiCCT5

[0058] 2 Extraction and Reverse Transcription of Foxtail Millet RNA

[0059] RNA Extraction: The total RNA of foxtail millet was extracted using the RNA prep pure plant kit with the product number DP432 from Tiangen Biochemical Technology (Beijing) Co., Ltd. The tissue samples used for RNA extraction were quickly put into tin foil after sampling in the field, frozen in liquid nitrogen, and then brought back to the Beijing laboratory for RNA extraction. The specific steps for RNA extraction are shown in the instruction manual.

[0060] RNA Reverse Transcription: The FastKing cDNA First Strand Synthesis Kit (Genome Removing) with the product number KR116 from Tiangen Biochemical Technology (Beijing) Co., Ltd. was used and the operation was carried out according to the instruction manual. The specific method is as follows:

[0061] A 20 μL reaction system can be established with 50 ng - 2 μg total RNA. Thaw the template RNA on ice; thaw 5×gDNA Buffer, FQ-RT Primer Mix, 10×King RT Buffer, and RNase-Free ddH 2 O at room temperature and quickly place them on ice after thawing. Vortex and mix each solution well before use and briefly centrifuge to collect the liquid remaining on the tube wall. The following operation steps need to be carried out on ice.

[0062] The gDNA removal reaction system is 2 μL of 5×gDNA Buffer; 50 ng - 2 μg of RNA; RNase-Free ddH 2 O to make up to 10 μL. Incubate at 42°C for 3 min, then place on ice.

[0063] The reverse transcription reaction system is 2 μL of 10×King RT Buffer; 1 μL of FastKing RT Enzyme Mix; 2 μL of FQ-RT Primer Mix; RNase-Free ddH 2 O to make up to 10 μL.

[0064] Add the Mix in the reverse transcription reaction to the reaction solution in the gDNA removal step and mix well. Incubate at 42°C for 15 min. Incubate at 95°C for 3 min and then place on ice. The obtained cDNA can be used for subsequent experiments or stored at low temperature.

[0065] 3 SiCCT5 Construction of Gene Overexpression Vector

[0066] The primers SiCCT5-CDS-F and SiCCT5-CDS-R were designed using the primer design software DNAMAN. The specific nucleotide sequences of SiCCT5-CDS-F / R are as follows:

[0067] SiCCT5-CDS-F: TTGTAGGTAGAAGAGGTACCCGGGATGGCGCTCGCCTTCGATGA (SEQ ID NO.5);

[0068] SiCCT5-CDS-R: GCATGCCTGCAGGTCGACTCTAGATCAGTATTCGGATGGTGAGA (SEQ ID NO.6).

[0069] Using the obtained cDNA as a template, with SiCCT5-CDS-F and SiCCT5-CDS-R as primers, the high-fidelity enzyme PrimeSTAR HS DNA Polymerase with GC Buffer (TaKaRa product number: R044A) purchased from Beijing Liuhetong Economic and Trade Co., Ltd. was used for PCR amplification to obtain the PCR product.

[0070] The obtained PCR product was separated by agarose gel electrophoresis, the gel was cut, and the gel was recovered. The recovered product was recombined with the pCam23A vector digested with Sma I and Xba I using the recombinase In-Fusion Snap Assembly Master Mix (TaKaRa product number: 638948). After sequencing, the recombinant vector pCam23A-SiCCT5 was the vector obtained by replacing the nucleotides of the CDS sequence of the foxtail millet SiCCT5 gene with the Sma I and Xba I sites of the pCam23A vector ( Figure 1 -C). The arrow in the figure indicates the SiCCT5 gene sequence.

[0071] 4 Preparation of overexpressed SiCCT5 gene Nipponbare rice lines

[0072] The prepared recombinant vector pCam23A-SiCCT5 was introduced into Agrobacterium tumefaciens AGL1 to obtain the recombinant bacterium AGL1 / pCam23A-SiCCT5. After digestion verification, the positive recombinant bacterium was obtained.

[0073] The recombinant bacterium AGL1 / pCam23A-SiCCT5 was transformed into Nipponbare rice by rice genetic transformation to obtain the T 0 generation overexpressed SiCCT5Gene Nipponbare rice strain. The specific operation steps are as follows:

[0074] (1) Seed sterilization and callus induction stage: Shell the mature seeds, select 300 plump and intact shelled rice seeds and put them into a 50 mL sterilized centrifuge tube, and wash the seeds three times with sterilized ultrapure water. Then surface disinfect with 40 mL of 75% ethanol for 5 min, sterilize with 40 mL of 50% sodium hypochlorite for 5 min, repeat the sterilization once, and finally wash with sterile water 10 times until the water is clear. Blot the sterilized seeds dry with sterile filter paper, place them on the induction medium, 20 seeds per dish, and culture them under dark conditions at 28°C for 28 days until callus the size of fallen millet grains grows out.

[0075] (2) Subculture and pre-culture stage of callus: Transfer the well-conditioned embryogenic callus to the MS medium. About 100 pieces can be placed in each dish. Culture under dark conditions at 28°C for 7 days. The selected callus can be put back for continued culture, and 3-4 selections can be made from one batch of induction. At the same time, large pieces of non-detached callus can be placed on a new MS medium to make them detach again and then be selected again.

[0076] (3) Preparation of Agrobacterium: One day in advance, plate and culture Agrobacterium on the YEP medium (or LB medium) added with the corresponding screening resistance, kanamycin (50 mg / mL), and rifampicin (25 mg / mL); scrape the Agrobacterium colonies on the original culture dish with a spreading rod and evenly smear them on the new medium. If it is a bacterial solution, pour the bacterial solution into the medium, a small amount is enough, and smear it evenly with a spreading rod. Mark the carrier number, place it upside down, and culture it in an incubator at 28°C overnight.

[0077] It is not necessary to perform an activation treatment. The transferred colonies can be directly cultured for 4-5 days. Before shaking the bacteria, use a sterilized spoon to smear all the colonies evenly, and then scrape an appropriate amount of bacteria for shaking. The bacteria stored at 4°C must be activated.

[0078] (4) OD value adjustment: Scrape the Agrobacterium with a key, put the bacteria into the AAM liquid medium, and culture them on a shaker at 28°C at 200 rpm for 2 h. Then adjust the OD value of the bacterial solution to 0.12-0.15 with the AAM liquid medium.

[0079] (5) Infection and co-culture: Collect about 100 embryogenic calli into a 100 mL Erlenmeyer flask; pour the Agrobacterium with the adjusted concentration into the conical flask for infection, and shake it on a shaker at 100 g for 20 min. After shaking, pour out the infection solution, blot the callus dry with filter paper, transfer the infected callus to the co-culture medium, cover it with sterile filter paper, and ensure that all the callus touches the filter paper surface on the filter paper. Culture in the dark at 22°C for 4 d.

[0080] (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.

[0081] (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.

[0082] (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.

[0083] (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.

[0084] (10) Overexpression SiCCT5 Genetic rice T 1 Creation of next-generation strains: SiCCT5 Gene overexpression vector was transferred into wild-type Nipponbare rice through Agrobacterium to obtain multiple millets SiCCT5 Overexpression of genes in Nipponbare rice 0 Generation strain, T 0 The T 1 generation SiCCT5For gene overexpression plants, SiCCT5-OE#1 and SiCCT5-OE#2 were randomly selected and used for subsequent analysis.

[0085] (11) The formulations of the media used are as follows:

[0086] N6 medium: The solutes are potassium nitrate 2830 g / L, ammonium sulfate 463 g / L, calcium chloride (CaCl 2 ·2H 2 O) 166 g / L, magnesium sulfate (MgSO 4 ·7H 2 O) 185 g / L, potassium dihydrogen phosphate 400 g / L, ferrous sulfate (FeSO 4 ·7H 2 O) 27.8 g / L, manganese sulfate (MnSO 4 ·H 2 O) 4.4 g / L, zinc sulfate (ZnSO 4 ·7H 2 O) 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, nicotinic acid 0.5 g / L, glycine 2.0 g / L, and the solvent is deionized water.

[0087] Induction medium: Based on the N6 medium, add 2,4-D at a final concentration of 2.5 mg / L, casein hydrolysate 0.8 g / L, proline 0.3 g / L, sucrose 30 g / L, and phytagel 3 g / L.

[0088] Suspension medium: Based on the N6 medium, add 2,4-D at a final concentration of 2.5 mg / L, casein hydrolysate 0.8 g / L, proline 0.3 g / L, sucrose 30 g / L, glucose 10 g / L, and acetosyringone 100 μM.

[0089] Co-culture medium: Based on the N6 medium, add 2,4-D at a final concentration of 2.5 mg / L, proline 0.3 g / L, sucrose 30 g / L, glucose 10 g / L, acetosyringone 100 μM, and agar powder 8 g / L.

[0090] Selection medium: Based on the N6 medium, add 2,4-D at a final concentration of 2.5 mg / L, proline 0.3 g / L, G418 50 mg / L, ticarcillin 200 mg / L, sucrose 30 g / L, and agar powder 8 g / L.

[0091] MS medium: The solute is CaCl2 ·2H 2 O 440 mg / L, KH 2 PO 4 170 mg / L, MgSO 4 ·7H 2 O 370 mg / L, NH 4 NO 3 1650 mg / L, KNO 3 1900 mg / L, KI 0.83 mg / L, CoCl 2 ·6H 2 O 0.025 mg / L, H 3 BO 4 6.2mg / L, Na 2 MoO 4 ·7H 2 O 0.25 mg / L, MnSO 4 ·4H 2 O 22.3 mg / L, CuSO 4 ·5H 2 O 0.025 mg / L, ZnSO 4 ·7H 2 O 8.6 mg / L, FeSO 4 ·7H 2 O 27.8 mg / L, Na 2 EDTA 37.3 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxine hydrochloride 0.5mg / L, nicotinic acid 0.5 mg / L, inositol 100 mg / L, glycine 2.0 mg / L, sucrose 30000mg / L, agar powder 7000 mg / L, with the balance being deionized water.

[0092] 1 / 2MS medium: The final concentration of the solutes in MS is halved.

[0093] Differentiation medium: On the basis of MS medium, add 2 mg / L KT, 0.2 mg / L NAA, 2 mg / L 6 - BA, 0.2mg / L IAA, 0.8 g / L casein hydrolysate, 0.3 g / L proline, 30 g / L sucrose and 3 g / L phytagel, with the balance being sterile water.

[0094] 5 Overexpression SiCCT5 Phenotypic analysis of the overexpressed

[0095] Identification was performed on wild - type rice WT, SiCCT5 - OE#1 and SiCCT5 - OE#2, and DNAMAN software was used to design for SiCCT5A pair of primers SiCCT5-RT-F and SiCCT5-RT-R for gene quantification, and primers RICE-actin-F and RICE-actin-R for the rice reference gene ACTIN The primer sequences are as follows:

[0096] SiCCT5-RT-F: GTTCAGACCTGCATGACAACTC (SEQ ID NO.7);

[0097] SiCCT5-RT-R: TCGAATGGGCATGTTAGGATGG (SEQ ID NO.8).

[0098] RICE-actin-F: TGCTATGTACGTCGCCATCCAG (SEQ ID NO.9);

[0099] RICE-actin-R: AATGAGTAACCACGCTCCGTCA (SEQ ID NO.10).

[0100] RNA was extracted from wild-type rice WT, SiCCT5-OE#1 and SiCCT5-OE#2, reverse transcribed into cDNA, and fluorescence quantitative PCR experiments were carried out. The specific steps were the same as those for the extraction and reverse transcription of foxtail millet RNA above. The test results are shown in Figure 1 -D, demonstrating that SiCCT5 the gene was successfully overexpressed in the SiCCT5-OE#1 and SiCCT5-OE#2 lines.

[0101] The mesocotyl development of wild-type rice WT, SiCCT5-OE#1 and SiCCT5-OE#2 seeds cultured in continuous darkness for 7 days was analyzed. The test results are shown in Figure 1 -E and Figure 1 -F, showing that the mesocotyl lengths of the two overexpression lines SiCCT5-OE#1 and SiCCT5-OE#2 were significantly increased.

[0102] 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. The test results are shown in Figure 1 -G and Figure 1 -H, showing that the cell lengths of the mesocotyls of the two overexpression lines SiCCT5-OE#1 and SiCCT5-OE#2 were significantly increased, that is, the increase in cell length was the main reason for the elongation of the mesocotyl. The above results demonstrated that the foxtail millet SiCCT5 gene has the function of regulating the mesocotyl development of rice.

[0103] Test Example 2 RiceOsCCT5 Gene cloning and functional analysis

[0104] 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. OsCCT5 The 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.

[0105] The amino acid sequence of OsCCT5 protein is as follows: MALAFDEFGRPFIILREQEKKSRLRGLDAQKANIAAGKAVARILRTSLGPKGMDKMLQSPDGDVTITNDGATILEQMDVDNQIAKLMVELSCSQDYEIGDGTTGVVVMAGSLLEQAEKLLERGIHPIRIAEGYELASRIAFDHLEHISHKFEFSATNIEPLVQTCMTTLSSKIVNRCKRTLAEIAVKAVLAVADLERKDVNLDLIKVEGKVGGKLEDTELVYGIIVDKDMSHPQMPKRIEDAKIAILTCPFEPPKPKTK HKVDIDTVEKFQMLREQEQKYFDEMVQKCKDVGATLVICQWGFDDEANHLLMHRNLPAVRWVGGVELELIAIATGGRIVPRFQELSPEKLGKAGIVREKSFGTTKDRMLYIEQCANSRAVTIFIRGGNKMMIEETKRS LHDALCVARNLIRNNSIVYGGGSAEISCSVAVEAAADRYPGVEQYAIRSFADALDAIPLALAENSGLSPIDTLTAVKSQQVKESNPHCGIDCNDVGTNHMKEQNVFETLIGKQQQILLATQVVKMILKIDDVISPSDY (SEQ ID NO. 1).

[0106] OsCCT5

[0107] 1 Overexpression OsCCT5 Preparation of the Gene-Overexpressing Nipponbare Rice Lines

[0108] Using the same method as in Test Example 1, extract the RNA of Nipponbare rice and reverse-transcribe it into cDNA, which is used as a template for amplification OsCCT5 of the CDS sequence of the gene.

[0109] Use the primer design software DNAMAN to design primers OsCCT5-OE-F and OsCCT5-OE-R, and the nucleotide sequences of the primers are as follows:

[0110] OsCCT5-OE-F: TTGTAGGTAGAAGAGGTACCCGGGATGGCGCTGGCCTTCGAC (SEQ ID NO.11);

[0111] OsCCT5-OE-R: GCATGCCTGCAGGTCGACTCTAGATCAATAGTCAGAAGGCGAGATAAC (SEQ ID NO.12).

[0112] Using the obtained Nipponbare rice cDNA as a template, OsCCT5-OE-F and OsCCT5-OE-R as primers, and using the same method as in Test Example 1, construct the recombinant vector pCam23A-OsCCT5, that is, by inserting the nucleotide sequence shown in SEQ ID No.2 between the Sma I and Xba I sites of the pCam23A vector to obtain the recombinant vector pCam23A-OsCCT5 shown in Figure 3 -A, and the arrow in the figure indicates the OsCCT5 gene sequence.

[0113] Using the same method as in Test Example 1, transfer the constructed rice OsCCT5 gene overexpression vector pCam23A-OsCCT5 into wild-type Nipponbare rice through Agrobacterium to obtain multiple rice OsCCT5 gene overexpression T 0 generation lines in Nipponbare rice, and self-cross the T 0 generation plants to obtain the T 1 generation OsCCT5 gene overexpression plants, and randomly select OsCCT5-OE#1 and OsCCT5-OE#2 for subsequent analysis.

[0114] Identify wild-type rice WT, OsCCT5-OE#1 and OsCCT5-OE#2, and use the DNAMAN software to design for OsCCT5A pair of primers OsCCT5-RT-F and OsCCT5-RT-R for gene quantification, and primers RICE-actin-F and RICE-actin-R for the rice reference gene ACTIN The primer sequences are as follows for the primers of the gene RICE-actin-F and RICE-actin-R

[0115] OsCCT5-RT-F: CCAATATAGAGCCTCTGGTGCA (SEQ ID NO.13);

[0116] OsCCT5-RT-R: ATGCTTTGTCTTAGGCTTCGGG (SEQ ID NO.14).

[0117] RICE-actin-F: TGCTATGTACGTCGCCATCCAG (SEQ ID NO.15);

[0118] RICE-actin-R: AATGAGTAACCACGCTCCGTCA (SEQ ID NO.16).

[0119] Using the same method as in Test Example 1, RNA of wild-type rice WT, OsCCT5-OE#1 and OsCCT5-OE#2 was extracted, reverse-transcribed into cDNA, and a fluorescence quantitative PCR experiment was carried out. The results are as Figure 3 shown in -B, OsCCT5 The gene was successfully overexpressed in the OsCCT5-OE#1 and OsCCT5-OE#2 lines.

[0120] 2 OsCCT5 Experiment on gene expression pattern analysis

[0121] To clarify OsCCT5 the tissue expression pattern of the gene, real-time fluorescence quantitative PCR was used to detect the expression level of the OsCCT5 gene in the roots, stems, leaves and panicles of different lengths of rice. The test results are as Figure 3 shown in -C, OsCCT5 the gene had the highest expression level in the roots, followed by a relatively high expression level in the panicles, especially the expression level in the 5 cm panicles was higher than that in the panicles at other stages. By performing GUS staining on the plants with GUS expression driven by the OsCCT5 promoter, as shown by the arrows in Figure 3 -D, Figure 3 -E, it was found that OsCCT5 the gene was expressed not only in the roots and glumes, but also in the coleoptiles. Therefore, it is speculated that the gene can also play a role in regulating crop grain development.

[0122] 3 Overexpression OsCCT5 Experiment on increasing rice yield by overexpressing the gene

[0123] To observe OsCCT5 the traits of the gene overexpression lines, wild-type rice WT, OsCCT5-OE#1, and OsCCT5-OE#2 were planted separately, and the relevant traits were statistically analyzed. 15 rice plants were counted for each line.

[0124] The test results are as Figure 4 shown. Compared with wild-type rice WT, OsCCT5 the number of grains per panicle of the gene overexpression lines increased significantly ( Figure 4 -A, Figure 4 -D); the grain length increased significantly ( Figure 4 -B, Figure 4 -E); the 1000-grain weight increased significantly ( Figure 4 -F); the yield per plant increased significantly ( Figure 4 -G), and at the same time, the width of the lemma epidermal cells increased significantly ( Figure 4 -C, Figure 4 -H), and the length of the lemma epidermal cells increased significantly ( Figure 4 -C, Figure 4 -I). The above data indicate that overexpressing OsCCT5 the gene in rice has functions such as increasing the number of grains per panicle, grain size, or 1000-grain weight, and can significantly improve the rice yield.

Claims

1. CCT5 Gene, CCT5 protein, containing CCT5 Gene expression cassette or containing CCT5 The use of a recombinant plant expression vector of a gene in increasing rice yield; characterized in that: Will CCT5 Gene overexpression in rice improves CCT5 The expression amount or expression level of the gene; the said increasing rice yield is to increase the number of rice grains per panicle, grain size and thousand-grain weight; the said CCT5 The gene is the nucleotide sequence shown in SEQ ID NO.2 OsCCT5 Gene; the CCT5 protein is the OsCCT5 protein whose amino acid sequence is shown in SEQ ID NO.

1.

2. A method for increasing rice yield, characterized in that: include: Build contains CCT5 Gene overexpression recombinant plant expression vector; CCT5 Gene overexpression recombinant plant expression vector transformed rice, CCT5 The gene was overexpressed in rice, and the resulting transgenic rice had increased yield; The method for increasing rice yield is to increase the number of rice grains per ear, the size of grains and the thousand-grain weight; CCT5 The gene is the nucleotide sequence shown in SEQ ID NO.2 OsCCT5 Gene.

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