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

Abstract

The invention discloses an application of a CCT5 gene or an encoded protein thereof in regulation and control of plant growth. Through GWAS analysis, the SiCCT5 gene related to mesocotyl development 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 rice mesocotyl development. The homologous gene OsCCT5 gene of the SiCCT5 gene in the rice is further determined, the OsCCT5 gene is over-expressed in the rice, the OsCCT5 gene overexpressed Nipponbare rice shows phenotypes such as the length of a plant mesocotyl, the height of seedlings during direct seeding of plant seeds, the increase of the germination rate and the like, and it is proved that the rice OsCCT5 gene has the function of positively regulating and controlling growth during direct seeding of the plants. The invention has application prospects in the aspects of regulating and controlling plant growth and breeding plant varieties.

Description

Technical Field

[0001] The present invention relates to growth-related genes separated from Gramineae plants and uses thereof, and in particular to growth-related genes separated from Gramineae plants. CCT5 Use of a gene or its encoded protein in regulating plant growth, belonging to CCT5 Genes and their areas of use. Background Art

[0002] Direct rice sowing (direct seeding) refers to a cultivation method in which rice seeds are sown directly in the field without seedling raising and transplanting. Compared with traditional seedling raising and transplanting, direct seeding has significant advantages, namely, direct seeding can save a lot of manpower and time costs, reduce the spread of pests and diseases, and help increase yields.

[0003] The mesocotyl is an important structure of the embryo part of rice seeds, which is directly related to the germination and early growth of rice. During the direct seeding of rice, the development of the mesocotyl plays a vital role in the germination of seeds and the growth of seedlings. Under direct seeding conditions, seed germination requires good environmental conditions, and the normal development of the mesocotyl is one of the key factors to ensure germination. When the mesocotyl is well developed, it can break through the soil and unfold the primary leaves faster, promoting the growth and development of the seedlings. Therefore, optimizing the development characteristics of the mesocotyl, such as increasing the length of the mesocotyl, is one of the important ways to improve the production efficiency of direct-seeded rice. At the same time, studying the physiological characteristics of the mesocotyl can provide a theoretical basis for selecting rice varieties suitable for direct seeding and promote the further application and promotion of direct seeding technology for rice.

[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 by regulating the signal transduction pathway related to plant hormones. Exploring its function in plant growth and development, as well as the molecular mechanism of regulating mesocotyl elongation, has great potential for application in the cultivation of high-yield and economical direct-seeded rice. 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 growth.

[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 CCT5Recombinant plant expression vectors of genes are used to regulate plant growth.

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

[0008] In a preferred specific embodiment of the present invention, the regulating plant growth is promoting rice growth; wherein, the promoting rice growth includes increasing the length of the rice mesocotyl, increasing the germination rate of rice seeds during direct seeding or the height of rice seedlings.

[0009] For reference, the present invention provides an embodiment, that is, by overexpressing the gene encoding the CCT5 protein related to plant growth in the plant, the expression amount or activity of the CCT5 protein related to plant growth is increased, thereby promoting plant growth, thereby increasing the height of the plant seedlings, increasing the length of the plant mesocotyl, and increasing the germination rate of plant seeds during direct seeding.

[0010] A preferred specific embodiment of the present invention is a method for breeding a direct-seeding tolerant rice variety, comprising: overexpressing in rice 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 rice, so that CCT5 The gene is overexpressed in rice, and the direct seeding resistance of the resulting transgenic rice is improved, including: increasing the length of the rice mesocotyl, and increasing the germination rate or seedling height of rice seeds during direct seeding.

[0011] For reference, the present invention provides a CCT5 Gene plant recombinant expression vector, comprising: SiCCT5 Genes or derived from rice OsCCT5 The 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 plant growth regulating agent derived from millet or rice. 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 growth.

[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 plant is a grass plant.

[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 growth.

[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, overexpression OsCCT5 Genetic Nipponbare rice showed an increase in the length of the mesocotyl, increased seedling height and germination rate when direct-seeding rice seeds, that is, the growth performance during direct-seeding was significantly improved, proving that rice OsCCT5 The gene has the function of positively regulating the growth performance of plants during direct seeding. The present invention further analyzes and obtains the OsCCT5 The excellent haplotype whose gene is related to plant mesocotyl elongation, namely haplotype B, indicates that this haplotype has application potential in breeding new rice varieties suitable for direct-seeding.

[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 is wild type 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 mesocotyl length of rice lines with the gene cultured in continuous darkness for 7 days. The mesocotyl is the part from the arrow to the seed, and the arrow indicates the coleoptile node; 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 Wild-type rice WT and overexpression OsCCT5 Phenotypic analysis of rice lines expressing genes; 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 represents wild-type rice WT and overexpression OsCCT5 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 3 -D represents wild-type rice WT and overexpression OsCCT5 Statistical analysis results of mesocotyl length of rice lines with the gene cultured in continuous darkness for 7 days; Figure 3 -E represents wild-type rice WT and overexpression OsCCT5 Longitudinal sections of mesocotyls of genetic rice lines; Figure 3 -F is wild type rice WT and overexpression OsCCT5 Statistical analysis data of mesocotyl cell length in rice lines with the gene; Figure 3 -G is a picture of the germination test in which wild-type rice WT, OsCCT5-OE#1 and OsCCT5-OE#2 were planted at a depth of 2 cm in the soil; Figure 3 -H is the observation results of the mesocotyl of wild-type rice WT, OsCCT5-OE#1 and OsCCT5-OE#2 seedlings planted at a depth of 2 cm in the soil for germination experiments. The arrows indicate the coleoptile nodes, and the mesocotyl is the part between the arrow and the seed. Figure 3 -I is the statistical analysis results of germination rate in germination test of wild-type rice WT, OsCCT5-OE#1 and OsCCT5-OE#2 planted at a depth of 2 cm in the soil, where 1-9D represents 1-9 days after sowing; Figure 3 -J is a picture of the germination test in which wild-type rice WT, OsCCT5-OE#1 and OsCCT5-OE#2 were planted at a depth of 5 cm in the soil; Figure 3 -K is the observation results of the mesocotyl of wild-type rice WT, OsCCT5-OE#1 and OsCCT5-OE#2 seedlings planted at a depth of 5 cm in the soil for germination experiments. The arrows indicate the coleoptile nodes, and the mesocotyl is the part between the arrow and the seed. Figure 3 -L is the statistical analysis results of germination rate in germination test of wild-type rice WT, OsCCT5-OE#1 and OsCCT5-OE#2 planted at a depth of 5 cm in the soil, where 1-9D represents 1-9 days after sowing; Figure 4 is the haplotype analysis of OsCCT5 gene; Figure 4 -A represents the five haplotypes analyzed. SNP is the abbreviation of single nucleotide polymorphism, which means the replacement of a single base. The red ones are the bases or amino acids that have changed compared to haplotype A. The corresponding amino acids and numbers in brackets indicate the positions of the SNPs. Figure 4 -B is the sample quantity and subspecies type information of the five haplotypes; Figure 4 -C is the significance analysis result of mesocotyl length in 510 germplasm resources and 5 haplotypes, and n represents the number of germplasm resources with this haplotype. 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 test materials 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; 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, then dilute to 10 mL with methanol; or dilute to 10 mL with DMSO, 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 cloning 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 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 genetically modified 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 by rice genetic transformation to obtain the T0 generation Nipponbare rice strain that overexpressed the SiCCT5 gene. The specific operation 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) Transplantation 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] 1 / 2MS medium: The final concentration of solutes in MS is halved.

[0076] 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).

[0077] RICE-actin-F: TGCTATGTACGTCGCCATCCAG (SEQ ID NO.9); RICE-actin-R:AATGAGTAACCACGCTCCGTCA (SEQ ID NO. 10).

[0078] 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.

[0079] 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 the two overexpression lines SiCCT5-OE#1 and SiCCT5-OE#2.

[0080] The longitudinal sections of the mesocotyls of wild-type rice WT, SiCCT5-OE#1 and SiCCT5-OE#2 seeds cultured in the dark for 7 days were analyzed. Figure 1 -G and Figure 1 -H, it can be seen that the cell length of the mesocotyl 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 mesocotyl. SiCCT5 The gene has the function of regulating the development of rice mesocotyl.

[0081] 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( S. italica Seita.2G010200.1) and rice OsCCT5 Gene( O. sativaLOC Os06g36700.1). 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.

[0082] The amino acid sequence of OsCCT5 protein is as follows: MALAFDEFGRPFIILREQEKKSRLRGLDAQKANIAAGKAVARILRTSLGPKGMDKMLQSPDGDVTITNDGATILEQMDVDNQIAKLMVELSCSQDYEIGDGTTGVVVMAGSLLEQAEKLLERGIHPIRIAEGYELASRIAFDHLEHISHKFEFSATNIEPLVQTCMTTLSSKIVNRCKRTLAEIAVKAVLAVADLERKDVNLDLIKVEGKVGGKLEDTELVYGIIVDKDMSHPQMPKRIEDAKIAILTCPFEPPKPKTK HKVDIDTVEKFQMLREQEQKYFDEMVQKCKDVGATLVICQWGFDDEANHLLMHRNLPAVRWVGGVELELIAIATGGRIVPRFQELSPEKLGKAGIVREKSFGTTKDRMLYIEQCANSRAVTIFIRGGNKMMIEETKRS LHDALCVARNLIRNNSIVYGGGSAEISCSVAVEAAADRYPGVEQYAIRSFADALDAIPLALAENSGLSPIDTLTAVKSQQVKESNPHCGIDCNDVGTNHMKEQNVFETLIGKQQQILLATQVVKMILKIDDVISPSDY (SEQ ID NO. 1).

[0083] OsCCT5

[0084] 1 Overexpression OsCCT5 Preparation of genetically modified 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.

[0085] 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).

[0086] 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.

[0087] 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.

[0088] Wild-type rice WT, OsCCT5-OE#1 and OsCCT5-OE#2 were identified and designed using DNAMAN software. 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).

[0089] RICE-actin-F: TGCTATGTACGTCGCCATCCAG (SEQ ID NO. 15); RICE-actin-R:AATGAGTAACCACGCTCCGTCA (SEQ ID NO. 16).

[0090] 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.

[0091] 2 Overexpression OsCCT5 Experiment on improving plant growth under direct seeding The mesocotyl development of wild-type rice WT, OsCCT5-OE#1 and OsCCT5-OE#2 seeds was analyzed after 7 days of continuous dark culture. Figure 3 -C and Figure 3 As shown in Figure 5-D, a significant increase in the length of the embryonic axis was observed in both OsCCT5-OE#1 and OsCCT5-OE#2 overexpression lines.

[0092] The longitudinal sections of the mesocotyls of wild-type rice WT, OsCCT5-OE#1 and OsCCT5-OE#2 seeds were observed after 7 days of continuous dark culture. Figure 3 -E and Figure 3 As shown in Figure 5-F, it can be seen that the length of mesocotyl cells in the two overexpression lines OsCCT5-OE#1 and OsCCT5-OE#2 increased significantly, that is, OsCCT5 The elongation of mesocotyl cells in gene overexpression lines is the main reason for mesocotyl elongation.

[0093] In order to detect the application of OsCCT5 gene in direct seeding of rice, wild-type rice WT, OsCCT5-OE#1 and OsCCT5-OE#2 were planted at a depth of 2 cm in the soil for germination. It can be observed that compared with wild-type rice WT, the seedling height of OsCCT5-OE#1 and OsCCT5-OE#2 increased significantly ( Figure 3 -G), mesocotyl length increased significantly ( Figure 3 -H), the germination rate was also significantly increased compared with the wild-type rice WT ( Figure 3 -I).

[0094] Wild-type rice WT, OsCCT5-OE#1, and OsCCT5-OE#2 were planted at a depth of 5 cm in the soil for germination. It was observed that compared with wild-type rice WT, OsCCT5-OE#1 and OsCCT5-OE#2 also showed a significant increase in seedling height ( Figure 3 -J), mesocotyl length increased significantly ( Figure 3 -K), the germination rate was also significantly increased compared with the wild-type rice WT ( Figure 3 -L).

[0095] The above results show that rice OsCCT5 The gene can significantly improve the growth status and germination rate of rice under direct seeding.

[0096] Test Example 3 OsCCT5 Excellent haplotype analysis of genes related to mesocotyl development Based on the database and analysis software such as rice 3K sequencing (snp-seek.irri.org), the OsCCT5 Among the 6 SNP sites in the gene coding region, 5 were identified in the 4711 rice data sets. OsCCT5 Gene haplotype ( Figure 4 -A). Five types of rice germplasm resources OsCCT5 The size of the fan-shaped area represents the number of samples of each haplotype, and different colors represent different subspecies of rice. OsCCT5 The natural variation of genes is mainly composed of two haplotypes: haplotype A (SNP type of rice Nipponbare) and haplotype B ( Figure 4 -B).

[0097] Based on the data of seedling mesocotyl length of 510 cultivated rice germplasm resources retrieved, combined with 5 haplotype types for significance analysis, it was found that OsCCT5 Haplotype B of the gene was significantly associated with increased mesocotyl length compared to haplotype A (wild type WT type) ( Figure 4 -C), indicating OsCCT5Natural variation in the gene is associated with differences in mesocotyl elongation, and haplotype B is an important gene resource with the potential for improved direct-seeding application in rice.

Claims

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

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

3. The use according to claim 2, characterized in that: The method for promoting rice growth includes increasing the length of the rice mesocotyl and improving the germination rate or seedling height of rice seeds during direct seeding.

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 1 or 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 growth.

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 breeding direct-seeding tolerant rice varieties, characterized in that: include: Build contains CCT5 Gene overexpression recombinant plant expression vector; Will CCT5 Gene overexpression recombinant plant expression vector transformed rice, CCT5 The gene is overexpressed in rice, and the growth performance of the resulting transgenic rice during direct seeding is improved, including: increasing the length of the rice mesocotyl, increasing the germination rate or seedling height of rice seeds during direct seeding; 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.

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