Rice grain number per ear regulation promoter pTCP17 and application thereof

Abstract

The invention discloses a promoter pTCP17 for regulating and controlling the number of grains per ear of rice and application of the promoter pTCP17. In particular discloses a DNA (deoxyribonucleic acid) molecule with a nucleotide sequence of SEQ ID No.1 and application of the DNA molecule as a promoter. The invention further discloses application of the promoter and related biological materials thereof in regulation and control of the grain number per ear of rice. The DNA molecule (SEQ ID No.1) provided by the invention has the function of a promoter, and mononucleotide mutation in a DNA sequence of the DNA molecule specifically regulates the expression quantity of a target gene in a panicle, and is specifically expressed in a rice stem base and a young panicle. The invention has important theoretical and practical significance on research of a rice grain number regulation molecular mechanism and rice molecular breeding, the rice quality is improved through rice grain number improvement molecular breeding, different requirements of consumers can be met, the competitiveness of products in the market is improved, and the application prospect is wide. Wide application and market prospects are realized in the agricultural field.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to promoters that regulate grain number in rice panicles. pTCP17 And its applications. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is one of the world's three most important food crops, with more than half of the world's population relying on it for sustenance. Therefore, food security plays a crucial role in maintaining social stability and the development of civilization. Rice has a long history of cultivation and consumption in China, providing the daily nutritional needs of over 60% of the population. Increasing its yield is vital to addressing the ever-growing demand for food, and developing high-yielding rice varieties to increase rice production is one of the most effective strategies for ensuring food security.

[0003] Cultivated rice evolved from wild rice through a long process of domestication. Due to the bottleneck effect of domestication and artificial selection, cultivated rice lost a large number of alleles during the domestication process, resulting in a richer genetic resource compared to cultivated rice. Therefore, exploring the abundant allele resources in wild rice and applying them to improve cultivated rice varieties, thus broadening the genetic background of existing breeding materials, has significant theoretical and practical implications for breaking through the genetic bottleneck of yield in rice breeding and achieving further high yields. Summary of the Invention

[0004] One objective of this invention is to provide a promoter for regulating grain number in rice panicles. pTCP17 And its applications. The technical problems to be solved are not limited to the technical topics described herein, and those skilled in the art will clearly understand other technical topics not mentioned herein through the following description.

[0005] To achieve the above objectives, the present invention first provides a DNA molecule, the nucleotide sequence of which may be as shown in SEQ ID No. 1.

[0006] The present invention also provides the application of the DNA molecule as a promoter.

[0007] Furthermore, the application can drive (initiate) the expression of the target gene in rice.

[0008] In the above applications, the DNA molecule may be derived from rice.

[0009] Furthermore, the DNA molecule may be derived from Niwara wild rice ( Oryza nivara ).

[0010] In the above applications, the DNA molecule can be a rice tissue-specific promoter.

[0011] Furthermore, the rice tissue specificity can be specific to the rice stem base and young panicle.

[0012] The present invention also provides a biomaterial, which may be any of the following: A1) An expression cassette containing the DNA molecule; A2) A recombinant vector containing the DNA molecule described above; A3) Recombinant microorganisms containing the DNA molecule described above; A4) Recombinant host cells containing the DNA molecule.

[0013] All of the biological materials can express the DNA molecule.

[0014] The recombinant vector described in A2) can be a recombinant expression vector. Existing plant expression vectors can be used to construct recombinant expression vectors containing the DNA molecule described. These plant expression vectors include, but are not limited to, binary expression vectors (such as the pBI series vectors (e.g., pBI121), pBIN series vectors (e.g., pBin19), pCAMBIA series vectors (e.g., pCAMBIA1300 vector), pPZP series vectors, pGreen series vectors, pBIBAC series vectors, pSKI015 vector, pSKI074 vector, pRI101-AN vector, etc.) and co-integration vectors (which can be constructed by inserting a segment homologous to the Ti plasmid or its segment into an intermediate vector via homologous recombination or cloning). When introducing the promoter of this invention using the Agrobacterium method, expression vectors suitable for the Agrobacterium method are preferably used, such as binary vectors or modified vectors thereof. Examples of these plant expression vectors include, but are not limited to, pBI121, pBIN19, pSMAB704, pCAMBIA series vectors, and pGreen series vectors. In one or more embodiments of the present invention, the plant expression vector used is pCAMBIA1300.

[0015] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed by adding genes that can be expressed in plants, including but not limited to genes encoding enzymes or luminescent compounds that produce color changes (GUS gene, luciferase gene, GFP gene, etc.), antibiotic resistance genes (kanamycin resistance gene kanr, neomycin resistance gene neo, hygromycin resistance gene hyg, chloramphenicol resistance gene cat, streptomycin resistance gene str, bleomycin resistance gene ble, etc.), or herbicide resistance genes (bar gene, glyphosate resistance marker gene epsps, chlorothalonil resistance marker gene als, etc.). For safety reasons, transgenic plants can also be screened directly without adding any selective marker genes.

[0016] The recombinant expression vector may also include a target gene (e.g., molecule) operably linked downstream of the DNA molecule (as a promoter). TCP17 Gene or reporter gene ） .

[0017] This invention also provides for the use of any of the DNA molecules or biological materials described herein in any of the following: B1) Application in regulating the number of grains per panicle in rice; B2) Application in driving the specific expression of target genes in the base of rice stems and young panicles; B3) Application in cultivating rice with altered grain number per panicle; B4) Application in molecular breeding for improving grain number per panicle in rice or in the improvement of rice germplasm resources related to grain number per panicle.

[0018] The change in the number of grains per ear can be an increase or a decrease.

[0019] Furthermore, the application can be driven in rice using the promoter (SEQ ID No. 1) described in this invention. TCP17 It is achieved through genes.

[0020] This invention also provides the application of any of the DNA molecules or biological materials described herein in increasing the expression level of target genes in rice.

[0021] In the above applications, increasing the expression level of the target gene in rice can refer to increasing the expression level of the target gene during the development of young rice panicles.

[0022] In the above applications, increasing the expression level of the target gene in rice can refer to increasing the expression level of the target gene in the base of the rice stem and in the young panicle.

[0023] The present invention also provides a method for cultivating transgenic rice, the method comprising using the DNA molecule as a promoter to drive the expression of a target gene in the target rice to obtain transgenic rice with altered grain number per panicle.

[0024] Furthermore, the method can be implemented by introducing the DNA molecule as a promoter into the target rice.

[0025] In this article, the target gene may be: TCP17 Genes or reporter genes.

[0026] Furthermore, the method includes any of the following: C1) In rice TCP17 The natural promoter of the gene is replaced with the DNA molecule described above; C2) In rice TCP17A second promoter, wherein the second promoter is the DNA molecule, is operatively linked to the gene. C3) The DNA molecule is operably linked as a promoter element to... TCP17 Upstream of the gene coding region, they are jointly introduced into rice.

[0027] Furthermore, the methods described in this article include, but are not limited to: Agrobacterium-mediated transformation, plant virus vector-mediated transformation, gene gun method (also known as microparticle bombardment method or biological missile method), chemical stimulation method, electric shock method, liposome-mediated method, microinjection method, laser microbeam method, pollen tube channel method, ultrasound method, air gun method, and eddy current method.

[0028] Furthermore, the method of introduction can be Agrobacterium-mediated transformation.

[0029] Further, the Agrobacterium-mediated method may include the following steps: (1) operably linking the DNA molecule (SEQ ID No. 1) as a promoter element to... TCP17 Upstream of the gene coding region, a recombinant DNA molecule is obtained; (2) the recombinant DNA molecule is cloned into a plant expression vector to obtain a recombinant expression vector; (3) the recombinant expression vector is introduced into Agrobacterium (such as Ca ion-induced transformation, polyethylene glycol-mediated transformation, metal cation-mediated transformation, electroporation transformation, phage transduction, etc.) to obtain recombinant Agrobacterium, and the recombinant Agrobacterium infects the callus or explant of the target rice; after identification, the obtained positive callus or explant is induced and cultured to obtain regenerated plants.

[0030] The explants include, but are not limited to, seeds, roots, leaves, petioles, cotyledons, cotyledonary petioles, hypocotyls, stem segments, shoot apical meristems, epidermal parenchyma cells, tubers, stolons, embryogenic suspension cells, and protoplasts.

[0031] The screening and identification methods are known to those skilled in the art. For example, transformed transgenic plants (including transgenic progeny materials) can be identified by techniques such as PCR detection, Southern hybridization, immunoblotting, Northern hybridization, enzyme-linked immunosorbent assay (ELISA), functional identification (testing for the presence of selection marker genes and target genes), and / or in situ hybridization.

[0032] The number of grains per panicle is a crucial factor affecting rice yield and quality. Increasing the number of grains per panicle can improve rice yield, while decreasing the number of grains per panicle not only helps concentrate nutrients, making each grain fuller and thus improving the nutritional value of rice, but also enhances yield stability and lodging resistance. Improving rice quality through molecular breeding to optimize grain number per panicle can meet diverse consumer needs and enhance product competitiveness in the market.

[0033] This invention discloses a rice panicle grain number regulating promoter. pTCP17 Its applications. The DNA molecule (SEQ ID No. 1) provided by this invention has the function of a promoter. A single nucleotide mutation in its DNA sequence specifically regulates the expression level of the target gene in the panicle, and specifically expresses it in the rice stem base and young panicle. This invention has important theoretical and practical significance for the study of the molecular mechanism of rice panicle grain number regulation and for rice molecular breeding, and will have broad application and market prospects in the agricultural field.

[0034] Terminology Definition In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0035] The term "expression cassette" generally refers to a nucleic acid construct containing sufficient nucleic acid elements to express a target gene. A typical expression cassette includes a promoter, a multiple cloning site (MCS), and a terminator. Expression cassettes may also include the target gene, marker genes (such as TK, DHFR, CAT, and NEO genes), ribosome recognition and binding sites (SDs), transcription factor binding sites (TFBSs), enhancers, silencers, repressors, introns, poly(A) signal sequences, and / or mRNA splicing signal sequences. Elements within an expression cassette can be directly linked or indirectly linked through adapters.

[0036] The term "vector" generally refers to a vector capable of delivering exogenous DNA or a target gene into host cells for amplification and / or expression. This vector can be a cloning vector or an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be amplified and / or expressed within the host cells. Those skilled in the art can select appropriate vectors based on the purpose of genetic engineering and the properties of the recipient cells. The vectors include, but are not limited to: plasmids, phages (such as λ phage or M13 phage), cosmids (i.e., Cosmids), phagemids, shuttle vectors (such as yeast expression vectors), Ti plasmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), P1 artificial chromosomes (PAC), or Ti plasmid artificial chromosomes (TAC)), and viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, papillomaviruses (such as SV40), and herpesviruses (such as herpes simplex virus)). A vector may contain multiple elements controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication origin site.

[0037] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasmas, chlamydiae, spirochetes, algae, etc. For example, the bacteria mentioned could be from the genus *Escherichia* (…). Escherichia sp. (such as Escherichia coli), Erwinia spp. Erwinia sp. ), Agrobacterium ( Agrobacterium sp. (such as Agrobacterium tumefaciens), Flavobacterium spp. ( Flavobacterium sp. Alcaligenes ( ) Alcaligenes sp. ), Pseudomonas spp. Pseudomonas sp. ) and Bacillus spp. ( Bacillus sp. (e.g., Bacillus). The viruses may include rotavirus, baculovirus, retrovirus (e.g., lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papillomavirus (e.g., SV40), and herpesvirus (e.g., herpes simplex virus). The fungi may be derived from yeasts (e.g., Bacillus). Saccharomyces sp. (such as Saccharomyces cerevisiae, Saccharomyces methylbenzene, Pichia pastoris), Fusarium genus ( Fusarium sp. ), Rhizoctonia spp. ( Rhizoctonia sp. Verticillium ( Verticillium sp. ), Penicillium ( Penicillium sp. Aspergillus ( ) Aspergillus sp. ) and Cephalosporin ( Cephalosporium sp. The actinomycetes may originate from the genus Streptomyces (…).Streptomyces sp. (e.g., Streptomyces). The algae may originate from the phylum Cyanophyta (e.g., cyanobacteria), genus Fucus (e.g., fucus vesiculosus). Fucus sp. ), genus *Cyclocarya* ( Achnanthes sp. ), genus *Codonopsis* ( Amphiprora sp. ), genus Dipterocarpa ( Amphora sp. ), Fiber Algae ( Ankistrodesmus sp. ), genus *Stellaria* ( Asteromonas sp. ) and the genus *Golden Color Algae* ( Boekelovia sp. )wait.

[0038] The term "host cell," also known as the recipient cell, generally refers to any type of cell that can be used to introduce a vector, such as plant and animal cells. The term "host cell" can be understood not only to the specific recipient cell but also to its offspring, which, due to natural, accidental, or intentional mutations and / or alterations, may not necessarily be identical to the original parent cell but are still included within the scope of the host cell.

[0039] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by linking a foreign target gene to a vector in vitro. It can be constructed in any suitable way, as long as the constructed recombinant vector can carry the foreign target gene into the recipient cell and provide the foreign target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0040] The term "recombinant microorganism" generally refers to a recombinant microorganism whose genes have been manipulated and modified to obtain a functionally altered microorganism. This can be achieved by introducing a foreign target gene or recombinant vector into the target microorganism, or by directly editing the endogenous genes of the target microorganism.

[0041] The term "recombinant host cell" generally refers to a recombinant host cell whose genes have been manipulated and modified to obtain a recombinant host cell with altered function. This can be achieved by introducing a foreign target gene or recombinant vector into the host cell, or by directly editing the host cell's endogenous genes.

[0042] The term "promoter" usually refers to the site that RNA polymerase specifically recognizes and binds to. It is located upstream of the transcription start site of a structural gene, has strict directionality, and initiates transcription.

[0043] The term "operably linked" generally refers to the physical and / or functional connection of one DNA segment to another in a way that allows the segment to function in the intended manner.

[0044] The term "target gene" generally refers to a nucleic acid molecule that encodes a target protein (any protein of interest). In gene cloning, the target gene can be the gene that is to be isolated, purified, cloned, and transformed into an organism to produce the desired phenotypic trait (such as insect resistance or herbicide tolerance). The target gene can be native to the organism itself or derived from a different organism. Methods for obtaining the target gene are well-known to those skilled in the art. For example, a complex organism's genome can be digested with enzymes to construct a genomic library, from which DNA fragments carrying the target gene can be isolated; or reverse transcription can be used to obtain cDNA from mRNA as the target gene; the target gene can also be artificially synthesized using enzymatic or chemical methods; or the target gene can be directly amplified in vitro from the donor organism's genome or an existing clone of the target gene using PCR technology.

[0045] The term "reporter gene" generally refers to a group of genes encoding easily detectable proteins or enzymes. The reporter gene's coding sequence is fused with a gene expression regulatory sequence, or fused with other target genes, and expressed under the control of the regulatory sequence. The expression regulation of the target gene is "reported" by detecting the expression product of the reporter gene. Reporter genes include β-glucuronidase (GUS) genes, luciferase genes, chloramphenicol acetyltransferase (CAT) genes, β-galactosidase (β-gal) genes, secretory human placental alkaline phosphatase (SEAP) genes, yellow fluorescent protein (YFP) genes, and green fluorescent protein (GFP) genes, among others.

[0046] The term "introduction" generally refers to the transfer of a foreign gene into a recipient cell, such as a eukaryotic or prokaryotic recipient cell. There are no particular restrictions on the method of introduction; any known transformation method that can transfer the target gene into the recipient cell is acceptable. The method of introduction may include any of the following: (1) chemical transformation (such as Ca...). 2+ (1) Induced transformation, polyethylene glycol-mediated transformation, or metal cation-mediated transformation, etc.) or physical transformation (such as electroporation transformation) to introduce the target gene or a recombinant vector containing the target gene into the host bacteria. (2) Transducing the target gene into the host bacteria through phage transduction. (3) Directly transferring the target gene into plant recipient cells through physical or chemical methods, such as chemical stimulation, electroporation, liposome-mediated transformation, microinjection, gene gun, laser microbeam, pollen tube channel, ultrasound, air gun, and eddy current. (4) Transforming the target gene into plant recipient cells using a vector as a medium, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector systems and binary vector systems) mediated transformation.

[0047] The term "explant" generally refers to a part of a plant used as in vitro culture material in plant tissue culture, which, after appropriate treatment and under suitable conditions, can regenerate into a whole plant. In practice, those skilled in the art select suitable explants for transformation based on different plants. Explants include seeds, roots, leaves, petioles, cotyledons, cotyledonary petioles, hypocotyls, stem segments, shoot apical meristems, epidermal parenchyma cells, tubers, stolons, embryogenic suspension cells, and protoplasts, etc.

[0048] The term "callus" generally refers to the new tissue that forms on the surface of a wound after a localized injury to the original plant. It consists of living parenchyma cells and can originate from living cells in various tissues within any organ of the plant. In plant tissue culture, it can refer to a cluster of disordered, rapidly dividing parenchyma cells formed from an explant. Cultivating callus on a suitable culture medium can induce the formation of a whole plant. Attached Figure Description

[0049] Figure 1 A comparison of panicle phenotypes between the introgression line IL60 and the indica rice variety 93-11.

[0050] Figure 2 To identify the expression levels of the TCP17 gene at different time points and in different tissue sites in the Niwara wild rice introgression line IL60 and the indica rice variety 93-11 using real-time quantitative PCR.

[0051] Figure 3 For penetration into IL60 and 93-11 TCP17 Comparison of gene DNA sequences.

[0052] Figure 4 The results of promoter activity detection were obtained from a dual-luciferase transient expression experiment.

[0053] Figure 5 This study compares the ear phenotypes of transgenic plants and Teqing plants.

[0054] Figure 6 For transgenic plants and special young spikelets TCP17 Comparison of gene expression levels. Detailed Implementation

[0055] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0057] The following examples used R (version v4.4.0) statistical software to process the data. The experimental results are expressed as mean ± standard deviation. t The t-test method is used, where P < 0.05 (*) indicates a statistically significant difference, P < 0.01 (**) indicates a statistically significant difference, and P < 0.001 (***) indicates a highly statistically significant difference. Unless otherwise specified, the quantitative experiments in the following examples are performed in triplicate, and the results are averaged.

[0058] The primer synthesis and sequencing work used in the following examples were all completed by Qingke Biotechnology Co., Ltd.

[0059] The biomaterials involved in the following embodiments: The rice variety “Teqing” is described in the following literature: Jiang LY, Ma X., Zhao SS, Tang Y.Y., Liu FX, Gu P., Fu YC, Zhu ZF, Cai HW, Sun CQ and Tan LB (2019) The APETALA2-like transcription factor SUPERNUMERARY BRACT controls rice seed shattering and seed size. Plant cell , 31: 17–36. The rice variety “93-11” is described in the following literature: Jiang LY, Ma X., Zhao SS, Tang Y.Y., Liu FX, Gu P., Fu YC, Zhu ZF, Cai HW, Sun CQ and Tan LB (2019) The APETALA2-like transcription factor SUPERNUMERARY BRACT controls rice seed shattering and seed size. Plant cell , 31: 17–36. The introgression line IL60 is described in the following literature: Ma X., Fu YC, Zhao XH, Jiang LY, Zhu Z.F., Gu P., Xu WY, Su Z., Sun CQ and Tan LB (2016) Genomic structure analysis of a set of Oryza nivara introgression lines and identification of yield-associated QTLs using whole-genome resequencing. Sci Rep , 6:27425. The plant expression vector pCAMBIA1300 is the same as the binary vector pCAMBIA-1300 in NCBI. It is a circular plasmid, and its nucleotide sequence is indexed in GenBank as AF234296.1. The total length of the plant expression vector pCAMBIA1300 is 8958 nucleotides. Nucleotides 5338 to 6132 are the aadA gene (kanamycin resistance gene), and nucleotides 6873 to 7898 are the hptII gene (hygromycin resistance gene).

[0060] The vector pGreenII 0800-LUC was purchased from Ubisoft Biotech, catalog number VT8124.

[0061] The following examples TCP17 The gene's GenBank accession number is EU702407.1.

[0062] Example 1: Rice panicle grain number regulation promoter ​ The discovery Firstly, a phenotypic survey of a wild rice introgression line population using the indica rice variety 93-11 as the recurrent parent revealed a line IL60 with a reduced number of grains per panicle compared to 93-11. Compared to the recurrent parent 93-11, IL60 exhibited a decrease in both the number of grains per panicle and yield. ​ Using map-based cloning, only one target gene is located within the defined region, i.e. ​ Genes. Next, real-time quantitative PCR was used to detect... ​ The expression levels of genes in IL60 and 93-11 at different time points and in different tissue sites were found. ​ The gene is specifically expressed only at the base of the stem and in the young spikelet, and its expression is highest in IL60 when the young spikelet is 1 to 3 mm in length. ​ The gene expression level was significantly higher than that of 93-11 ( ​). And in IL60 and 93-11 ​ The results of genome sequence comparison showed that SNP mutations in the coding region did not alter the amino acid sequence; only the promoter region showed differences in SNPs and InDels. ​ Based on the above, the judgment is... ​ Mutations in the gene promoter region lead to ​ The gene expression levels differed between IL60 and 93-11 when the panicle length was 1–3 mm, and this difference in expression level was the direct cause of the difference in grain number per panicle between IL60 and 93-11. Therefore, we have discovered a rice grain number regulating promoter, and the DNA molecule shown in SEQ ID No. 1 is named... ​ .

[0063] Example 2, Promoter ​ Functional verification I. IL60 and 93-11 types ​ promoter activity analysis 1. Using genomic DNA from introgression lines IL60 and 93-11 as templates, PCR amplification was performed using primer pairs consisting of primers LUCF and LUCR, respectively, to obtain PCR products PCR-IL60pro-TCP and PCR-93-11pro-TCP.

[0064] Primer LUCF: 5'-ACTCACTATAGGGCGAATTGGGTACCAGATATATACTGTGTCTCAC-3', Primer LUCR: 5'-GAACTAGTGGATCCCCCGGGCTGCAGGGTGGTCGTAGGGTTTGGAG-3'.

[0065] PCR reaction conditions: 1) 94℃ for 5 minutes; 2) 94℃ for 30 seconds, 58℃ for 30 seconds, 72℃ for 2 minutes, for a total of 34 cycles; 3) Extension at 72℃ for 10 minutes. PCR products were stored at 4℃.

[0066] 2. Using restriction endonucleases ​ I and ​ The pGreenII 0800-LUC vector was digested with enzyme I, and the linearized vector backbone was recovered using an agarose gel recovery kit.

[0067] 3. The PCR product from step 1 and the linearized vector from step 2 were subjected to homologous recombination using a homologous recombinase to obtain the recombinant plasmid TCP17-pro. IL60 and TCP17-pro 93-11 Based on the sequencing results: Recombinant plasmid TCP17-pro IL60 It is the pGreenII 0800-LUC vector ​ I and ​ The fragment (small fragment) between the I recognition sites is replaced with the nucleotide sequence shown in SEQ ID No. 1 of the sequence listing (IL60 type). ​ The promoter sequence of the gene was obtained by keeping the other nucleotide sequences of the pGreenII 0800-LUC vector unchanged.

[0068] Recombinant plasmid TCP17-pro 93-11 It is the pGreenII 0800-LUC vector ​ I and ​ The fragment (small fragment) between the I recognition sites is replaced with a nucleotide sequence that is the DNA fragment (type 93-11) shown in SEQ ID No. 2 of the sequence listing. ​ The promoter sequence of the gene was obtained by keeping the other nucleotide sequences of the pGreenII 0800-LUC vector unchanged.

[0069] 4. Obtaining rice protoplasts and plasmid transformation (1) Select plump seeds with no abnormal color spots on the surface and no open opening. After removing the seed shell with a rice huller, put them into a 100 mL Erlenmeyer flask, add 75% ethanol for 3 min to disinfect, and then continue to disinfect with 20% sodium hypochlorite solution for 30 min.

[0070] (2) Pour out the sodium hypochlorite solution in the clean bench, being careful not to spill the seeds. Add sterile ultrapure water to wash the seeds several times until the water no longer has the smell of sodium hypochlorite. Spread the washed seeds evenly on sterile absorbent paper to air dry, and then place them on MS medium (about 20 seeds per bottle). Incubate at 28°C for 10 days, avoiding light during this period to produce etiolated seedlings.

[0071] (3) Cut off the roots and leaves of the etiolated seedlings, leaving only the central stem. Use a sharp blade to cut the stem into segments of about 0.5 mm. Place the cut stem segments into a 200 mL Erlenmeyer flask (wrapped in aluminum foil) and add 50 mL of enzymatic hydrolysis solution. The relevant reagents and formulas are as follows:

[0072]

[0073] (4) After shaking slowly at 40 rpm for 4 h at 28℃, cover the bottle mouth with a single layer of Miracloth filter cloth and pour out the enzymatic hydrolysate. Add 50 mL of W5 resuspension and shake slowly at 40 rpm for 1 h at 28℃ to release the protoplasts.

[0074] (5) Cover the mouth of the culture dish with a single layer of Miracloth filter cloth, slowly pour in the liquid from (4), discard the residue on the Miracloth filter cloth, and gently aspirate the filtered liquid into a 50 mL centrifuge tube with a blue pipette tip that has been cut off. Each tube can hold a maximum of 25 mL of liquid. After balancing, centrifuge at 1000 rpm (speed increase 1, speed decrease 0) for 10 min to enrich the protoplasts at the bottom of the tube.

[0075] (6) Gently aspirate the supernatant, as protoplasts are very easy to resuspend. It is necessary to leave 5 mL of liquid in this case. Add 10 mL of W5 resuspension and gently rotate the centrifuge tube to resuspend the protoplasts. After balancing, centrifuge at 1000 rpm (speed 1 for rising and speed 0 for falling) for 10 min to enrich the protoplasts at the bottom of the tube.

[0076] (7) Gently aspirate the supernatant, avoiding resuspending the protoplasts as much as possible, leaving approximately 1 mL of liquid that cannot be completely aspirated. Add the calculated volume of 200 μL of Mmg resuspension to the protoplasts according to the dosage for each reaction, and gently rotate the centrifuge tube to resuspend the protoplasts. The relevant reagents and formulations are as follows:

[0077] (8) The recombinant plasmid (TCP17-pro) obtained in step 3 IL60 and TCP17-pro 93-11 Pipette 10 μg into the bottom of a sterile 1.5 mL centrifuge tube and add 200 μL of protoplasts.

[0078] (9) Add 220 μL of 40% (w / v) PEG solution, gently invert to mix until no layering is visible. Incubate at room temperature in the dark for 20 min.

[0079]

[0080] (10) Add 1 mL of W5 solution (see Table 4) and mix gently. Centrifuge at 1000 rpm (speed 1, speed 0) for 5 min. After removing 1 mL of supernatant, add 1 mL of W5 resuspension.

[0081] (11) Incubate at room temperature for 14 h, away from light.

[0082] 5. Detect promoter activity using a dual-luciferase transient expression assay. After incubation in the dark for 14 h in step 4, protoplasts were centrifuged at 1000 rpm (acceleration 1, deceleration 0) for 5 min. The supernatant was aspirated, leaving only 200 μL of protoplasts. 100 μL of cell lysis buffer (Promega) was added, and the cells were vigorously shaken for 30 s to fully lyse the cells. Subsequently, LUC and REN values ​​were measured using a multi-functional microplate reader and a LUC dual-luciferase assay kit (Promega). Each sample was measured three times.

[0083] The results of the dual-luciferase transient expression assay to detect promoter activity indicated that IL60 type ​ Gene promoter (TCP17-pro) IL60 The promoter activity of (SEQ ID No. 1) is higher than that of type 93-11. ​ Gene (TCP17-pro) 93-11 promoter activity of SEQ ID No. 2 ​ ).

[0084] II. Constructing genome-complementing transgenic plants to validate promoters ​ Functions 1. pCAMBIA1300- ​ :: ​ Obtaining recombinant plasmids 1-1. Using the genomic DNA of the introgression line IL60 as a template, PCR amplification was performed using primers consisting of HBF and HBR to obtain the PCR product.

[0085] Primer HBF: 5'-ATGATTACGAATTCGAGCTCGGTACCAGATATATACTGTGTCTCAC-3', Primer HBR: 5'-TTGCATGCCTGCAGGTCGACTCTAGACTAATATTGCATACCGTCCAAG-3'.

[0086] PCR reaction conditions: 1) 94℃ for 5 minutes; 2) 94℃ for 30 seconds, 58℃ for 30 seconds, 72℃ for 2 minutes, for a total of 34 cycles; 3) 72℃ for 10 minutes extension. PCR products were stored at 4℃.

[0087] 1-2. Using restriction endonucleases ​ I and ​ The plant expression vector pCAMBIA1300 was digested with enzyme I, and the linearized vector backbone was recovered using an agarose gel recovery kit.

[0088] 1-3. The PCR product from step 1-1 and the linearized vector from step 1-2 are subjected to homologous recombination using a homologous recombinase to obtain the recombinant plasmid pCAMBIA1300-​ :: ​ (This carrier is used for) ​ (A genome complementation vector constructed from the promoter of the gene itself). According to sequencing results, the recombinant plasmid contains... ​ I and ​ A DNA molecule (promoter) as shown in SEQ ID No. 1 was inserted between the I restriction sites. ​ )and ​ A recombinant fragment consisting of a gene coding region sequence (SEQ ID No. 3).

[0089] 2. Obtaining transgenic plants with genome complementation 2-1. The recombinant plasmid pCAMBIA1300- ​ :: ​ Agrobacterium EHA105 was transformed to obtain the recombinant vector pCAMBIA1300- ​ :: ​ Agrobacterium EHA105, also known as Agrobacterium EHA105 / pCAMBIA1300- ​ :: ​ .

[0090] 2-2. Preparation of mature embryo callus from the indica rice variety Teqing (indica conventional rice, hereinafter referred to as TQ) (1) Select unopened rice seeds with no obvious fungal spots on the seed surface and place them in a 42℃ oven for 2 days to break dormancy.

[0091] (2) Remove the outer shell of the rice seeds with a threshing machine, and select seeds with normal endosperm and no bacterial spots on the surface and place them in a 100mL Erlenmeyer flask.

[0092] (3) Add 75% alcohol for disinfection for 3 min, pour out the 75% alcohol, then add 15% sodium hypochlorite solution and disinfect in a shaker at 37℃ for 20 min.

[0093] (4) Pour out the sodium hypochlorite solution in the laminar flow hood, rinse the seeds 5-6 times with sterile deionized water until the water used to wash the seeds is relatively clear, and place them in a petri dish lined with 6 layers of sterile filter paper to air dry.

[0094] (5) Place the dried seeds on NB medium with a spoon, and spread them evenly on NB medium with tweezers, about 30-50 seeds per dish.

[0095] (6) Cultured in the dark in a constant temperature incubator at 28℃ for 5 days until pale yellow callus tissue grows. Peel off the induced callus and place it on NB basic culture medium to recover for 2 days to obtain mature embryo callus tissue.

[0096] The composition of NB culture medium is shown in Table 5:

[0097] 2-3. Agrobacterium infection Agrobacterium EHA105 / pCAMBIA1300- ​ :: ​ The mature embryo callus tissue of the above-prepared indica rice variety Teqing was infected. The callus tissue needed to undergo three screening cultures, with the amount of hygromycin in the screening medium increasing each time. For the first screening culture, 300 μL of 50 mg / mL hygromycin was added to 1 L NB medium, and the screening time was 25–30 days. For the second screening culture, 500 μL of 50 mg / mL hygromycin was added to 1 L NB medium, and the screening time was about 20 days. For the third screening culture, 700 μL of 50 mg / mL hygromycin was added to 1 L NB medium, and the screening time was about 20 days (the screening time can be adjusted according to the state of the callus tissue). After screening, the resistant callus (dense, hard, large granular or blocky callus tissue) was transferred to predifferentiation medium and cultured for 10 days, and then transferred to differentiation medium for differentiation for about 20 days. After differentiation, the seedlings were transferred to rooting medium and cultured for 10–15 days until they grew into seedlings, thus obtaining complementary transgenic positive lines.

[0098] The above transgenic lines were identified by PCR using hygromycin primer sequences and target gene fragment sequences, yielding T0 generation complementary transgenic positive plants (EHA105 / pCAMBIA1300-). ​ :: ​ Twelve plants (obtained through infection) were named Teqing. CTP .

[0099] The compositions of the predifferentiation medium, differentiation medium, and rooting medium are shown in Tables 6, 7, and 8:

[0100]

[0101]

[0102] 3. PCR identification and phenotypic identification of transgenic rice plants Materials preparation: Prepare the Teqing obtained above. CTP Two T0 generation positive transgenic plants were self-crossed, and two T1 generation complementary transgenic positive plant families were obtained after seed sowing. These families were named Teqing. CTP -1 T1 generation complementary transgenic positive line (abbreviated as Teqing) CTP -1 T1 series) and Teqing CTP-2 T1 generation complementary transgenic positive line (abbreviated as Teqing) CTP -2 T1 series), and continued self-pollination to harvest Teqing. CTP -1 T1 series and Teqing CTP -2 Seeds from a positive-positive plant in the T1 series were further sown to obtain Teqing. CTP -1 T2 generation complementary transgenic positive line (abbreviated as Teqing) CTP -1 T2 series) and Teqing CTP -2 T2 generation complementary transgenic positive line (abbreviated as Teqing) CTP -2 T2 series). In Teqing CTP -1 T2 series and Teqing CTP -2 Phenotypic identification was performed on at least 10 positive plants from each of the T2 lines, and phenotypic identification was also performed on the recipient material Teqing. Teqing was found to be positive. CTP -1T2 series and Teqing CTP -2 The number of grains per ear in positive plants of the T2 line was significantly less than that of Teqing ( ​ ).

[0103] 4. Genetically modified rice ​ Gene expression level detection For Teqing CTP -1 T2 series and Teqing CTP -2 Young panicles of positive plants in the T2 line were sampled, and RNA was extracted and reverse transcribed into cDNA. Using the rice UBI2 gene as an internal control (amplification primers UBIF / UBIR), the above-mentioned Teqing... CTP -1 T2 series and Teqing CTP -2 T2 line positive plant spikelets ​ Gene expression levels were detected (using amplification primers TCP17F / TCP17R).

[0104] Primer UBIF: 5'-CTGTCAACTGCCGCAAGAAG-3' Primer UBIR: 5'-GGCGAGTGACGCTCTAGTTC-3' Primer TCP17F: 5'-AAAGGAGACGCGGACAAGG-3' Primer TCP17R: 5'-TCCTTCTCCTCCGGTGTTGT-3' PCR amplification: The reaction volume was 10 μL, including: 1 μL cDNA template, 5 μL 2×M5 HiPer SYBRPremix EsTaq, and 0.4 μL 10 μM primers. The amplification program was as follows: 95℃ for 3 min; 95℃ for 10 s, 58℃ for 30 s, 72℃ for 45 s, 39 cycles; 72℃ for 5 min; melting curves were plotted at 0.5℃ intervals from 65℃ to 95℃.

[0105] like ​ As shown, in Teqing CTP -1 T2 series and Teqing CTP -2 T2 line positive plant spikelets ​ The expression level of the gene was significantly increased. Combined with the promoter activity analysis results of pTCP17, it is shown that the promoter of the present invention can not only effectively initiate the expression of the target gene in plants, but also significantly increase the expression level of the target gene, has good activity, and has a regulatory function on the agronomic trait of ear grain number.

[0106] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A DNA molecule, characterized in that, The nucleotide sequence of the DNA molecule is shown in SEQ ID No.

1.

2. The application of the DNA molecule as a promoter as described in claim 1.

3. The application according to claim 2, characterized in that, The DNA molecules were derived from rice.

4. The application according to claim 2 or 3, characterized in that, The DNA molecule in question is a rice tissue-specific promoter.

5. A biomaterial, characterized in that, The biomaterial is any one of the following: A1) An expression cassette containing the DNA molecule of claim 1; A2) A recombinant vector containing the DNA molecule of claim 1; A3) Recombinant microorganisms containing the DNA molecule described in claim 1; A4) A recombinant host cell containing the DNA molecule of claim 1.

6. The use of the DNA molecule according to any one of claims 1-4 or the biological material according to claim 5 in any of the following: B1) Application in regulating the number of grains per panicle in rice; B2) Application in driving the specific expression of target genes in the base of rice stems and young panicles; B3) Application in cultivating rice with altered grain number per panicle; B4) Application in molecular breeding for improving grain number per panicle in rice or in the improvement of rice germplasm resources related to grain number per panicle.

7. The use of the DNA molecule described in any one of claims 1-4 or the biomaterial described in claim 5 in increasing the expression level of the target gene in rice.

8. A method for cultivating transgenic rice, characterized in that, The method includes using the DNA molecule of claim 1 as a promoter to drive the expression of the target gene in the target rice to obtain transgenic rice with altered grain number per panicle.

9. The method according to claim 8, characterized in that, The method is achieved by introducing the DNA molecule described in claim 1 as a promoter into the target rice.

10. The application according to claim 6 or 7, or the method according to claim 8 or 9, characterized in that, The target gene is TCP17 Genes or reporter genes.

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