Application of BrHCYSTM1 gene in regulation and control of plant leaf size development

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a BrHCYSTM1 gene in regulation and control of plant leaf size development. The invention discovers that the BrHCYSTM1 gene positively regulates the plant leaf size development for the first time. The expression quantity of the BrHCYSTM1 gene is improved through the indigenous and heterologous overexpression of the BrHCYSTM1 gene, and the plant leaf is obviously larger than that of a wild type. And a theoretical basis and a gene source are provided for cultivating new germplasm of high-yield and excellent crops.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering technology and specifically relates to BrHCYSTM1 The application of genes in regulating plant leaf size development. Background Art

[0002] Chinese cabbage ( Brassica rapa L. ssp. pekinensis Known as the "national dish," Chinese cabbage plays an irreplaceable and significant role in ensuring the "vegetable basket" of food and stabilizing people's lives. Chinese cabbage leaves are divided into outer leaves and head, which serve as the primary photosynthetic organ, nutrient storage organ, and edible organ, respectively. Leaf size is a key factor influencing Chinese cabbage yield. Exploring the key functional genes associated with leaf size development in Chinese cabbage and analyzing their mechanisms will provide important theoretical basis and technical support for the development of high-yield, high-quality Chinese cabbage varieties using molecular breeding techniques.

[0003] Based on the whole genome sequencing technology of Chinese cabbage and modern molecular biology technology, researchers have discovered a series of functional genes related to the development of Chinese cabbage leaves, and the regulatory mechanism of Chinese cabbage leaf development has gradually revealed its mystery. BrERF4 、 BrANT-1 、 BrARGOS and BrGRF Overexpression of these genes in Arabidopsis thaliana can induce leaf enlargement, but their mechanisms of action are different: BrERF4 By driving cell expansion, BrANT-1 、 BrARGOS and BrGRF The significant increase in leaf area is achieved by regulating cell proliferation; similarly, negative regulatory factors BrrTCP4 Affects leaf size by inhibiting cell proliferation, BrARP1 、 BrDRM1 It does this by inhibiting cell expansion, which results in smaller leaves.

[0004] HCYSTM This gene family encodes a class of small cysteine-rich proteins. Studies have shown that members of this family play key roles in plant defense, signal transduction, and host-parasite interactions. However, the functions of this gene family members in leaf size development have not been reported. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the present invention aims to provide BrHCYSTM1 The application of genes in regulating plant leaf size development.

[0006] The purpose of the present invention can be achieved through the following technical solutions: The first aspect of the present invention provides BrHCYSTM1 The application of genes in regulating plant leaf size development.

[0007] The second aspect of the present invention provides BrHCYSTM1 The use of genes in breeding plants with altered leaf size.

[0008] The third aspect of the present invention provides BrHCYSTM1 Application of genes in molecular breeding for plant leaf improvement or improvement of germplasm resources related to plant leaves.

[0009] A fourth aspect of the present invention provides a biomaterial comprising at least one of the following (a1) to (a4): (a1) Contains BrHCYSTM1 expression cassette of the gene; (a2) a recombinant vector containing the expression cassette described in (a1); (a3) transforming or transfecting the host cell as described in (a2); (a4) culturing the mixture obtained by culturing the host cell described in (a3); described BrHCYSTM1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0010] A fifth aspect of the present invention provides a product for enlarging plant leaves, comprising auxiliary materials and the biological material of the present invention.

[0011] A sixth aspect of the present invention provides the use of the biomaterial or product of the present invention in positively regulating the size of plant leaves.

[0012] A seventh aspect of the present invention provides a method for cultivating plants with enlarged leaves, the method comprising increasing the BrHCYSTM1 The expression level of the gene is increased to obtain a plant with leaves larger than those of the target plant.

[0013] Compared with the existing technology, the above technical solution has the following beneficial effects: (1) This invention is the first to discover BrHCYSTM1 Genes that positively regulate plant leaf size development. Through native and heterologous overexpression BrHCYSTM1 Genes, improve BrHCYSTM1 The gene expression level in plant leaves is significantly greater than that in wild-type plants, providing a theoretical basis and gene source for breeding new high-yield and excellent crop germplasm.

[0014] (2) The present invention found through physiological and biochemical experiments that overexpression BrHCYSTM1 The leaf area of ​​the transgenic Chinese cabbage plants BrHCYSTM1-OE-1 and BrHCYSTM1-OE-2 was significantly larger than that of the wild-type Chinese cabbage; heterologous expression in Arabidopsis BrHCYSTM1 gene, also caused the large-leaf phenotype of the transgenic lines; BrHCYSTM1 Homologous genes AtHCYSTM1 Under the condition of lack, overexpression BrHCYSTM1 The study found that the BrHCYSTM1 gene in Chinese cabbage can completely compensate for the defective phenotype of the mutant. This indicates that the BrHCYSTM1 gene in Chinese cabbage has the function of regulating the development of plant leaf size, providing new ideas for using this gene to cultivate new crop germplasms with high yield and excellent traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] 图1 In the embodiment of the present invention BrHCYSTM1 The relative expression levels and phenotypes of the genes in wild-type Chinese cabbage and overexpression transgenic lines; WT is wild-type Chinese cabbage; BrHCYSTM1-OE-1 and BrHCYSTM1-OE-2 are overexpression BrHCYSTM1 Transgenic Chinese cabbage lines after the gene; 图2 In the embodiment of the present invention BrHCYSTM1 Relative expression levels and phenotypes of genes in wild-type Arabidopsis and overexpression transgenic lines; WT is wild-type Arabidopsis; 35S::BrHCYSTM1-1 and 35S::BrHCYSTM1-2 are heterologous overexpression lines. BrHCYSTM1 Arabidopsis transgenic lines after gene; 图3 In the embodiment of the present invention, AtHCYSTM1 Overexpression of genes in Arabidopsis mutants BrHCYSTM1 The expression levels and phenotypes of related genes in transgenic plants of the gene; WT is wild-type Arabidopsis; athcystm-1 and athcystm-2 are knockout AtHCYSTM1 Arabidopsis mutants after the AtHCYSTM1 gene; athcystm1 / BrHCYSTM1-1 and athcystm1 / BrHCYSTM1-2 are transgenic lines overexpressing BrHCYSTM1 in the AtHCYSTM1 gene deletion background. DETAILED DESCRIPTION

[0016] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0017] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0018] In one embodiment of the present invention, there is provided BrHCYSTM1 The application of genes in regulating plant leaf size development.

[0019] In some embodiments of the present invention, the BrHCYSTM1 The nucleotide sequence of the gene is as shown in SEQ ID NO.1 or has 95% or more identity or identity with the nucleotide sequence shown in SEQ ID NO.1 and expresses the same functional protein.

[0020] In some embodiments of the present invention, the application is overexpression BrHCYSTM1 Gene that makes plants' leaves larger.

[0021] In another embodiment of the present invention, there is provided BrHCYSTM1 The use of genes in breeding plants with altered leaf size.

[0022] In another embodiment of the present invention, there is provided BrHCYSTM1 Application of genes in molecular breeding for plant leaf improvement or improvement of germplasm resources related to plant leaves.

[0023] In some embodiments of the present invention, the plant includes an herbaceous plant or a crop; the herbaceous plant includes Arabidopsis thaliana; the crop includes corn, rice, tomato, potato, peanut, soybean, cotton, tobacco, cucumber, melon, watermelon, Chinese cabbage, rapeseed, baby cabbage, spinach and / or radish.

[0024] In some embodiments of the present invention, the plant is Arabidopsis thaliana or Chinese cabbage.

[0025] In another embodiment of the present invention, a biomaterial is provided, comprising at least one of the following (a1) to (a4): (a1) contains BrHCYSTM1 expression cassette of the gene; (a2) a recombinant vector containing the expression cassette described in (a1); (a3) transforming or transfecting the host cell as described in (a2); (a4) culturing the mixture obtained by culturing the host cell described in (a3); described BrHCYSTM1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0026] The nucleic acid described in the present invention may be DNA, RNA, cDNA or PNA. In an embodiment of the present invention, the nucleic acid is in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. Nucleic acid may include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). Nucleic acid may be linear or circular in topology. Nucleic acid may be part of a vector (e.g., an expression or cloning vector) or a fragment. The nucleic acid may be obtained directly from a natural source, or may be prepared with the assistance of recombination, enzymatic methods or chemical techniques. The RNA form may be mRNA obtained by gene transcription, etc.

[0027] In the present invention, the nucleic acid may be optimized or unoptimized; the optimization includes but is not limited to: codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, mRNA secondary structure, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning, etc.

[0028] The expression cassette described herein refers to a DNA sequence beginning with a promoter and ending with a terminator. Regulatory elements may also be included on either side of or between the promoter and terminator. These regulatory elements may include a promoter, enhancer, transcription termination signal, polyadenylation sequence, replication origin, nucleic acid restriction sites, and homologous recombination sites operably linked to the nucleic acid sequence, such as a promoter enhancer and poly(A) signal.

[0029] The source of the backbone of the recombinant vector of the present application includes plants, animals, bacteria, fungi, bacteriophages, or viruses, which are not limited by the present application. The viral vector includes an adenovirus vector, an adeno-associated virus (AAV) vector, a retrovirus vector, a lentivirus vector, or a tobacco mosaic virus, etc. The bacteriophage vector includes a phagemid and a helper vector, and the phagemid includes but is not limited to pBluescript II-KS(+), pcomb3XSS, pCANTAB5E, or pKK233.3. The animals include mammals and non-mammals, and the mammalian expression vector includes but is not limited to pcDNA 3.1, pIRES, pTT3, pCEP4, pATX1, or pCHO1.0. The bacterial vector includes but is not limited to pET28a, pET16b, pET26b, pET28a, pET31b, pBAD, pBADHis, pTrc99a, pTrcHis, pACYCduet-1, pET duet-1, pCDFduet-1, pColdI, pColdII, etc. The fungal vector includes but is not limited to pYES2, pYES3, pYES6, pAUR23, etc.

[0030] In some embodiments of the present application, the vector includes but is not limited to a pCAMBIA1300 vector, a pCAMBIA1301 vector, a pCAMBIA1303 vector, a pCAMBIA1304 vector, a pBI121 vector, and a pCAMBIA3300-GFP vector.

[0031] The recombinant vector of the present application refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences or elements necessary for the expression of the operably linked coding gene in a specific host organism or for the completion of the desired operation.

[0032] The present application provides a host cell transformed or transfected with the recombinant vector. The host cell is transformed or transfected with a vector constructed using recombinant DNA technology, so that the transformed host cell has the ability to replicate the vector encoding the protein, express the desired protein, or perform the desired function.

[0033] Furthermore, the transformation method includes chemical transformation and electroporation, and the transfection method includes calcium phosphate co-precipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0034] In some embodiments of the present application, the starting strain of the host cell includes Escherichia coli and Agrobacterium.

[0035] In another embodiment of the present invention, a product for enlarging plant leaves is provided, comprising an auxiliary material and the biomaterial of the present invention.

[0036] In another embodiment of the present invention, there is provided the use of the biomaterial or product of the present invention in positively regulating the size of plant leaves.

[0037] In another embodiment of the present invention, a method for cultivating plants with enlarged leaves is provided, the method comprising increasing the amount of BrHCYSTM1 The expression level of the gene is increased to obtain a plant with leaves larger than those of the target plant.

[0038] Furthermore, the improvement of target plants BrHCYSTM1 Gene expression can be achieved by at least one of the following methods: (b1) Increase the BrHCYSTM1 Gene copy number; (b2) BrHCYSTM1 The gene is expressed under the drive of a strong promoter; (b3) Increase the BrHCYSTM1 Gene regulatory elements enable overexpression, including enhancer elements, elements that improve mRNA stability, elements that enhance translation efficiency, and / or elements that enhance protein secretion; (b4) Add the following BrHCYSTM1 ribosome binding sites of genes; (b5) BrHCYSTM1 The genes were codon optimized; (b6) Upregulating gene expression by altering epigenetic modifications such as DNA methylation or histone acetylation.

[0039] Furthermore, the (b2) can be achieved by BrHCYSTM1 This can be achieved by replacing the natural promoter of the gene with a strong promoter, or by BrHCYSTM1 This is achieved by genetically operably linking a second promoter.

[0040] The strong promoter includes but is not limited to T7 promoter, CaMV promoter, SV40 promoter, SFFV promoter, ubq promoter, ubi promoter, RBCS promoter, Actin promoter, Emu promoter, CYP450 promoter, Adhl promoter and pinⅡ promoter.

[0041] The enhancers include, but are not limited to, CMV enhancer, SV40 enhancer and RSV enhancer.

[0042] In the above method, the improvement of the target plant BrHCYSTM1 The expression level of the gene can be BrHCYSTM1 This is achieved by introducing the gene into the target plant.

[0043] In the above method, the BrHCYSTM1 The nucleotide sequence of the gene can be shown as SEQ ID NO.1.

[0044] Furthermore, the improvement of the target plant BrHCYSTM1 The expression level of the gene can be achieved by introducing the DNA molecule shown in SEQ ID NO. 1 into the target plant.

[0045] The method for cultivating plants with enlarged leaves may include the following steps: (1) Construct the code containing the BrHCYSTM1 Recombinant vector of the nucleic acid molecule of the gene; (2) introducing the recombinant vector constructed in step (1) into the target plant; (3) Obtain transgenic plants through screening and identification.

[0046] Furthermore, the above method may further include step (4) after step (3): hybridizing the transgenic plant with the plant to be improved to obtain an offspring transgenic plant, wherein the offspring transgenic plant has a phenotype substantially identical to that of the transgenic plant (i.e., the transgenic plant serving as the parent).

[0047] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0048] Purchase Arabidopsis thaliana from the Arabidopsis Mutant Library (http: / / www.arashare.cn). AtHCYSTM1 The T-DNA insertion mutant seeds of the gene (SALK_124375C) were identified by sequencing as having a T-DNA insertion at 300 bp of the gene.

[0049] Example 1 BrHCYSTM1 Construction of gene overexpression vector 1. Chinese cabbage BrHCYSTM1 Gene cloning Obtained from the Brassica Database (http: / / www.brassicadb.cn / # / ) BrHCYSTM1 CDS sequence of the gene, and design the gene cloning primers based on the sequence ( BrHCYSTM1- F and BrHCYSTM1- R), PCR amplification was performed using the cDNA of the Chinese cabbage variety "Improved Guangdong Early" as a template. The primer sequences are as follows: BrHCYSTM1- F: 5′-ATGAGCCAGTACAACAACC-3′; BrHCYSTM1- R: 5′-TTAGAAGCAGATGTCCAGG-3′.

[0050] After agarose gel electrophoresis, the PCR product obtained was ligated to the T vector and transformed into competent E. coli DH5α cells. After overnight culture, single clones were selected for PCR identification and sequence verification. Colonies with correct sequencing were preserved and plasmids were extracted for later use.

[0051] 2. Construction and identification of overexpression vectors Using homologous recombination, BrHCYSTM1 The gene was inserted into the plant expression vector pCAMBIA3300- GFP (miaoling plasmid, P27596) was constructed BrHCYSTM1 Gene overexpression vector.

[0052] First, use restriction enzymes Bam H1 (NEB, R0136V) was used to cut pCAMBIA3300-GFP. The reaction system was shown in Table 1. After incubation at 37°C for 0.5 h, electrophoresis was performed to recover the linearized plasmid fragment.

[0053] Table 1 Enzyme digestion reaction system

[0054] At the same time, PCR amplification was performed using the correctly sequenced plasmid as a template, and the primer sequences were as follows: 3300-GFP-BrHCYSTM1-F5′-cgggggactctagagATGAGCCAGTACAACAACCAATCTG-3′; 3300-GFP-BrHCYSTM1-R5′-gctcgaattcggatcTTCTTCTTCTTCTTCTTCGAAGCAG ATGTCCAGGGCACAA-3′.

[0055] The PCR products were recovered after electrophoresis.

[0056] Ligation reaction using ClonExpress ® II kit (Vazyme, C112), the reaction system is shown in Table 2, and the ligation was completed at 50°C for 30 min.

[0057] Table 2 Ligation reaction system

[0058] The ligation product was transformed into competent E. coli DH5α cells. After overnight culture, single clones were selected for PCR identification and sequence verification. Colonies with correct sequencing were considered to carry the successfully constructed fusion expression vector. The cells were then maintained and the plasmid was extracted.

[0059] The constructed fusion expression vector was transformed into Agrobacterium GV1301 competent cells, and the bacterial solution was spread on K + 、Rif + On the surface of the double-antibody YEP solid culture medium, after 3 days, single clones were picked for PCR identification. The colonies with the correct band size were the Agrobacterium that had successfully transformed with the fusion expression vector. The bacteria were stored at -80℃ for later use.

[0060] Example 2 Overexpression of Chinese Cabbage BrHCYSTM1 Genetic plants and identification 1. Agrobacterium Transformation into Chinese Cabbage The plant material used in this experiment was the pure line of Chinese cabbage "Improved Guangdong Early", which is a germplasm resource preserved by the Vegetable Research Institute of Shandong Academy of Agricultural Sciences.

[0061] In this experiment, the inflorescence infection method was used to create a transgenic line of Chinese cabbage overexpressing. The seeds of the wild type "Improved Guangdong Early" were taken and placed in a culture dish pre-added with an appropriate amount of water. After the seeds turned white, the culture dish was placed in a 4 ℃ refrigerator for 14 days of vernalization. Subsequently, the seedlings were transplanted into a flower pot filled with substrate and cultured in a tissue culture room at 20 ± 2 ℃ with a light cycle of 16 hours of light / 8 hours of darkness until the flowering stage. Select the unopened buds, gently peel them open to expose the stigma, apply fresh pollen on the stigma, and pollinate. After 30 minutes, immerse the pollinated inflorescence in the Agrobacterium suspension (OD 600 = 0.8) for 30 seconds. Transformed plants were grown overnight in the dark. The next day, the plants were removed and cultivated under normal conditions until the seeds matured.

[0062] 2. Identification of transgenic lines 2.1 Using qRT-PCR method, select BrHCYSTM1 Transgenic lines with significantly upregulated gene expression.

[0063] RNA was extracted from wild-type Chinese cabbage and transgenic lines using the following method: Weigh 2 g of plant leaves in a liquid nitrogen-chilled mortar and pestle, then quickly grind in liquid nitrogen. Transfer the powder to a centrifuge tube pre-filled with 1 mL of Trizol and let it sit at room temperature for 30 minutes to allow for complete lysis. Centrifuge at 12,000 rpm at 4°C for 20 minutes, and transfer the supernatant to a new RNase-free EP tube. Add 200 μL of chloroform, vortex to mix, and let it sit at room temperature for 15 minutes. Centrifuge at 12,000 rpm at 4°C for 20 minutes. Transfer 500 μL of the supernatant to a new EP tube, add 500 μL of pre-chilled isopropanol, and let it sit at -20°C for 30 minutes. Centrifuge at 12,000 rpm at 4°C for 10 minutes, and discard the supernatant. The pellet is total RNA. Add 1 mL of 75% ethanol to the precipitate, gently invert and wash the precipitate, centrifuge at 8,000 rpm for 5 min at 4°C, repeat twice; discard the supernatant, dry the precipitate at room temperature, dissolve the precipitate in 50 μL of DEPC water, and store in a -80°C ultra-low temperature freezer until use.

[0064] The total RNA of wild-type Chinese cabbage and its corresponding transgenic plants were obtained respectively by this method.

[0065] Reverse transcription PCR amplification (two-step method) was performed using the total RNA sample as a template. The specific reverse transcription PCR amplification system is shown in Tables 3 and 4.

[0066] Table 3 First step amplification system

[0067] Reaction conditions: 42°C, 2 min, stored at 4°C.

[0068] Table 4 Second step amplification system

[0069] Reaction conditions: 37°C for 15 min, 85°C for 5 s, and storage at 4°C.

[0070] Total cDNA of wild-type Chinese cabbage and related transgenic plants was obtained using this method. The reagents in Step 1 and Step 2 were from the TAKALA (PrimeScript™ II 1st Strand cDNA Synthesis Kit).

[0071] Chinese cabbage ACTIN1 The gene was used as an internal reference gene and the LightCycler ® 480 Real time PCR amplification instrument and fluorescent quantitative PCR kit (SYBR ® Premix EX Taq TMThe total cDNA obtained in this example was subjected to real-time fluorescence quantitative PCR using a TAKARA (II) PCR instrument. The procedure was based on the LC480 instrument manual and the kit instructions. Real-time fluorescence quantitative PCR primer design adhered to the following principles: primer length: 18–22 bp; Tm value: 55–65°C; GC content: 40%–60%; product length: 80–250 bp; and primers were designed to avoid primer dimers and hairpin structures.

[0072] The qRT-PCR primer sequences are as follows: BrACTIN-F:5′-CGTACTACCGGTATTGTGCT-3′; BrACTIN-R:5′-GAGCTGGATTTGGAAGTCTC-3′; BrHCYSTM1-qF: 5′-ATGAGCCAGTACAACAACCAATC-3′; BrHCYSTM1-qR:5′-GCAACACATGGCCGCAAGAC-3′.

[0073] The PCR reaction system (set up on ice) is shown in Table 5.

[0074] Table 5 PCR reaction system

[0075] The standard procedure for amplification (two-step method) is as follows: Stage 1: Pre-denaturation, 95°C, 30 s, 20°C / s, 1 cycle; Stage 2: PCR reaction, 95°C, 5 s, 20°C / s; 60°C, 20 s, 20°C / s, 40 cycles; Stage 3: Melting curve analysis, 95℃, 0 s, 20℃ / s; 65℃, 15 s, 20℃ / s; 95℃, 0 s, 0.1℃ / s.

[0076] Three biological replicates were taken for each sample, and 2 -ΔΔCt The relative expression levels were calculated.

[0077] Select the strains with significantly increased relative expression levels and continue to culture them until the next generation of seeds is harvested.

[0078] 2.2 Detection of GFP-BrHCYSTM1 fusion protein expression by Western Blot Total protein was extracted from wild-type Chinese cabbage and transgenic lines using the following method: Take an appropriate amount of plant leaves and freeze in liquid nitrogen. Transfer the leaves to a pre-chilled mortar and pestle, grind them into a powder, and add 2 ml of pre-chilled IP buffer. Centrifuge at 14,000 rpm at 4°C for 30 minutes. Aliquot 100 μl into tubes and store at -80°C until needed. The IP buffer recipe is shown in Table 6.

[0079] Table 6 IP buffer recipe

[0080] Thaw the stored protein sample on ice, add the appropriate amount of loading buffer, and perform SDS-PAGE gel electrophoresis. After electrophoresis, cut the gel near the target protein fragment and transfer the protein from the SDS-PAGE gel to an Amersham Hybond™-P membrane (GE Healthcare, RPN303B) using the Mini PROTEAN Tetra system (Bio-Rad). Wash the membrane with TBST buffer (TBST) containing a final concentration of 20 mM Tris-Cl, 150 mM NaCl, and 0.05% Tween 20. Block the membrane with 5% (w / v) skim milk in TBST (TBS™) for 4 hours at room temperature. Incubate overnight at 4°C with anti-GFP antibody (abcam, ab290) at a final concentration of 1:5000 in TBS™. After washing the membrane with TBST, the membrane was incubated in TBS™ supplemented with Goat Anti-Rabbit IgG H&L (HRP) antibody (abcam, ab6721) at a final concentration of 1:7500 for approximately 3 hours at room temperature. After washing six times with TBST, ECL substrate (Thermo, 32132) was added to the membrane, and protein bands were visualized using a Bio-01 Multi-Function Fluorescence Imaging System (Guangzhou Guangyi Biotechnology Co., Ltd.).

[0081] 3. Phenotypic Observation of Transgenic Chinese Cabbage Wild-type Chinese cabbage seeds and overexpression BrHCYSTM1 Seeds of transgenic T3 plants were germinated in glass culture dishes pre-lined with filter paper. After 3 days of growth, the seedlings were transferred to a culture medium and grown for 20 days. Germination and culture were performed in a tissue culture room with a photoperiod of 20 ± 2°C and 16 h light / 8 h dark. Phenotypes were observed after 20 days of growth in the culture medium.

[0082] The results are as follows 图1 As shown in (A), the leaf area of ​​the transgenic Chinese cabbage overexpressing BrHCYSTM1-OE-1 and BrHCYSTM1-OE-2 plants was significantly larger than that of the wild-type Chinese cabbage, indicating that the overexpression of BrHCYSTM1 Significant changes occurred in the development of posterior lobe organs.

[0083] 4. Overexpression lines BrHCYSTM1 Gene expression detection The experimental method is the same as step 2.1 in Example 2, using the qRT-PCR method to select BrHCYSTM1 The results are as follows: 图1 (B) As shown, the transgenic Chinese cabbage lines BrHCYSTM1-OE-1 and BrHCYSTM1-OE-2 overexpressing BrHCYSTM1 compared with wild-type Chinese cabbage. BrHCYSTM1 The gene expression level increased significantly.

[0084] 5. Detection of GFP-BrHCYSTM1 Fusion Protein Expression in Overexpression Strains The experimental method was the same as step 2.2 in Example 2, "Detection of expression of GFP-BrHCYSTM1 fusion protein by Western Blot". The results are as follows: 图1 As shown in (C), BrHCYSTM1-OE-1 and BrHCYSTM1-OE-2 have bands of GFP-tagged fusion proteins compared with wild-type Chinese cabbage, so both transgenic lines in the figure are effective mutants.

[0085] Example 3 Arabidopsis overexpression BrHCYSTM1 Genetic plants and identification 1. Agrobacterium Transformation of Arabidopsis Wild-type Arabidopsis seeds germinate and grow on normal 1 / 2 MS medium for 10 days before being transferred to a substrate and grown for 15 days. Culture conditions: After 10 days of vertical culture in an incubator with a photoperiod of 20 ± 2°C, 16 hours light / 8 hours dark, the seeds are transferred to a substrate and grown in a tissue culture room at 20 ± 2°C, 16 hours light / 8 hours dark until flowering. Inflorescences are directly dipped into the Agrobacterium suspension described in Example 1 (OD600 = 0.8) for 30 seconds. Transformed plants are grown overnight in the dark. The next day, the plants are removed and cultivated under normal conditions until the seeds mature.

[0086] 2. Identification of transgenic lines The Arabidopsis mutant seeds obtained in step 1 were plated and grown on 1 / 2MS medium containing 10 mg / mL PPT. Seedlings with green leaves and normal rooting were transferred to a medium and grown until the seeds were harvested.

[0087] Wild-type Arabidopsis seeds and T2 mutant seeds were germinated and grown on normal 1 / 2 MS medium for 10 days, then transferred to a substrate and grown for 15 days. Culture conditions were as described in step "1. Agrobacterium transformation of Arabidopsis thaliana." RNA was extracted from samples and analyzed by qRT-PCR using the same method as described in "2. Identification of transgenic lines" in Example 2. The qRT-PCR primer sequences are as follows: AtACTIN2-qF: 5′-GGTAACATTGTGCTCAGTGGTGG-3′; AtACTIN2-qR: 5′-AACGACCTTAATCTTCATGCTGC-3′; BrHCYSTM1-qF: 5′-ATGAGCCAGTACAACAACCAATC-3′; BrHCYSTM1-qR: 5′-GCAACACATGGCCGCAAGAC-3′.

[0088] The identified transgenic plants were cultured continuously until T3 generation seeds were harvested.

[0089] 3. Phenotypic Observation of Transgenic Arabidopsis Wild-type Arabidopsis seeds and Arabidopsis mutant T3 seeds were germinated and grown on normal 1 / 2 MS medium for 10 days, and then transferred to a matrix for growth for 15 days.

[0090] The results are as follows 图2 As shown in (A), the leaves of Arabidopsis mutants 35S::BrHCYSTM1-1 and 35S::BrHCYSTM1-2 that heterologously express BrHCYSTM1 are significantly larger than those of wild-type Arabidopsis, further indicating that BrHCYSTM1 Heterologous expression of the gene in Arabidopsis thaliana resulted in significant changes in the growth and development of Arabidopsis leaves.

[0091] 4. Overexpression lines BrHCYSTM1 Gene expression detection The experimental method is the same as step 2.1 in Example 2, using the qRT-PCR method to select BrHCYSTM1 The results were from the transgenic lines with significantly upregulated gene expression. 图2 As shown in B, the Arabidopsis overexpressing transgenic lines are compared with wild-type Arabidopsis. BrHCYSTM1 The gene expression level increased significantly, so the two transgenic lines in the figure are both effective mutants.

[0092] 5. Detection of GFP-BrHCYSTM1 Fusion Protein Expression in Overexpression Strains The experimental method was the same as step 2.2 in Example 2, "Detection of GFP-BrHCYSTM1 fusion protein expression by Western Blot". 图2 As shown in C, the Arabidopsis overexpressing transgenic line has a band with a GFP-tagged fusion protein compared to the wild-type Arabidopsis, so both transgenic lines in the figure are effective mutants.

[0093] Example 4 In Arabidopsis mutants athcystm1 Overexpression BrHCYSTM1 Transgenic plants and identification of genes In this example, the Arabidopsis mutant athcystm1 Arabidopsis thaliana was purchased from the Arabidopsis mutant library (http: / / www.arashare.cn). AtHCYSTM1 The T-DNA insertion mutant seeds of the gene (SALK_124375C) were identified by sequencing as having a T-DNA insertion at 300 bp of the gene.

[0094] 1. Agrobacterium Transformation The experimental method was the same as step 1. Transformation of Arabidopsis thaliana by Agrobacterium in Example 3.

[0095] 2. Identification of transgenic lines The experimental method was the same as step 2. Identification of transgenic lines in Example 3.

[0096] 3. Phenotypic Observation of Transgenic Arabidopsis Wild-type Arabidopsis seeds, Arabidopsis mutant athcystm1, and T3 generation Arabidopsis mutant seeds heterologously expressing BrHCYSTM1 in a mutant background were obtained, and culture conditions were the same as step “3. Phenotypic Observation of Transgenic Arabidopsis” in Example 3.

[0097] The results are as follows 图3 As shown, Arabidopsis mutants athcystm1 The leaves of Arabidopsis thaliana were significantly smaller than those of the wild type, indicating that the functional loss of AtHCYSTM1, a homologous gene of BrHCYSTM1, led to a significant reduction in the leaves of Arabidopsis thaliana. athcystm1 / BrHCYSTM1-1 and athcystm1 The leaves of / BrHCYSTM1-2 were significantly larger than those of the wild type, which indicated that BrHCYSTM1 Heterologous expression of the gene in Arabidopsis completely compensated for the defective phenotype caused by loss of AtHCYSTM1 function, further demonstrating that BrHCYSTM1 The gene has the function of regulating the size and development of plant leaves.

[0098] (4) Detection of BrHCYSTM1 gene expression in overexpression lines The experimental method is the same as step 2.1 in Example 2, using the qRT-PCR method to select BrHCYSTM1 The qRT-PCR primer sequences for transgenic lines with significantly upregulated gene expression are as follows: AtACTIN2-qF: 5′-GGTAACATTGTGCTCAGTGGTGG-3′; AtACTIN2-qR: 5′-AACGACCTTAATCTTCATGCTGC-3′; ATHCYSTM1-qF: 5′-ATGAGCCAGTACGATCACAACC-3′; ATHCYSTM1-qR: 5′-TCACCCTTAGACTTGGTTTCCAC-3′; BrHCYSTM1-qF: ATGAGCCAGTACAACAACCAATC-3′; BrHCYSTM1-qR: GCAACACATGGCCGCAAGAC-3′.

[0099] Depend on 图3 B and C show that in athcystm1 mutant AtHCYSTM1 Compared with wild-type Arabidopsis, the gene expression level was significantly decreased, indicating that the mutant was an effective mutant; in the transgenic plants overexpressing BrHCYSTM1 in this mutant background, the expression of BrHCYSTM1 in the transgenic plants was significantly decreased. AtHCYSTM1 The expression level was significantly lower than that of the wild type. BrHCYSTM1 The expression level of was significantly higher than that of the wild type, which shows that the heterologous expression strain in the mutant background is also an effective mutant.

[0100] (5) Detection of GFP-BrHCYSTM1 fusion protein expression in overexpression lines The experimental method was the same as step 2.2 "Detection of GFP-BrHCYSTM1 fusion protein expression by Western Blot" in Example 2. 图3 D shows that athcystm1 / BrHCYSTM1-1 and athcystm1 Compared with the wild type, / BrHCYSTM1-2 has a band of fusion protein with a GFP tag, so the two transgenic lines in the figure are effective mutants.

[0101] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or to replace portions thereof with equivalents. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. BrHCYSTM1 The application of genes in regulating plant leaf size development.

2. The use according to claim 1, characterized in that The application is overexpression BrHCYSTM1 Gene that makes plants' leaves larger.

3. BrHCYSTM1 The use of genes in breeding plants with altered leaf size.

4. BrHCYSTM1 Application of genes in molecular breeding for plant leaf improvement or improvement of germplasm resources related to plant leaves.

5. The use according to any one of claims 1 to 4, characterized in that The plants include herbaceous plants or crops; the herbaceous plants include Arabidopsis thaliana; the crops include corn, rice, tomato, potato, peanut, soybean, cotton, tobacco, cucumber, melon, watermelon, Chinese cabbage, rapeseed, baby cabbage, spinach and / or radish.

6. Biomaterial, characterized in that Including at least one of the following (a1) to (a4): (a1) Contains BrHCYSTM1 expression cassette of the gene; (a2) a recombinant vector containing the expression cassette described in (a1); (a3) transforming or transfecting the host cell as described in (a2); (a4) culturing the mixture obtained by culturing the host cell described in (a3); described BrHCYSTM1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

7. The biomaterial according to claim 4, wherein The starting strain of the host cell is Agrobacterium.

8. A product for enlarging plant leaves, characterized in that: The invention comprises auxiliary materials and the biomaterial according to any one of claims 4 to 5.

9. Use of the biomaterial according to any one of claims 4 to 5 or the product according to claim 6 in positively regulating plant leaf size.

10. A method for cultivating a plant with enlarged leaves, characterized in that: The method comprises increasing BrHCYSTM1 The expression level of the gene is increased to obtain a plant with leaves larger than those of the target plant.

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