Chinese cabbage ACA10 gene and application of Chinese cabbage ACA10 gene in improvement of plant calcium ion enrichment operation capability

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a Chinese cabbage ACA10 gene and application thereof to improvement of plant calcium ion enrichment and operation capacity. The Chinese cabbage ACA10 gene comprises a nucleotide sequence as shown in SEQ ID NO: 18 or SEQ ID NO: 19. Researches find that the gene is expressed on a cell membrane and participates in calcium ion transport, and the calcium ion absorption, enrichment and transport capabilities of yeast and arabidopsis thaliana can be effectively improved by overexpression of the ACA10 gene, so that the plant growth or stress resistance is improved.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a Chinese cabbage ACA10 gene and its application in improving the ability of plants to accumulate and transport calcium ions. Background Technology

[0002] Chinese cabbage is one of the most important and widely cultivated vegetables in China and East Asia. It is rich in vitamins, minerals, and dietary fiber, making it highly nutritious. As a cruciferous vegetable, Chinese cabbage has a high calcium content. Calcium is essential for plant growth and development, participating in processes such as cell wall construction and signal transduction. For Chinese cabbage, sufficient calcium can effectively promote its growth and development and enhance its disease resistance.

[0003] The ACAs gene family is a subfamily of the P-type ATPase superfamily, playing a crucial role in calcium ion transport and cell signaling. The self-inhibition mechanisms and biological functions of ACAs are of significant research value in multiple species. Currently, members of the ACAs gene family have been identified in several species, including soybean, wheat, and Arabidopsis thaliana. For example, Zhao et al. (2021), using whole-genome bioinformatics and expression analysis, found 105 P-type ATPase genes in the soybean genome, of which the ACAs gene family contains 47 members; Aslam et al. (2017), through genome analysis, found 9 ACAs family members in wheat. Furthermore, studies have demonstrated that some members of the ACAs gene family have calcium ion transport functions. For example, ACA2 has been identified as a calmodulin-regulated calcium ion pump located in the endoplasmic reticulum. Hwang et al. (2000) used recombinant enzymes expressed in yeast to transport calcium ions and determined that phosphorylation of the N-terminal regulatory domain of calcium-dependent protein kinases could inhibit basal activity and calmodulin stimulation. ACA2 binds to calmodulin, and its activated state becomes resistant to phosphorylation inhibition.

[0004] ACA10 is a member of the ACAs gene family, and its main characteristics and research findings are primarily derived from studies on the model plant Arabidopsis thaliana. However, the role of ACA10 in calcium transport and accumulation in Chinese cabbage remains unclear. Summary of the Invention

[0005] In view of this, one of the objectives of the present invention is to provide an ACA10 gene and its variants.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A gene comprising the nucleotide sequence shown in SEQ ID NO:18, said gene being derived from cabbage.

[0008] A gene comprising the nucleotide sequence shown in SEQ ID NO:19, said gene being derived from cabbage.

[0009] A gene comprising the nucleotide sequence shown in SEQ ID NO:18 or SEQ ID NO:19, said gene being expressed on the cell membrane and involved in calcium ion transport.

[0010] Preferably, the gene, when overexpressed, can enhance calcium ion transport capacity in yeast or Arabidopsis thaliana.

[0011] Preferably, the gene is the ACA10 gene or a variant thereof.

[0012] A second objective of this invention is to provide a vector containing the aforementioned genes.

[0013] A third objective of this invention is to provide a host cell containing the aforementioned carrier.

[0014] The fourth objective of this invention is to provide a protein expressed through the aforementioned host cells.

[0015] To achieve the above objectives, the present invention adopts the following technical solution:

[0016] A protein expressed by the aforementioned host cells.

[0017] Preferably, the amino acid sequence of the protein is shown in SEQ ID NO:20 or SEQ ID NO:21.

[0018] The fifth objective of this invention is to provide a method for cloning the aforementioned gene.

[0019] To achieve the above objectives, the present invention adopts the following technical solution:

[0020] A method for cloning the aforementioned gene includes the following steps:

[0021] 1) Extract genomic DNA from Chinese cabbage;

[0022] 2) PCR amplification is performed using specific primers to obtain the amplification product;

[0023] 3) Purify the amplification product and ligate it into a vector;

[0024] 4) Transform the vector into the host cell;

[0025] The primers include the forward primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.2.

[0026] Preferably, in step 2), the PCR amplification reaction system is as follows:

[0027] Component Name System quantity / ul cDNA temple (100ng / ul) 1.5 Forward Primer (10µM) 2 Reverse Primer (10µM) 2 2X PCR Buffer for KOD FX 20 2mM dNTPs 4 KOD FX 0.5 <![CDATA[dd H2O]]> Up to 40ul .

[0028] Preferably, the PCR amplification reaction conditions in step 2) are as follows:

[0029]

[0030] Preferably, in step 3), the following steps are used: The gel was purified using a Gel Extraction Kit.

[0031] Preferably, in step 3), the carrier is the PEASY-BluntZero Cloning Kit carrier.

[0032] Preferably, in step 4), the host cell is Escherichia coli, more preferably Escherichia coli DH5α.

[0033] The sixth objective of this invention is to provide a method for expressing the aforementioned gene.

[0034] To achieve the above objectives, the present invention adopts the following technical solution:

[0035] A method for expressing the aforementioned gene includes the following steps:

[0036] S1: Transform the aforementioned vector into host cells;

[0037] S2: Culture the host cells in a suitable culture medium;

[0038] S3: Collect the expression products.

[0039] Preferably, the method is a method of overexpressing the ACA10 gene or a variant thereof.

[0040] Preferably, the vector is a yeast overexpression vector of the ACA10 gene or an Arabidopsis thaliana overexpression vector.

[0041] Preferably, the carrier is a PYES2 carrier or a PBI121 carrier.

[0042] More preferably, the vector is obtained by linearizing the PYES2 vector with XbaI and BamHI restriction endonucleases, and then ligating ACA10 into the PYES2 vector via homologous recombination; or the vector is obtained by linearizing the PBI121 vector with XbaI and BamHI restriction endonucleases, and then ligating ACA10 into the PBI121 vector via homologous recombination.

[0043] Preferably, the host cell is a yeast cell and / or an Agrobacterium cell.

[0044] More preferably, the Agrobacterium cells are Agrobacterium tumefaciens GV3101.

[0045] Preferably, the yeast cells are Saccharomyces cerevisiae strain BY4741.

[0046] As a preferred technical solution, the ACA10 gene or its variant is overexpressed in yeast:

[0047] The yeast overexpression vector of the ACA10 gene was transformed into yeast cells to obtain yeast strains that overexpress ACA10.

[0048] As a preferred technical solution, the ACA10 gene or its variant is overexpressed in Arabidopsis thaliana:

[0049] The Arabidopsis thaliana overexpression vector of the ACA10 gene was transformed into Agrobacterium, and then the Agrobacterium was used to infect the target plants. Positive transgenic plants were screened to obtain transgenic Arabidopsis thaliana plants overexpressing ACA10.

[0050] The seventh objective of this invention is to provide a method for silencing the aforementioned genes using virus-induced gene silencing technology.

[0051] To achieve the above objectives, the present invention adopts the following technical solution:

[0052] A method for silencing the aforementioned gene using virus-induced gene silencing technology includes the following steps:

[0053] a. Design specific primers targeting the aforementioned genes;

[0054] b. Amplify the target sequence using the specific primers and insert it into the VIGS vector;

[0055] c. Transform the VIGS vector containing the target sequence into Agrobacterium;

[0056] d. Introduce the VIGS vector into Chinese cabbage cells via Agrobacterium infection;

[0057] e. Observe and detect the gene silencing effect.

[0058] Preferably, the specific primer sequences are shown in SEQ ID NO.14 and SEQ ID NO.15.

[0059] As a preferred option, the target site of VIGS in the Chinese cabbage ACA10 gene is shown in SEQ ID NO.9.

[0060] Preferably, in step b, the amplification product is ligated into the pTRV2 vector treated with restriction endonucleases BamHI and SamI to obtain ACA10-pTRV2.

[0061] Preferably, the Agrobacterium is Agrobacterium tumefaciens GV3101.

[0062] The eighth objective of this invention is to provide an application of the aforementioned gene in enhancing the calcium ion transport capacity and calcium ion enrichment capacity of plants and / or yeast.

[0063] To achieve the above objectives, the present invention adopts the following technical solution:

[0064] The aforementioned genes are used to enhance the calcium ion transport capacity and calcium ion accumulation capacity of plants and / or cells.

[0065] Preferably, it is used to improve the calcium ion transport capacity and the ability to absorb and accumulate calcium ions in Arabidopsis thaliana and / or yeast.

[0066] As a preferred method, overexpression of the ACA10 gene enhances the plant's ability to absorb and transport calcium ions, thereby improving plant growth or stress resistance.

[0067] As a preferred method, the ACA10 gene overexpression vector is transferred into the plant genome, thereby overexpressing the ACA10 gene in the transgenic plant and improving the plant's ability to absorb and transport calcium ions.

[0068] The beneficial effects of this invention are as follows: Attached Figure Description

[0069] Figure 1 Sequence alignment diagram of amino acid ACA10 for amplified genes in two Chinese cabbage varieties;

[0070] Figure 2 A schematic diagram of the ACA10 gene protein domain;

[0071] Figure 3 A phylogenetic tree diagram of the ACA10 protein and other ACA10 homologs;

[0072] Figure 4 A diagram showing the predicted interactions between the ACA10 protein and proteins;

[0073] Figure 5 A prediction map of ACA10 protein enrichment in biological processes;

[0074] Figure 6 A prediction map of functional enrichment of ACA10 protein molecules;

[0075] Figure 7 A partial map of the ACA10 subcellular localization vector;

[0076] Figure 8 A diagram showing the subcellular localization of ACA10;

[0077] Figure 9 This is a graph showing the growth of gene-modified Saccharomyces cerevisiae on a plate.

[0078] Figure 10 Growth curves for ACA10-5-6 and wild-type yeast;

[0079] Figure 11 Growth curves for ACA10-6-8 and wild-type yeast;

[0080] Figure 12 Graph showing the determination of calcium content in yeast;

[0081] Figure 13 A graph showing the relative expression levels of the ACA10 gene in Arabidopsis thaliana;

[0082] Figure 14 A graph showing the calcium content in Arabidopsis leaves under different calcium application treatments;

[0083] Figure 15 The graph shows the changes in ACA10 gene expression and the detection results of ACA10 gene silencing efficiency under different treatments at three different time periods; among them, Figure 15 -A shows the changes in ACA10 gene expression levels under different treatments during the three periods of ACA10-5-6; Figure 15 -B is a graph showing the silencing efficiency of the ACA10 gene under different treatments in three time periods (ACA10-5-6).

[0084] Figure 16 The graph shows the changes in ACA10 gene expression and the detection results of ACA10 gene silencing efficiency under different treatments at three different time periods; among them, Figure 16 -A shows the changes in ACA10 gene expression levels under different treatments during the three periods of ACA10-6-8; Figure 16 -B is a graph showing the silencing efficiency of the ACA10 gene under different treatments at three different time points;

[0085] Figure 17 A graph showing the calcium content of ACA10-5-6 under different treatments at three different time periods;

[0086] Figure 18 The graph shows the calcium content of ACA10-6-8 under different treatments at three different time periods. Detailed Implementation

[0087] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0088] In this embodiment of the invention, the relevant materials, reagents, and instruments used in the bioinformatics analysis experiment of ACA10 are as follows:

[0089] (1) Plant materials

[0090] The cabbage samples of "Black Leaf May Slow" and "Shanghai June Slow" collected for the pot experiment were provided by our laboratory.

[0091] (2) Vectors and strains

[0092] The PEASY-BluntZero Cloning Kit vectors were purchased from TransGen Biotech Ltd. (Beijing), and E. coli DH5α was purchased from Beijing Qingke Biotechnology Co., Ltd. The subcellular localization vector PAN580 was provided by our laboratory.

[0093] (3) Enzymes and kits

[0094] The Plasmid Maxi Kit (Omega Bio-tek) was purchased from Chongqing Pengguang Biotechnology Co., Ltd. Cellulase R-10 (CELLULASE "ONOZUKA" R-10) (Yakult) and chromogenic enzyme R-10 (MACEROZYME R-10) (Yakult) were both purchased from Shanghai Haoye Biotechnology Co., Ltd. Mannitol, potassium chloride, calcium chloride, sodium chloride, and magnesium chloride were all purchased from Chongqing Wubai Biotechnology Co., Ltd. Bovine serum albumin, glucose, PEG4000, and MES were all purchased from Chongqing Jinxique Technology Development Co., Ltd.

[0095] (4) Preparation methods of culture medium and antibiotics

[0096] 100 mg / mL Kan stock solution: Weigh 1.0 g Kan, slowly pour 10 mL of sterile deionized water into a clean bench, filter and sterilize using a 0.22 μm organic phase filter membrane sterilized by autoclaving and a 1 mL sterile syringe, then dispense 200 μL into each centrifuge tube and store in a -20°C freezer.

[0097] 100 mg / mL Ampicillin Stock Solution: Weigh 1 g of ampicillin sodium salt, add 10 mL of sterile water to a clean bench, filter and sterilize using a 0.22 μm organic phase filter membrane sterilized by autoclaving and a 1 mL sterile syringe, then dispense 200 μL into each centrifuge tube and store in a -20°C freezer.

[0098] 100 mg / mL Gen stock solution: Weigh 1.0 g Gen, slowly pour 10 mL of pre-sterilized deionized water into a clean bench, filter and sterilize using a 0.22 μm aqueous phase filter membrane sterilized by autoclaving and a 1 mL sterile syringe, then aliquot into 200 μL per centrifuge tube and store in a -20°C freezer.

[0099] 100 mg / mL Rif stock solution: Weigh 0.5 g Rif, slowly pour DMSO into it in a clean bench, filter it with a 0.22 μm aqueous phase filter membrane sterilized by autoclaving and a 1 mL sterile syringe for sterilization, then dispense 200 μL into each centrifuge tube and store in a -20°C freezer.

[0100] (5) Test instruments

[0101] PCR amplification instrument, high-speed refrigerated centrifuge, electrophoresis apparatus, high-speed benchtop centrifuge, constant temperature shaker, gel imaging system, DH6000 electric thermostatic incubator, -80℃ ultra-low temperature freezer, clean bench, HWS24 electric thermostatic water bath, nucleic acid DNA / RNA protein ultra-micro spectrophotometer, high temperature and high pressure sterilizer, pH meter, laser confocal microscope (TCS SP8 X, Leica, Southwest University).

[0102] In this embodiment of the invention, the relevant materials, reagents, and instruments used in the functional verification experiment of ACA10 are as follows:

[0103] (1) Plant materials

[0104] Cabbage leaves treated with 0 and 0.4 g / kg CaSO4·2H2O were provided by our laboratory.

[0105] (2) Vectors and strains

[0106] VIGS vectors pTRV1, pTRV2, and PDS; Arabidopsis thaliana overexpression vector PBI121; and yeast expression vector PYES2.0 were all provided by our laboratory. Yeast strains BY4741 and Agrobacterium GV3101 were purchased from Shanghai Weidi Biotechnology Co., Ltd. Escherichia coli strain E. coli DH5a was purchased from Beijing Qingke Biotechnology Co., Ltd.

[0107] (3) Enzymes and kits

[0108] The restriction endonuclease XbaI was purchased from Beijing NEB Technology Development Co., Ltd. qPCR Master Mix (Promega) was purchased from Chongqing Runchen Biotechnology Co., Ltd. DO Supplement-Ura (deficient amino acids), yeast competent cell preparation and transformation kit were purchased from Promega Biotechnology Co., Ltd.

[0109] (4) Culture medium preparation

[0110] Three-antibiotic solid culture medium: 10g sodium chloride, 5g yeast extract, 10g tryptone, double-distilled water to a final volume of 1L, pH 7.0, with rifampin, gentamicin and kanamycin added in proportion.

[0111] Three-antibiotic solid culture medium: 10g sodium chloride, 8.5g agar powder, 5g yeast extract, 10g tryptone, double-distilled water to a final volume of 1L, pH 7.0, with rifampin, gentamicin and kanamycin added in proportion.

[0112] YPDA liquid culture medium: 10g yeast extract, 20g tryptone, 22g glucose, 0.08g adenine, and double-distilled water to a final volume of 1L.

[0113] YPDA solid culture medium: 10g yeast extract, 20g tryptone, 22g glucose, 0.08g adenine, 20g agar powder, and double-distilled water to a final volume of 1L.

[0114] SD / -Ura liquid medium: SD / -ura 1.3g, yeast nitrogen source 6.7g, glucose 22g, double-distilled water to a final volume of 1L, adjust pH to 5.8.

[0115] SD / -Ura solid medium: SD / -ura 1.3g, yeast nitrogen source 6.7g, glucose 22g, agar powder 20g, double distilled water to a final volume of 1L, adjust pH to 5.8.

[0116] Arabidopsis transformation buffer: MES 0.5 g / L, MS 2.2 g / L, sucrose 50 g / L, mix well and adjust pH to 5.7, add Silweet L-77300 μL just before use.

[0117] 1 / 2MS medium: 2.215g MS, 10g sucrose, add sterile distilled water to a final volume of 1L, adjust pH to 5.5-6.0, and add 8.5g agar powder.

[0118] In this embodiment of the invention, ACA10-5-6 refers to "Black Leaves in May" and ACA10-6-8 refers to "Shanghai in June".

[0119] Example 1. Bioinformatics Analysis of ACA10

[0120] I. Experimental Methods

[0121] 1. Cloning of the ACA10 gene

[0122] (1) Polymerase chain reaction

[0123] The two ends of the ACA10 gene in the Arabidopsis thaliana genome database were used as the amplification start and focus, and the primer sequences and names are shown in Table 1.

[0124] Table 1 Primer sequences and names

[0125] Primer name Primer sequence (5'-3') ACA10-F SEQ ID NO.1 ACA10-R SEQ ID NO.2 M13-F SEQ ID NO.3 M13-R SEQ ID NO.4

[0126] The cDNA sequence of the ACA10 gene was obtained by polymerase chain reaction (PCR) using KOD FX. The reaction system is shown in Table 2.

[0127] Table 2 ACA10 PCR amplification reaction system

[0128] Component Name System quantity / ul cDNA temple (100ng / ul) 1.5 Forward Primer (10µM) 2 Reverse Primer (10µM) 2 2X PCR Buffer for KOD FX 20 2mM dNTPs 4 KOD FX 0.5 <![CDATA[dd H2O]]> Upto40ul

[0129] After thoroughly mixing the above-mentioned system, the mixture was briefly centrifuged and then placed in a PCR instrument for amplification. The amplification conditions are shown in Table 3.

[0130] Table 3 ACA10 PCR amplification conditions

[0131]

[0132]

[0133] Detection of PCR products by agarose gel electrophoresis.

[0134] (2) Purification of the target fragment

[0135] Using Omega Biotechnology Co., Ltd. The purification process using the Gel Extraction Kit is as follows:

[0136] 1) Cut off the stripe with a knife, aliquot it into 1.5ml centrifuge tubes, add Binding Buffer to the centrifuge tubes until the colloid is completely submerged.

[0137] 2) Dissolve in a 55℃ water bath for 10-15 minutes (until the colloid is completely dissolved).

[0138] 3) Wait for the centrifuge tube to cool to room temperature, then transfer the entire solution to the pink column and centrifuge at 22°C, 12000xg for 1 min. Discard the waste liquid.

[0139] 4) Add 300ul Binding Buffer to the column and centrifuge for 1 minute under the same centrifugation conditions.

[0140] 5) Add 700ul of Spw Buffer to the pink column (Spw Buffer has been diluted with the correct amount of anhydrous ethanol), place it in a centrifuge at 22°C, centrifuge at 12000xg for 1 min, and discard the waste liquid.

[0141] 6) Repeat step 4).

[0142] 7) Centrifuge the empty tube for 3 minutes without adding solution to dry the matrix.

[0143] 8) Add 30ul of Elution Buffer vertically and let stand for 2 minutes (be careful not to puncture the membrane in the middle of the column with the pipette tip).

[0144] 9) Centrifuge at 22℃, 12000xg for 1 min, and discard the waste liquid.

[0145] 10) Repeat steps 8) and 9) to obtain more DNA yield.

[0146] 11) Keep the bottom filtrate at -20℃ for later use.

[0147] (3) Vector cloning and transfection of E. coli

[0148] The following steps were taken using the PEASY-Blunt Zero Cloning Kit vector purchased from Beijing TransGen Biotechnology Co., Ltd., and E. coli DH5α purchased from Beijing Qingke Biotechnology Co., Ltd.:

[0149] 1) Pre-control the water temperature in the ice box to around 14℃.

[0150] 2) Take a sterilized 200uL microcentrifuge tube and add 4uL of the target gene (i.e., the purified PCR product).

[0151] 3) Add 1 μL of T vector (B-Zero), gently mix the whole system, and place the system in a PCR instrument at 25°C for 10 min.

[0152] 4) Transfer the system to an ice box and incubate for 2 minutes.

[0153] 5) The ligation product can be used to transform competent E. coli cells.

[0154] 6) Take out a tube (100μL) of competent bacteria from the -80℃ ultra-low temperature freezer, immediately warm it with your finger to melt it, and then insert it into the ice bath for 5-10 minutes.

[0155] 7) Add 5 μL of the ligated plasmid mixture (DNA content not exceeding 100 ng), gently shake, and place on ice for 20 min.

[0156] 8) After gently shaking, insert into a 42℃ water bath for 90 seconds to induce heat shock, then quickly return to ice and let stand for 3 minutes.

[0157] 9) In a clean bench, add 800 μL of LB medium (without antibiotics) to each of the above tubes and mix gently. Then fix them on the spring frame of the shaker and revive at 37°C and 220 r / min for 1-1.5 h.

[0158] 10) In a clean bench, take 200 μL of the above transformation mixture and drop it into solid LB agar plates containing a suitable antibiotic (Amp 100 mg / L). Then, add 40 μL of 20 mg / ml X-gal and 7 μL of 200 mg / ml IPTG to the plates and spread them evenly with a glass spreader that has been heated with an alcohol lamp. (Note: One plate without antibiotics serves as a control group. After the alcohol on the glass spreader is extinguished, wait a moment for it to cool before spreading. When spreading the bacterial solution, avoid repeated back-and-forth spreading, as the cell walls of competent bacteria have changed. Excessive mechanical squeezing during spreading can cause cell rupture and affect the transformation rate.)

[0159] 11) Mark the coated petri dish, place it in a 37°C incubator for 30 minutes until the liquid on the surface has penetrated into the culture medium, then invert it and place it in a 37°C incubator overnight.

[0160] 12) Spray 70% ethanol on the bacteria-contaminated desktop and wipe it dry.

[0161] 13) Observe the colony clones that grow on the plate. It is best if the colonies can be separated from each other. Pay attention to the white spots.

[0162] (4) Bacterial suspension detection

[0163] Table 4. Bacterial PCR amplification reaction system

[0164] Component Name System quantity / ul M13ForwardPrimer 1 M13ReversePrimer 1 GreenTaqMix 10 bacterial solution 2 <![CDATA[ddH2O]]> Upto20ul

[0165] After thoroughly mixing the system in Table 4, centrifuge briefly and then place it in a PCR instrument for amplification. The amplification conditions are shown in Table 5.

[0166] Table 5. Conditions for PCR amplification of bacterial culture

[0167]

[0168] PCR products were detected by agarose gel electrophoresis, and after correct alignment, they were sent to Shanghai BioBioTech Co., Ltd. for sequencing analysis.

[0169] 2. Structural prediction and physicochemical property analysis of ACA10 protein

[0170] The amino acid sequence of the amplified ACA10 gene was analyzed using the online SMART software. The protein sequence of ACA10 was downloaded from the Ensembl Plants database, and the secondary structure of the ACA10 protein was predicted using the online software SOPMA. Finally, the protein's amino acid number, molecular weight, isoelectric point, and hydrophilicity / hydrophobicity were obtained from the ExPASy website.

[0171] 3. Construction of the ACA10 protein phylogenetic tree

[0172] To analyze the phylogenetic relationship between ACA10 and its homologous proteins, BLAST homology searches and sequence alignment were performed on the NCBI database using ACA10 as the seed sequence. Sequences with high sequence identity and an E-value less than 1×10⁻⁶ were selected. -53 As a standard for homologous sequences in ACA10, a phylogenetic tree was constructed using MEGA11 with the Neighbor-Joining (NJ) method. The resulting phylogenetic tree file was then uploaded to the online software iTOL for enhancement.

[0173] 4. Construction of protein-protein interaction networks

[0174] The obtained ACA10 protein sequence was used to predict the protein interaction network diagram using the online software STRING in the existing protein-protein interaction database, and then further analyzed using Cytoscape software to finally obtain the protein interaction network diagram.

[0175] 5. Construction of ACA10 protein subcellular localization vector

[0176] (1) Acquisition of target fragment

[0177] 1) Based on the ACA10 gene sequence, specific primers were designed using PrimerPremier6.0 software, as shown in Table 6.

[0178] Table 6 Primer sequences and names

[0179] Primer name Primer sequence (5'-3') ACA10-F SEQ ID NO.5 ACA10-R SEQ ID NO.6 PAN580-F SEQ ID NO.7 PAN580-R SEQ ID NO.8

[0180] 2) Polymerase chain reaction and purification of the target fragment.

[0181] (2) Carrier linearization processing

[0182] The PAN580 vector was linearized using Sma I, Cut Smart Buffer, and BamHI-HF. The reaction system is shown in Table 7.

[0183] Table 7 PCR reaction system for PAN580 vector

[0184] Component Name System quantity / ul PAN580 Plasmid 8 Cut Smart Buffer 5 Sma I 1 Bam HI-HF 1 <![CDATA[ddH2O]]> Up to 50ul

[0185] Mix the above system thoroughly, centrifuge briefly, and then react in a water bath at 37°C for 30 minutes. Use Omega Biotechnology Co., Ltd. Gel Extraction Kit purification kit.

[0186] (3) Carrier construction

[0187] After removing the stop codon from the full-length ACA10 gene sequence, it was constructed into the vector PAN580-GFP using homologous recombination (ClonExpressII OneStep Cloning Kit). The target fragment was located between the N-terminus of the GFP-encoding gene in the vector and between Sma I and BamHI downstream of the CaMV 35s promoter. The reaction system is shown in Table 8.

[0188] Table 8. Reaction system of homologous recombination ACA10-PAN580

[0189] Component Name System quantity / ul PAN580 Plasmid 4 Exnase Multis 2 5×CE Multis Buffe 4 Purpose of the fragment 5 The following is a fragment of the purpose 5

[0190] Mix the above system thoroughly, centrifuge briefly, and react at 37°C for 30 minutes. After the reaction is complete, immediately place it on ice.

[0191] (4) Escherichia coli transformation

[0192] 1) Place the system in a 4°C refrigerator for 3 minutes.

[0193] 2) Quickly transfer the entire system solution into competent E. coli cells (the E. coli should be in a state similar to ice cream; prolonged exposure to room temperature will affect the survival rate of E. coli).

[0194] 3) Slowly aspirate and mix the entire system, place it on an ice box, and put it in a 4°C refrigerator for 30 minutes (the target fragment, vector, and E. coli will bind at this time).

[0195] 4) Place the system in a 42°C water bath for 45 seconds (the temperature rises, and the binding is interrupted).

[0196] 5) Place the system in a 4°C refrigerator for 3 minutes.

[0197] 6) Transfer to a clean bench for testing. Add 800 μL of LB culture medium to the system and mix thoroughly by pipetting.

[0198] 7) Use a shaker (37℃) to revive the entire system for 1.5 hours.

[0199] 8) 12000xg at room temperature, 1min.

[0200] 9) In a clean bench, spread the bacterial solution evenly on LB medium containing ampicillin (be as gentle as possible when spreading to prevent E. coli from being crushed).

[0201] 10) Incubate at 37℃ for 12-16 hours.

[0202] (5) Bacterial suspension detection

[0203] Table 9. Bacterial PCR Amplification Reaction System

[0204] Component Name System quantity / ul M13 Forward Primer 1 M13 Reverse Primer 1 Green Taq Mix 10 bacterial solution 2 <![CDATA[dd H2O]]> Up to 20ul

[0205] After thoroughly mixing the system in Table 9, centrifuge briefly and then place it in a PCR instrument for amplification. The amplification conditions are shown in Table 10.

[0206] Table 10 Conditions for PCR amplification of bacterial culture

[0207]

[0208] PCR products were detected by agarose gel electrophoresis, and after correct alignment, they were sent to Shanghai BioBioTech Co., Ltd. for sequencing analysis.

[0209] (6) Plasmid extraction

[0210] First, activate the correctly sequenced bacterial culture. Using a 50ml centrifuge tube, transfer 100µl of the bacterial culture, then add 15ml of LB broth containing ampicillin and incubate at 37°C with gentle shaking for one day. Second, perform large-scale propagation of the bacterial culture. Transfer 1ml of the bacterial culture from the 50ml centrifuge tube to a 250ml Erlenmeyer flask, add 100ml of LB broth containing ampicillin, and incubate at 37°C with vigorous shaking for one day (do not overdo it, as this will affect bacterial activity). Then, extract plasmids using the Plasmid MaxiKit (Omega Bio-tek) plasmid extraction kit. The specific steps are as follows:

[0211] 1) Collect 100ml of bacterial culture (in two 50ml centrifuge tubes) by centrifugation at 4000xg for 10 minutes at room temperature.

[0212] 2) Discard the culture medium and add 12 ml of Solution I / RNase A mixture to the precipitate. Mix well by pipetting to completely resuspend the cells. (When adding Solution I / RNase A mixture, first add 4 ml to resuspend the cells, then add 8 ml and gently invert to mix.)

[0213] 3) Add 12ml Solution II and gently invert the container 8-10 times to mix (when adding the solution, add it gently along the wall; after mixing, you can see a stringy consistency when you open the cap).

[0214] 4) Add 16ml of Solution Ⅲ, gently invert the centrifuge tube several times to mix. You will see the formation of white flocculent matter during the process. Let it stand at room temperature for 3 minutes.

[0215] 5) Dispense the flocculent material and liquid into 2ml round-bottom centrifuge tubes.

[0216] 6) Centrifuge at 4℃, 14000xg for 10min.

[0217] 7) Insert the DNAMaxi Column into a 50ml collection tube (start with the empty tube). Add 3 ml of 3M NaOH Buffer to DNAMaxiColumn, let stand for 4 min, centrifuge at 4000 x g for 3 min at room temperature, and remove waste liquid.

[0218] 8) Transfer no more than 20 ml of supernatant to DNAMaxi Column: Centrifuge at 4000xg for 5 minutes at room temperature and discard the waste liquid (be careful not to aspirate proteins when collecting the supernatant, as this will cause plasmid contamination).

[0219] 9) Put Insert the DNA Maxi Column into the same collection tube, add 10 ml of HBC Buffer (diluted with isopropanol) to... In DNAMaxi Column, centrifuge at 4000xg for 5 min at room temperature and discard the waste liquid.

[0220] 10) Put Insert the DNAMaxi Column into the same collection tube, add 15 ml of DNAWash Buffer (diluted with anhydrous ethanol), centrifuge at 4000 x g for 5 minutes at room temperature, and discard the waste liquid.

[0221] 11) Repeat step 10).

[0222] 12) Centrifuge with an empty tube, and... Insert the DNAMaxi Column into the same collection tube and centrifuge at 4000xg for 10 min at room temperature.

[0223] 13) will Insert the DNAMaxi Column into a new 50ml collection tube, add 800ul of Elution Buffer to the binding column matrix, and let it stand at room temperature for 5 minutes.

[0224] 14) Centrifuge at 4000xg for 5 minutes at room temperature to elute DNA.

[0225] 15) Repeat steps 13) and 14) to increase production.

[0226] 16) Discard the column and store the plasmid DNA at -20°C.

[0227] (7) Plasmid Concentration

[0228] 1) Transfer the eluted plasmid to a 2ml centrifuge tube.

[0229] 2) Add 50 μL of 3M NaAC (pH = 5.2) and 350 μL of isopropanol, and let stand at room temperature for 10 minutes.

[0230] 3) Centrifuge at 4℃, 15000xg for 20min, remove the supernatant and collect the precipitate.

[0231] 4) Add 1 ml of 70% ethanol to wash the DNA precipitate.

[0232] 5) Centrifuge at 4℃, 15000xg for 10min, remove the supernatant and collect the precipitate.

[0233] 6) Place the plasmid in a clean bench to dry until the liquid is completely air-dried.

[0234] 7) Dissolve the plasmid in 50 μL of Elution Buffer.

[0235] 8) Detect plasmid concentration and store at -20℃.

[0236] 6. Extraction and transformation of protoplasts from Arabidopsis thaliana leaves

[0237] (1) Select wild-type Arabidopsis seedlings with well-expanded cotyledons that have been growing for about 2 weeks.

[0238] (2) Cut the true leaves into small square pieces and remove the veins and lower epidermis with tape.

[0239] (3) Place the leaves into a round petri dish containing the prepared enzymatic hydrolysate.

[0240] (4) Add the enzyme hydrolysate to a final volume of 20 mL (ensure that the leaves are completely submerged in the enzyme hydrolysate). The preparation method of the enzyme hydrolysate is shown in Table 11.

[0241] Table 11 Preparation of Enzyme Hydrolysate (20 mL)

[0242] Component Name content 1% Cellulase R10 0.2g 0.25% Macerozyme R10 0.05g 0.4M mannitol 8ml 0.2M KCl 2ml 0.2M MES 4ml <![CDATA[0.1M CaCl2]]> 400ul 0.1% BSA 0.02g

[0243] Adjust the pH of the solution to 5.7, dilute to 20 ml with water, and then filter through a 0.22 μm filter membrane for sterilization before use.

[0244] (5) Wrap the petri dish with aluminum foil and place it stably in the dark at 22°C for about 3 hours (the cell walls of plant cells are decomposed by enzymes, and the remaining protoplasts are round and fragile, so handle them as gently as possible).

[0245] (6) Microscopic examination to determine the state of protoplasts.

[0246] (7) Filter the enzymatically hydrolyzed chloroplast solution through a 100-mesh plastic sieve (cut off the tip of the 1ml pipette tip with a blade beforehand to draw the enzymatic solution).

[0247] (8) Transfer the filtrate to a 50 mL round-bottom centrifuge tube and place the centrifuge tube on ice immediately.

[0248] (9) Balance the centrifuge, 4℃, 100g, centrifuge for 5min (the centrifuge's acceleration and deceleration speeds are both adjusted to 1).

[0249] (10) Discard the supernatant and add an equal volume of pre-cooled W5 solution. The preparation method of W5 solution is shown in Table 12.

[0250] Table 12 Preparation of W5 solution

[0251] Component Name content 1M NaCl 7.7ml <![CDATA[0.5M CaCl2]]> 12.5ml 0.2M KCl 1.25ml 0.1M MES 1ml 0.05M glucose 0.04504g

[0252] Mix the above system thoroughly, adjust the pH to 5.7, bring the volume to 50 ml with water, and then filter it through a 0.22 μm filter membrane for sterilization.

[0253] (11) Centrifuge at 4℃, 100g for 5min.

[0254] (12) Repeat steps (10) and (11).

[0255] (13) Remove the supernatant, add 5 ml of W5 solution, and place on ice for 30 min.

[0256] (14) Under the same centrifugation conditions, centrifuge again for 5 minutes.

[0257] (15) Remove the supernatant and add an appropriate amount of MMG solution along the wall. The preparation method of MMG solution is shown in Table 13.

[0258] Table 13 Preparation of MMG solution

[0259]

[0260]

[0261] Mix the above system thoroughly, adjust the pH to 5.7, bring the volume to 50 ml with ddH2O, and then filter it through a 0.22 μm filter membrane for sterilization.

[0262] (16) Examine under a microscope to see the dispersion of protoplasts (if the protoplasts are too dispersed, they need to be centrifuged again. Centrifugation conditions: 4℃, 100g, centrifugation for 5min, speed adjusted to 1; if many protoplasts are found to be stuck together, it means that the concentration of protoplasts is too high and an appropriate amount of MMG solution needs to be added).

[0263] (17) Add 15 μg of plasmid and 200 μl of protoplast to a 2 ml EP tube and gently tap the tube wall to mix.

[0264] (18) Add an equal volume of PEG4000 solution and mix well. The preparation method of PEG4000 solution is shown in Table 14.

[0265] Table 14 Preparation of PEG4000 solution

[0266] Components content 0.2M mannitol 2ml <![CDATA[0.1M CaCl2]]> 2ml PEG 4000 4g <![CDATA[ddH2O]]> 3ml

[0267] Mix the above system thoroughly and place it in a 65°C water bath until completely dissolved (to fully dissolve PEG4000, it is best to prepare the solution 1 hour before use. The solution can be left at room temperature for 5 days, but the conversion efficiency of the freshly prepared solution is relatively high).

[0268] (19) Place the centrifuge tubes in an Arabidopsis thaliana incubator and incubate for 10-15 minutes.

[0269] (20) Add 4 times the volume of W5 solution, gently tap the tube wall to mix, and terminate the conversion.

[0270] (21) Centrifuge at 4℃, 200g for 5 minutes, and set the centrifuge speed to 1 to remove any possible supernatant.

[0271] (22) Add 150 μL of W5 solution, mix gently, and place horizontally.

[0272] (23) Wrap the contents in aluminum foil and store them in an Arabidopsis thaliana incubator for 9-12 hours (the incubation time should be determined according to the characteristics of the target gene protein).

[0273] (24) Microscopic examination confirms the state and fragmentation of the protoplasts.

[0274] (25) Fluorescence signals were detected using a laser confocal microscope (TCS SP8 X, Leica, Southwest University).

[0275] II. Experimental Results

[0276] 1. Cloning and analysis of the ACA10 gene

[0277] Using the full-length ACA10 gene as a template, the cDNA extracted from two Chinese cabbage varieties was amplified using primer sequences and sequence alignment was performed. The results are as follows: Figure 1 As shown. The full-length gene sequence is 3222 bp. There were 15 differences between the amplified results of the two varieties and the gene alignment. Specifically, the nucleotide sequence of ACA10-5-6 (Black Leaf May Slow) is shown in SEQ ID NO:18, and the nucleotide sequence of ACA10-6-8 (Shanghai June Slow) is shown in SEQ ID NO:19. The amino acid sequence of ACA10-5-6 is shown in SEQ ID NO:20, and the amino acid sequence of ACA10-6-8 is shown in SEQ ID NO:21. Further comparison of the amino acid sequences of the two varieties revealed differences at two locations: 412 bp and 734 bp. At 412 bp and 734 bp, "Black Leaf May Slow" contains valine and aspartic acid, respectively, while "Shanghai June Slow" contains leucine and serine. Valine is a basic amino acid, aspartic acid is an acidic amino acid, while leucine and serine are neutral amino acids. This indicates that the acidity / basicity of these two amino acids has changed in both varieties. This is because natural variations accumulated during evolution among different varieties have led to differences in gene sequences, including single nucleotide polymorphisms and insertions / deletions, which in turn affect amino acid sequences and protein function. Simultaneously, different varieties have varying abilities to adapt to their environment, resulting in adaptive variations in ACA10 during evolution, leading to differences in CDS and amino acid sequences to optimize protein function.

[0278] 2. Physicochemical properties and structural analysis of ACA10 protein

[0279] The physicochemical properties of the protein obtained from ACA10 after transcription-translation and final modification were predicted using the online software Expasy-protparam. The results showed that 1073 amino acids are encoded by this gene. Among these amino acids, the most abundant are Val (9.9%), Leu (9%), Gly (7.9%), Ala and Ser (7.5%), and Ile (7.4%). The total number of negatively charged residues (Asp+Glu) is 119, and the total number of positively charged residues (Arg+Lys) is 111. Its molecular weight is 117064.72, and its molecular formula is C1. 5205 H 8366 N 1428 O 1552 S 40The total number of amino acid atoms is as high as 16,591. When all paired cysteine ​​residues form cystine, its extinction coefficient is 105,725; when all cysteine ​​residues are reduced, its extinction coefficient is 104,850. ACA10 has a theoretical isoelectric point of 6.23, is an acidic protein, and is also an unstable and hydrophobic protein. Its fatty acid coefficient is 100.17, and its average hydrophilicity is 0.036.

[0280] SMART analysis revealed a CaATP-NAI domain, a Pfam domain, located at positions 29-73 bp. This entry represents the N-terminal autorepressive calmodulin-binding domain characteristic of certain calcium-transporting ATPases. This domain binds calmodulin in a calcium-dependent manner and possesses a conserved RRFR sequence motif. This region extends from position 105 to position 114. There is also a Cation-ATPase-N domain, located at positions 140-211. This region extends from position 219 to position 238. Another region extends from position 851 to position 873. Finally, there is a Cation-ATPase-C domain, a Pfam domain located from positions 875 to 1051. P-ATPases (also known as E1-E2 ATPases) are present in bacteria and many eukaryotic plasma membranes and organelles. A schematic diagram of the ACA10 gene protein domains is shown below. Figure 2 As shown.

[0281] Transmembrane ATPases are membrane-bound enzyme complexes / ion transporters that drive proton transport across the membrane via ATP hydrolysis. Some transmembrane ATPases also work in the opposite direction, using the energy of the proton gradient and the ion flux through the ATPase proton channel to drive ATP synthesis.

[0282] ACA10 contains an N-terminal autorepressive calmodulin-binding domain, a Cation-ATPase-N domain, and a Cation-ATPase-C domain, enabling it to activate the calcium ion pump. This is related to the binding of the autorepressive domain to the catalytic core of the ACA protein. The basic amino acid residues (such as lysine and arginine) in the autorepressive domain can interact with the acidic residues of calmodulin. The results of this study indicate that ACA10 is an unstable acidic hydrophobic protein. The isoelectric point and hydrophilicity or hydrophobicity of ACA proteins are important physicochemical properties, significant for understanding protein function. The acidic isoelectric point of ACA proteins is adapted to the negatively charged environment of the cytoplasm, while the hydrophobicity of the transmembrane region is fundamental to ACA proteins' embedding in membrane structures and performing calcium ion transport functions.

[0283] 3. Phylogenetic analysis of ACA10 protein

[0284] By selecting homologous sequences for ACA10, we identified a total of 13 homologous proteins in the NCBI database, including those from radish, rapeseed, and Arabidopsis. Based on amino acid sequence uniformity, a phylogenetic tree showed that ACA10 and this homologous protein were divided into 5 branches, as shown below. Figure 3 As shown, ACA10 is most homologous to Arabidopsis thaliana AtACA10.1, with a sequence identity of 99% and an E-value of 2×10⁻⁶. -106 .

[0285] 4. ACA10 protein interaction network analysis

[0286] Using the STRING online tool, we predicted the interacting proteins of ACA10 (M4D3U0). The local clustering coefficient of this protein was 0.732, and the p-value for protein interaction network enrichment was 0.0277. The prediction results indicate that ACA10 interacts with proteins such as M4ESC3, and the M4ESC3 gene is a gene involved in calcium transport. (See details...) Figure 4 Therefore, it is speculated that the ACA10 protein may also be related to calcium transport.

[0287] Biological process enrichment prediction analysis of ACA10 protein revealed that the strongest signal (3.05) was observed for cellular calcium homeostasis, followed by chemical homeostasis at 2.22, and then gibberellin biosynthesis at 2.12. (See details...) Figure 5 Meanwhile, molecular function enrichment prediction analysis of the ACA10 protein showed that the strongest signal for P-type calcium transporter activity reached 5.17, while the weakest signal for metal ion binding was 0.64. (See details...) Figure 6 .

[0288] 5. Construction of subcellular localization vectors

[0289] The ACA10 gene fragment was ligated into the PAN580-GFP linearized expression vector as follows: Figure 7 As shown, the ligation product is transformed into E. coli, and single clones are picked for positive detection. After the positive single clone is correctly sequenced, the plasmid is extracted, and the construction of the ACA10 subcellular localization vector is completed.

[0290] 6. Subcellular localization analysis of ACA10 protein

[0291] Subcellular structures provide relatively independent sites for proteins to perform their functions, and studying subcellular localization helps to better understand the structure and function of cells and organisms. To further investigate the role of ACA10 in calcium transport, this study constructed an ACA10-GFP fusion protein expression vector. By transforming the ACA10-GFP fusion protein expression vector into Arabidopsis protoplasts, observation using laser confocal microscopy revealed a continuous bright fluorescent signal on the cell membrane of the recombinant vector, while the empty vector signal was distributed throughout the entire cell. (See details...) Figure 8 This result indicates that ACA10 is located on the cell membrane, suggesting that it may perform calcium ion transport functions on the cell membrane.

[0292] The cell membrane is the first barrier between the cell and its external environment, and calcium ions are important second messengers in plant cells, participating in various signal transduction processes. ACA10 can directly regulate the calcium ion concentration gradient between the cytoplasm and the extracellular environment, and its localization allows for rapid response to external stimuli. ACA10 maintains intracellular calcium ion homeostasis by pumping calcium ions out of the cytoplasm. Simultaneously, because ACA10 possesses multiple transmembrane helices, these domains are embedded in the cell membrane, forming calcium ion transport channels. These channels can provide energy through ATP hydrolysis to drive the transmembrane transport of calcium ions.

[0293] Example 2. Functional Verification of ACA10

[0294] I. Experimental Methods

[0295] 1. Construction of yeast expression vector

[0296] After linearizing the PYES2 vector with XbaI and BamHI restriction endonucleases, ACA10 was ligated into the PYES2 vector via homologous recombination.

[0297] 2. Preparation and transformation of yeast competent cells

[0298] (1) Preparation of yeast competent cells

[0299] 1) Take 150 μL of the Saccharomyces cerevisiae strain BY474 stored at -80℃ and inoculate it into 10 mL - 20 mL of LYPD liquid medium. Incubate overnight at 30℃ and 250 rpm (it is recommended to use a 50 mL centrifuge tube for shaking).

[0300] 2) Detect the OD600 of the bacterial solution. After calculation, take an appropriate amount of bacterial solution, dilute it to 50 mL, and culture in LYPD. The OD600 is 0.4. The calculation method is: required stock solution (mL) = 0.4 × OD / mL × 50 mL / measured OD / mL. Continue to culture for 2-4 hours.

[0301] 3) Transfer the culture medium to a 50 mL centrifuge tube and centrifuge at 3000 g (4000 rpm) for 5 min.

[0302] 4) Remove the supernatant, add 10 mL of 1×TE to resuspend the cells, and centrifuge at 3000g (4000rpm) for 5 min at room temperature.

[0303] 5) Resuspend the bacterial cells in 2 mL of 1×LiAc / 1×TE (1 mL each) and incubate at room temperature for 10 min.

[0304] 6) Take a 1.5 mL Eppendorf tube, add 1 μg (10 μL) of purified Maker plasmid with homologous arms, 100 μg (5 μL) of frog-fish sperm vector DNA (denatured DNA can be used directly), and centrifuge to mix.

[0305] 7) Add 100 μL of competent yeast cells and shake to mix.

[0306] 8) Add 800 μL of freshly prepared 40% PEG-3350 / 1×LiAc / 1×TE. Preparation method: 50% PEG-3350: 10×LiAc: 10xTE = 8:1:1, and mix well.

[0307] 9) Incubate at 30℃ and 250rpm for 45 minutes on a shaker.

[0308] 10) Add 88 μL of DMSO and mix by inverting (do not shake).

[0309] 11) Heat shock in a 42℃ water bath for 15 min, cool on ice for 2 min, centrifuge at 10000g (12000rpm) for 30 s, and discard the supernatant.

[0310] 12) Resuspend the bacterial cell in 1 mL of 1×TE, centrifuge at 10000 g (12000 rpm) for 30 s, and discard the supernatant.

[0311] 13) The bacterial cells were resuspended in 150 μL of 1×TE and spread on selective medium (SD-His).

[0312] 14) Incubate at 30℃ upside down, and colonies will generally grow in 2-5 days.

[0313] 15) Pick a single colony and add it to 800uL (SD-His) liquid medium with 2% D-Glucose added to the monosaccharide, keeping other components unchanged. Shake at 30℃ and 250rpm for 6-8 hours for further positive identification.

[0314] (2) Transformation of yeast competent cells

[0315] 1) Select yeast strains and culture them overnight at 30°C with shaking in 5ml LYPD liquid medium.

[0316] 2) Take 4 mL of overnight culture and add 16 mL of fresh culture medium to a 50 mL shaker tube. Shake and incubate at 30°C for another 3 hours.

[0317] 3) Centrifuge at 200g for 5 minutes at room temperature, carefully remove the supernatant with a pipette, and slowly add 5 mL of 1xTE to suspend the colonies along the tube wall; centrifuge again at 200g for 5 minutes, discard the supernatant and resuspend with 2 mL of lxTE.

[0318] 4) Take 500uL of the above suspension into a 1.5mL centrifuge tube, centrifuge briefly at 13000rpm, and discard the supernatant.

[0319] 5) Add 50uL Buffer I, 5uL plasmid, 2.5uL denatured salmon extract (100mg / mL), then add 300uL Buffer 2, mix well, and incubate at 30℃ on a shaker for 30min.

[0320] 6) Add 70uLDMSO and incubate at 42℃ for 15min.

[0321] 7) Centrifuge briefly at 13000 rpm, discard the supernatant, add 500 μL of 1x TE and mix well. Centrifuge briefly at 13000 rpm, discard the supernatant, add 300 μL of 1x TE and incubate at 30°C for 30 min. Spread evenly on SD-ura (pH=5.8) plates and incubate at 30°C for 3-4 days. Perform further positive identification.

[0322] 3. Yeast plate validation test

[0323] (1) Take 30 μL of bacterial culture stored in glycerol and spread it on (SD-Ura) solid plate until a single colony grows, or pick a single clone directly from the transformed plate and place it in about 8 mL of (SD-Ura) liquid medium. Shake overnight at 30°C and 250 rpm until the bacterial concentration OD600 = 0.6-0.8.

[0324] (2) Dilute the yeast culture with 0.9% NaCl solution to three concentration gradients: OD600 = 0.1, 0.01, and 0.001. The dilution method is the same as that used during transformation.

[0325] (3) Take 2uL of diluted yeast and spot it onto solid YPD plates containing 0, 200, 300 and 400mM CaCl2 respectively. Place the plates in a 30℃ incubator and incubate for 2 to 5 days. Observe the yeast growth and then take pictures.

[0326] 4. Yeast growth curve determination experiment

[0327] (1) Take 20 μL of the bacterial culture stored in glycerol and spread it on (SD-ura) solid plate until a single colony grows. Pick a single colony and put it into about 5 mL of (SD-Ura) liquid culture medium. Shake overnight at 30℃ and 200 rpm until the bacterial culture OD600 = 0.6-0.8.

[0328] (2) Re-inoculate 20 μL of yeast culture into 5 mL (SD-ura) liquid medium, shake overnight at 30°C and 200 rpm until the OD600 of the culture is 0.6-0.8, centrifuge and wash and resuspend with ultrapure water.

[0329] (3) Prepare YPD-Ca liquid culture medium by exogenously adding different concentrations of CaCl2 to the YPD-Ca liquid culture medium, so that it contains Ca 2+ The bacteria were expressed in 0 or 300 mM medium. The bacterial culture was diluted to OD600 = 0.05 in 12-well plates (2 mL of medium) and incubated at 30°C and 200 rpm. OD600 was measured every 6 hours, and a growth curve was plotted over 72 hours.

[0330] 5. Determination of Ca content in yeast cells

[0331] (1) Take 20 μL of bacterial culture preserved in glycerol and spread it on (SD-ura) solid plate until a single colony grows. Pick a single colony and put it into about 10 mL of (YPD) liquid culture medium. Shake at 30℃ and 200 rpm for 48 h and then take it out.

[0332] (2) Centrifuge at 5000 rpm for 15 min, remove the supernatant, add ultrapure water and centrifuge for 10 min. Repeat this process three times. Then put the bacterial precipitate into an oven and dry it at 65℃ until constant weight. Take it out and weigh it.

[0333] (3) Place the weighed sample into a 50ml conical flask and place a glass funnel at the mouth of the flask for adding nitric acid and heating the nitric acid to form vapor reflux.

[0334] (4) Nitrification: In a fume hood, pour 9 ml of concentrated HNO3 into the bottle and let it stand overnight. The next day, add 3 ml of concentrated HCl and place the conical flask containing the mixed acid on an electric stove to heat it to completely degrade the plant tissue. Heating to 220°C will cause a noticeable change in the color of the solution (gradually turning from grayish-yellow to transparent). Once the solution becomes transparent, it indicates that one round of digestion is complete.

[0335] (5) Volume adjustment: Dilute the remaining liquid in the conical flask with a small amount of pure water several times, and then transfer all the diluted nitrated liquid to the volumetric flask.

[0336] (6) Detection: The calcium content was determined by atomic absorption spectrophotometry (Perkin Elmer SIMMA6000, Norwalk, USA).

[0337] 6. Construction of Arabidopsis thaliana overexpression vector

[0338] After linearizing the PBI121 vector with XbaI and BamHI restriction endonucleases, ACA10 was ligated into the PBI121 vector via homologous recombination.

[0339] 7. Agrobacterium-mediated transformation

[0340] The plasmids extracted from correctly sequenced positive clones were transformed into Agrobacterium tumefaciens GV3101. The specific steps are as follows:

[0341] (1) Dissolve Agrobacterium competent cells on ice, add 5 μl (100 ng / μl) of plasmid, and mix the above system.

[0342] (2) The above system was subjected to ice bath for 5 min, liquid nitrogen quick freeze for 5 min, water bath at 37℃ for 5 min, and ice bath for 2 min in sequence.

[0343] (3) Add 500 μl of LB liquid culture medium and incubate at 28°C and 180 rpm for 2 h.

[0344] (4) Take 200 μl of bacterial culture and spread it onto solid LB medium containing kanamycin, rifampin and gentamicin triple antibodies. Incubate at 28°C with the medium inverted for 2 days.

[0345] (5) PCR detection of positive plaques. Plaques with correct bands were cultured overnight at 28°C with shaking in 10 ml LB liquid medium containing kanamycin, rifampin, and gentamicin antibodies at 180 rpm for propagation. Simultaneously, 20% glycerol was added to the bacterial culture, and the culture was stored at -80°C.

[0346] 8. Arabidopsis transformation and pureline verification

[0347] (1) Arabidopsis thaliana transformation

[0348] The triple-antibiotic LB liquid culture was incubated at 28℃ until the OD600 value was greater than 2.0. After centrifugation at 4000 rpm / min for 10 min, excess culture medium was discarded, and the culture was resuspended in transformation buffer. At this point, the OD600 value was approximately 1.1. Arabidopsis flowers were then immersed in transformation buffer for 2 min to infect the inflorescences. After infection, the inflorescences were cultured in the dark for 24 h, and then cultured under normal conditions until fruiting occurred. The harvested seeds were the T0 generation. Infection was repeated every 10 days, with 2-3 repeats to improve transformation efficiency. All siliques and exposed flowers should be removed during the initial infection.

[0349] (2) Screening and pure line validation of Arabidopsis thaliana

[0350] Arabidopsis seeds were soaked in isopropanol for 3 minutes, then in 84 disinfectant for 10 minutes, and finally rinsed 5 times with sterile water in a clean bench. Plump seeds were selected and sown on 1 / 2 MS medium using a pipette. The seeds were vernalized at 4°C for 1-2 days, then placed in a light incubator at 23-25°C and 16h / 8h (light / dark) with a light intensity of 2000 LUX until seedlings emerged. Uniformly growing Arabidopsis seedlings were transplanted into substrate soil (nutrient soil: vermiculite = 1:2), covered with plastic wrap, and cultured in a light incubator under the same conditions for three days. After the seedlings recovered, the plastic wrap was removed, and cultivation continued under the same conditions until flowering. The specific steps for screening positive Arabidopsis seedlings and identifying pure lines are as follows:

[0351] 1) Culture T0 generation seeds in 1 / 2 MS medium containing 50 mg / L kanamycin for 7-10 days, select positive seedlings with normal growth (good root growth of seedlings and corresponding transgenic phenotypic characteristics), transplant them into the culture medium, and harvest seeds from each plant, which are the T1 generation seeds.

[0352] 2) Continue culturing T1 generation seeds in 1 / 2 MS medium containing 50 mg / L kanamycin for 7–10 days. According to Mendel's laws of inheritance, screen plates with a resistant to non-resistant plant ratio of 3:1. The resistant lines on these plates are the single-copy transgenic lines. Screen 2–3 lines. Transplant these lines into a culture medium, and harvest seeds from each plant; these are the T2 generation seeds.

[0353] 3) Continue to culture the T2 generation seeds in 1 / 2 MS medium containing 50 mg / L kanamycin for 7–10 days, and select the lines that grow normally throughout the plate as pure lines.

[0354] 9. Identification of transgenic Arabidopsis plants

[0355] The expression level of the target gene was detected by qRT-PCR. cDNA from transgenic lines (OE-1, OE-2) and wild-type (Col-1) were used as templates, and Actin from Arabidopsis thaliana was used as an internal control. The relative expression level was calculated using a 2-1 timescale (CT) value. -△△CT Obtained through legal processing.

[0356] 10. Arabidopsis thaliana calcium content test

[0357] Three seedlings of homozygous transgenic lines (OE-1, OE-2) and wild-type Arabidopsis thaliana with similar growth were selected and transplanted into the same substrate soil. They were cultured for 21 days in nutrient solutions containing 0 and 20 mM CaCl2, respectively. Phenotypic data were recorded by photographing and calcium content was measured.

[0358] 11. Determination of calcium content in Arabidopsis thaliana leaves

[0359] Homozygous transgenic lines (OE-1, OE-2) and wild-type Arabidopsis thaliana were removed from the substrate soil, washed twice with pre-cooled double-distilled water, and residual moisture was blotted off with filter paper. The samples were then placed in petri dishes lined with filter paper and dried in an oven at 65°C for 5 hours. The dried samples were gently removed from the filter paper and placed in 50 ml Erlenmeyer flasks for nitrification. The calcium content was then determined using atomic absorption spectrophotometry.

[0360] 12. Construction of VIGS carrier

[0361] The test materials were the control (CK) and two treatment groups (0.4 g / kg calcium application) of 'Black Leaf May Slow' and 'Shanghai June Slow' varieties. Specific sites of ACA10, with a length of 200-300 bp, were screened using the online software SGN-VIGS (vigs.solgenomics.nett) as target sites for VIGS. A pair of specific primers was designed for PCR amplification. Relevant gene CDS sequences were searched using BLAST on the NCBI website. Primer parameters were set on the Primer3Puls page, and the sequences were copied to NCBI, selecting primer sequences with 100% gene matching. The primer sequences are shown in SEQ ID NO.14 and SEQ ID NO.15. PCR products of 200-300 bp in length were obtained and ligated into the pTRV2 vector treated with restriction endonucleases BamHI and SamI to generate ACA10-pTRV2.

[0362] The target site of VIGS in the Chinese cabbage ACA10 gene is shown in SEQ ID NO.9.

[0363] 13. Agrobacterium-mediated transformation

[0364] Plasmids from correctly sequenced positive clones and the helper vector pTRV1 were transformed into Agrobacterium tumefaciens GV3101.

[0365] 14. VIGS infection methods

[0366] (1) Take 100ul of bacterial culture into a 50ml centrifuge tube and 15ml of triple antibody culture. Recover at 28℃ and 220r / min in a shaker for 48h (one tube each of pTRV1, pTRV2 with the target gene fragment, and PDS).

[0367] (2) 1 ml of bacterial culture was added to 100 ml of triple-antibiotic culture medium in a 250 ml Erlenmeyer flask and cultured on a shaker at 220 r / min for 24 h to promote large-scale propagation (pTRV1: 3 flasks; PDS: 1 flask; pTRV2-10: 2 flasks).

[0368] (3) Take out the conical flask and pour it into a 50ml centrifuge tube in two batches. Centrifuge at 4000r / min for 10min and discard the supernatant.

[0369] (4) Add 45 ml of buffer solution to each centrifuge tube and adjust the OD value to 0.8-1.0 at 600 nm using a UV spectrophotometer. If the OD value is too high, continue to add buffer solution.

[0370] (5) PDS, pTRV2-10 and pTRV1 are mixed in equal volumes at a 1:1 ratio.

[0371] (6) Wrap in tin foil and incubate in the dark for 3 hours.

[0372] (7) For the CK and 0.4 g / kg treatment groups, the 5th and 6th true leaves were injected on the back. The experiment was repeated three times.

[0373] (8) Mark the injected leaves, cover them with black bags, and treat them in the dark for 24 hours before normal cultivation.

[0374] 15. Virus detection and qRT-PCR detection

[0375] Albino leaves treated with pTRV2-ACA10 and leaves from pTRV1 empty vector plants at the same location were used to extract RNA using a kit. The RNA was then reverse-engineered into cDNA. PCR amplification was performed using specific primers to detect the presence of the virus. qRT-PCR was then performed using ACA10-F / -R quantitative primers to detect changes in ACA10 gene expression. Primer sequences are shown in Table 15.

[0376] Table 15 Primer sequences and names

[0377] Primer name Primer sequence (5'-3') pTRV2-F SEQ ID NO.10 pTRV2-R SEQ ID NO.11 pTRV1-F SEQ ID NO.12 pTRV1-R SEQ ID NO.13 VACA10-F SEQ ID NO.14 VACA10-R SEQ ID NO.15 Actin-F SEQ ID NO.16 Actin-R SEQ ID NO.17

[0378] 16. Determination of Ca content in Chinese cabbage leaves

[0379] Leaves from albino plants treated with pTRV2-ACA10 and leaves from pTRV1 uncultivated plants at the same location were rinsed thoroughly with tap water, followed by three rinses with distilled water. The fresh leaves were blanched at 105℃ for 1-2 hours, then dried at 60-80℃ to constant weight. 0.2g of the dried sample was weighed and the calcium content was determined by atomic absorption spectrophotometry, with the experiment repeated in triplicate.

[0380] 17. Primer synthesis and sequencing

[0381] Primers were synthesized and sequenced by Chongqing Qingke Biotechnology Co., Ltd.

[0382] 18. Statistical Analysis

[0383] Statistical processing of the data was performed using Microsoft Excel 2019. One-way ANOVA and Fisher's least significant difference (LSD) test were used in IBM SPSS Statistics 26.0 to compare the significance of differences in the means of different treatments (P<0.05). Graphs were plotted using Origin 2022.

[0384] II. Experimental Results and Analysis

[0385] 1. Functional study of the ACA10 gene in yeast

[0386] ① Gene-based yeast plate test

[0387] Yeast is a single-celled eukaryotic organism. Due to its excellent expression system, ease of efficient transformation, rapid reproduction, and short growth cycle, it is widely used as a model organism to study gene function, protein-protein interactions, and signal transduction pathways. Yeast experiments can rapidly and efficiently verify the function and regulatory mechanisms of target genes. Depending on the experimental requirements, different yeast strains are used. In this study, BY4741, a derivative strain of the S288C background, was used.

[0388] Growth of gene-modified Saccharomyces cerevisiae on plates, as follows Figure 9 As shown, with increasing calcium concentration, wild-type yeast strains and overexpressing yeast strains exhibited different characteristics. Under 200 mM CaCl2 treatment, the cell counts of both overexpressing yeast strains increased compared to the control group, but the growth of wild-type yeast strains remained unchanged. Under 300 mM CaCl2 treatment, the yeast strains ACA10-5-6 (Black Leaf May Slow) and ACA10-6-8 (Shanghai June Slow) were no longer observable, while wild-type yeast strains still showed white cells even under 300 mM CaCl2 and even 400 mM CaCl2 treatments. These results indicate that ACA10 can increase calcium transport capacity and enhance the yeast's sensitivity to calcium.

[0389] When the ACA10 gene is expressed in yeast, the protein it encodes enhances the transport capacity of calcium ions, helping cells cope with high calcium environments. At the same time, ACA10 enhances the calcium transport capacity of yeast through mechanisms such as active calcium ion transport, regulation of gene expression, response to stress, and membrane localization.

[0390] ② Growth curve of gene-modified yeast

[0391] ACA10-5-6 and wild-type yeast growth as Figure 10 As shown, yeast growth was relatively slow before 18 hours. As the growth time increased to 24 hours, the growth rate of yeast in all treatment groups increased dramatically. Wild-type yeast in the calcium-free medium grew the fastest, followed by wild-type yeast in the calcium-added medium, then yeast in the ACA10-5-6 calcium-free medium, and finally, yeast in the ACA10-5-6 medium with exogenous calcium added grew the slowest. When the yeast growth time reached approximately 32 hours, wild-type yeast in the calcium-free medium still grew the fastest, but the growth rate of yeast in the ACA10-5-6 calcium-free medium surpassed that of wild-type yeast in the calcium-added medium.

[0392] ACA10-6-8 and wild-type yeast growth as Figure 11 As shown, similar to ACA10-5-6, the yeast growth rate in each treatment group was relatively slow before 18 hours. Around 24 hours into growth, the wild-type yeast in the calcium-free medium still grew the fastest, followed by the ACA10-6-8 yeast in the calcium-free medium, then the wild-type yeast in the calcium-added medium, and finally the yeast in the medium containing the overexpression vector and added exogenous calcium grew the slowest. The results indicate that after exogenous calcium addition, in the control groups, the growth of the overexpressed yeast strains was significantly slower than that of the wild-type yeast strains. The overexpression vector-containing yeast showed enhanced calcium absorption capacity, with the order being ACA10-5-6 > ACA10-6-8 > blank strain.

[0393] After yeast is inoculated into a new environment, it needs time to adapt, undergo metabolic adjustments and enzyme synthesis, and then begins to grow. In this study, the yeast strain began to multiply rapidly after 18 hours. The reason why yeast begins to grow rapidly after 18 hours is due to the combined effects of factors such as adaptation period, nutrient consumption, accumulation of metabolic products, oxygen supply, cell cycle synchronization, environmental changes, and gene expression regulation.

[0394] ③ Determination of calcium content in gene-modified yeast

[0395] Results of yeast calcium content determination are as follows Figure 12As shown, without the addition of exogenous calcium, the calcium content of WT, ACA10-5-6, and ACA10-6-8 was determined, and the differences in calcium content were not significant. The calcium content of ACA10-5-6 was 1.2 percentage points higher than that of WT. When exogenous calcium was added, significant differences appeared among the treatment groups. Both ACA10-5-6 and ACA10-6-8 showed significant differences compared to the wild-type yeast strain. The calcium contents of ACA10-5-6 and ACA10-5-6 were 37.62 mg / kg and 26.88 mg / kg, respectively, which were 6.61 times and 4.72 times that of the wild-type yeast strain. In the culture medium without exogenous calcium addition, there was no significant difference in calcium content between the strain with the overexpression vector and the wild-type yeast empty vector. However, after the addition of exogenous calcium, the calcium content of ACA10-5-6 and ACA10-6-8 increased significantly, with ACA10-5-6 > ACA10-6-8.

[0396] Calcium has a cumulative effect in yeast strains. This invention measured the calcium content of gene-expressing yeast. The results showed that, without the addition of exogenous calcium, there was no significant difference in calcium content between wild-type and overexpressing yeast strains; however, under high-calcium treatment, the calcium content of the overexpressing yeast strains was significantly higher than that of the wild-type yeast strains. The difference between wild-type and overexpressing yeast strains under these two conditions is mainly because the overexpressed gene may encode calcium ion transport proteins (such as P-type ATPase or calcium ion channels). These proteins have enhanced activity under high-calcium conditions, enabling more efficient transport of calcium ions into the cell or organelles. Simultaneously, the overexpressed gene may alter the yeast's metabolic pathways, indirectly affecting the distribution or storage of calcium ions, leading to greater accumulation of calcium ions within the cell. This study found differences in the calcium accumulation capacity of the two overexpressing yeast strains. Yeast ACA10-5-6 had a stronger calcium accumulation capacity than yeast ACA10-6-8. This may be due to a combination of factors, including functional differences in the overexpressed gene, differences in calcium ion transport efficiency, differences in calcium ion storage capacity, differences in calcium signaling pathway response, differences in cellular stress response, differences in metabolic pathways, or differences in gene expression levels between the two Chinese cabbage varieties. The specific mechanism requires further analysis.

[0397] 2. Functional study of the ACA10 gene in Arabidopsis thaliana

[0398] ① Overexpression gene detection and phenotypic analysis

[0399] Previous studies have shown that the ACA10 gene can enhance the calcium accumulation capacity of yeast strains. Therefore, this invention transforms the ACA10 gene into Arabidopsis thaliana to further investigate its effect on the calcium accumulation capacity of Arabidopsis. Using wild-type Arabidopsis as a control, the expression level of ACA10 in Arabidopsis OE-1 and OE-2 strains overexpressing the ACA10 gene was analyzed. The results are as follows... Figure 13As shown, the relative expression levels of the ACA10 gene in the overexpression lines OE-1 and OE-2 were 136.58 times and 322.79 times that of wild-type Arabidopsis, respectively, indicating a significant difference between the overexpression and wild-type ACA10 gene expression levels. In this study, WT, OE-1, and OE-2 strains with similar growth were cultured in the same substrate and irrigated with nutrient solutions containing 0 and 20 mM CaCl2, respectively, for 21 days. It was found that WT, OE-1, and OE-2 showed consistent growth in the nutrient solution without CaCl2. However, phenotypic differences were observed in the nutrient solution containing 20 mM CaCl2. WT grew normally, while the growth of OE-1 and OE-2 was inhibited, with smaller rosette leaf area and smaller and fewer leaves. Furthermore, this study found that the calcium accumulation capacity of Arabidopsis thaliana was significantly correlated with the relative expression level of the gene; increased gene expression enhanced the calcium accumulation capacity. In conclusion, overexpression of the ACA10 gene in Arabidopsis thaliana enhances its calcium accumulation and transport capacity.

[0400] Calcium is an essential nutrient for plants, but excessive calcium absorption can negatively impact plant growth and development, leading to problems such as malformed new leaves, increased flower and fruit abscission, and stunted growth. This study found that after exogenous calcium supplementation, the overexpressing Arabidopsis lines (OE-1 and OE-2) exhibited significantly inhibited growth and reduced rosette leaf area compared to wild-type Arabidopsis. This may be because excessive calcium content in the substrate soil disrupts cell cycle regulation (such as the CDK-cyclin complex), downregulates auxin gene expression, and consequently reduces meristematic cell division, thus inhibiting Arabidopsis growth and development.

[0401] ② Determination of calcium content in Arabidopsis thaliana

[0402] like Figure 14 As shown, there were differences in calcium content in Arabidopsis leaves when 0 mM CaCl2 was added. Without exogenous calcium, the calcium content of OE-1 and OE-2 was 1.06 times and 1.11 times that of WT, respectively, showing a significant difference between WT and overexpressing OE-1 and OE-2. When 20 mM CaCl2 was added, the calcium content in Arabidopsis overexpressing OE-2 reached a maximum of 34.85 mg / kg, an increase of 58.18% compared to WT, showing a significant difference. Arabidopsis overexpressing OE-1 also showed a 45 percentage point increase in calcium content compared to WT. This indicates that overexpression of the ACA10 gene in Arabidopsis can indeed enhance its calcium accumulation and transport capacity.

[0403] Under high-calcium conditions, the calcium content in the overexpressing Arabidopsis thaliana lines (OE-1 and OE-2) was significantly higher than that in wild-type Arabidopsis thaliana. This is because increased gene expression directly increases the mRNA level of the protein, which in turn leads to the synthesis of more calcium transport proteins through translation, thereby improving transmembrane transport efficiency, enhancing intracellular calcium processing capacity, and improving overall calcium uptake. This result further demonstrates that ACA10 has the function of enriching and transporting calcium.

[0404] 3. Functional study of the ACA10 gene in VIGS

[0405] Currently, methods for studying the genome function of Chinese cabbage are relatively scarce, and the application of some emerging and well-developed technologies in Chinese cabbage is limited by factors such as technology, cost, and genetic background. For example, the complex genome and high genetic diversity of Chinese cabbage make epigenetic and protein-protein interaction network analysis difficult, greatly increasing the challenge. Furthermore, Chinese cabbage is a polyploid plant with a large number of repetitive sequences and homologous genes in its genome. Gene knockout may not completely eliminate functional redundancy because homologous genes may compensate for the function of the knocked-out gene. Additionally, the polyploid nature of Chinese cabbage makes phenotypic changes after gene knockout inconspicuous, making accurate gene function analysis difficult. Researchers have found that virus-induced gene silencing (VIGS) technology is significantly superior to other techniques in studying gene function in Chinese cabbage. VIGS is an RNA-mediated reverse genetics technique that has become an indispensable method for analyzing gene function, offering advantages such as short cycle time, low cost, and high throughput, with obvious phenotypic symptoms after silencing. To further investigate the function of the ACA10 gene in calcium transport in Chinese cabbage, this invention uses VIGS technology to silence the ACA10 gene by injecting a virus into Chinese cabbage leaves. The test results are as follows:

[0406] ①Detection of ACA10 gene silencing

[0407] The changes in the relative expression level and silencing efficiency of the ACA10 gene in the leaves of 'Black Leaf May Slow' Chinese cabbage were detected on days 7, 12, and 17, respectively. Figure 15As shown in the figure, on day seven, the relative expression level of the virus vector-injected leaves in the non-calcium treatment was 14.12% higher than that in the control group, with a silencing efficiency of 10.57%. When the calcium application rate was 0.4 g / kg, the relative expression level of the virus vector-injected leaves was 1.22 times that of the control group, with a silencing efficiency of 22.07%. On day twelve, the expression level of ACA10 in the cabbage leaves under both the non-calcium and calcium treatments showed significant changes compared to the control group, with a significant decrease and the silencing efficiency reaching its maximum. Under treatment A, the expression level of ACA10 decreased by 37.99% compared to the control group, with a silencing efficiency of 77.64%; under treatment B, the expression level of ACA10 decreased by 66.44% compared to the control group, with a silencing efficiency of 87.90%. On day 17, there was a difference in the expression level of ACA10 in cabbage leaves under the non-calcium treatment and the calcium treatment. The gene expression level in the control group was 19.44% and 14.08% higher than that under the viral vector injection, respectively. The silencing efficiency of the ACA10 gene at this time was lower than that of the two treatment groups on day 12, by 16.50 and 28.50 percentage points, respectively.

[0408] The changes in the relative expression level and silencing efficiency of the ACA10 gene were detected in the leaves of "Shanghai June Slow" cabbage on days 7, 12, and 17. Figure 16 As shown in the figure, on day seven, the relative expression levels of the ACA10 gene in both treatments were higher than those in the control group, being 1.04 times and 1.09 times higher, respectively, than those injected with the viral vector. The silencing efficiency of the ACA10 gene injected with the viral vector was 5.01 percentage points higher than that in the control group. On day twelve, under treatment A, the relative expression level of the ACA10 gene injected with the viral vector was 1.16 times that of the control group, while the gene expression level in treatment B was the opposite of that in treatment A. The silencing efficiency of the ACA10 gene in both treatments reached its maximum on day twelve, at 83.93% and 90.00%, respectively. On day seventeen, the gene expression level in the leaves injected with the viral vector under treatment A was 20.66 percentage points lower than that in the control group, while the gene expression level in the leaves injected with the viral vector under treatment B was 1.11 times that of the control group. The gene silencing efficiency at this time was lower than that in the second period, but higher than that in the first period, indicating that day twelve was the time when the silencing efficiency of the ACA10 gene was the highest. The reason is that VIGS relies on the systemic spread of the virus. Only when the virus spreads to a sufficient number of cells and triggers the RNA silencing mechanism will the silencing efficiency of the ACA10 gene gradually increase. Day 12 may be the time point when the virus spreads and the silencing mechanism reaches its peak. At the same time, environmental factors are also important factors affecting virus silencing. Among them, environmental conditions such as temperature, light, and humidity may affect the rate of virus replication and spread, thus affecting the dynamic changes in silencing efficiency. Day 12 may be the optimal matching point between virus infection and plant growth status.

[0409] ② Determination of Ca content after ACA10 gene silencing

[0410] The calcium content of ACA10-5-6 under different treatments was observed in three periods, as follows: Figure 17 As shown in the figure, the calcium content of leaves at three different time points under different treatments showed similar trends between the control group and the virus-vector-injected group. Under the same treatment, the calcium content of leaves in the control group was higher than that in the virus-vector-injected group. On day seven, under treatment A, the calcium content of leaves injected with the virus vector was 75.66 mg / kg, which was 8.31 percentage points lower than that of the control group; under treatment B, the calcium content of leaves in the control group was 3.46% higher than that in the virus-vector-injected group. On day twelve, the calcium content of the control group under treatment A was 100.47 mg / kg, which was 1.11 times that of the experimental group; under treatment B, the calcium content of leaves in the control group was 11.54 mg / kg higher than that in the experimental group. On day seventeen, under the two treatments, the calcium content of the control group was 6.01% and 8.61% higher than that in the experimental group, respectively.

[0411] The calcium content of ACA10-6-8 under different treatments was observed in three periods. Figure 18 As shown in the figure. Overall, under the same treatment, the calcium content of the control group leaves was higher than that of the virus-vector-injected leaves. On day 7, under treatment A, the calcium content of the control group leaves was 1.05 times that of the virus-vector-injected leaves; under treatment B, the calcium content of the virus-vector-injected leaves was 74.47 mg / kg, which was 3.29 mg / kg lower than that of the control group. On day 12, under treatment A, the calcium content of the control group leaves was 89.49 mg / kg, which was 5.56% higher than that of the experimental group; under treatment B, the calcium content of the control group leaves was 1.06 times that of the experimental group. On day 17, the calcium content of the control group leaves under the two treatments was 99.68 mg / kg and 104.31 mg / kg, respectively, and the calcium content of the experimental group leaves was 4.98% and 4.70% lower than that of the control group, respectively.

[0412] The results above show that when ACA10 was silenced by the virus in cabbage leaves, the calcium content was significantly lower than that in the control group. This confirms that ACA10 is a calcium-containing compound. 2+ -ATPase, or ACA10, is responsible for transporting calcium ions from the cytoplasm to the extracellular space or into organelles, maintaining intracellular calcium homeostasis. When ACA10 is silenced by a virus, its function is inhibited, leading to a decrease in calcium ion transport capacity, accumulation of intracellular calcium ions, and reduced efflux, ultimately resulting in a decrease in calcium content in the leaves.

Claims

1. A gene comprising the nucleotide sequence shown in SEQ ID NO:18, characterized in that, The gene is derived from Chinese cabbage.

2. A gene comprising the nucleotide sequence shown in SEQ ID NO:19, characterized in that, The gene is derived from Chinese cabbage.

3. A gene comprising the nucleotide sequence shown in SEQ ID NO:18 or SEQ ID NO:19, characterized in that, The gene is expressed on the cell membrane and participates in calcium ion transport.

4. The gene according to claim 3, characterized in that, The gene, when overexpressed, can enhance calcium ion transport in yeast or Arabidopsis thaliana.

5. A vector comprising the gene according to any one of claims 1-4.

6. A host cell containing the vector of claim 5.

7. A protein expressed by the host cell of claim 6.

8. The protein according to claim 7, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO:20 or SEQ ID NO:

21.

9. A method for cloning the gene according to any one of claims 1-4, characterized in that, Includes the following steps: 1) Extract genomic DNA from Chinese cabbage; 2) PCR amplification is performed using specific primers to obtain the amplification product; 3) Purify the amplification product and ligate it into a vector; 4) Transform the vector into the host cell; The primers include the forward primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.

2.

10. A method for expressing the gene according to any one of claims 1-4, characterized in that, Includes the following steps: S1: Transform the vector according to claim 5 into host cells; S2: Culture the host cells in a suitable culture medium; S3: Collect the expression products.

11. A method for silencing the gene according to any one of claims 1-4 using virus-induced gene silencing technology, characterized in that, Includes the following steps: a. Design specific primers for the gene according to any one of claims 1-4; b. Amplify the target sequence using the specific primers and insert it into the VIGS vector; c. Transform the VIGS vector containing the target sequence into Agrobacterium; d. Introduce the VIGS vector into Chinese cabbage cells via Agrobacterium infection; e. Observe and detect the gene silencing effect.

12. The use of the gene according to any one of claims 1-4 in improving the calcium ion transport capacity and calcium ion accumulation capacity of plants and / or cells.

13. The application according to claim 12, characterized in that, It is used to improve the calcium ion transport capacity and the absorption and accumulation capacity of calcium ions in Arabidopsis thaliana and / or yeast.

14. The application according to claim 12, characterized in that, Overexpression of the ACA10 gene enhances the plant's ability to absorb and transport calcium ions, thereby improving plant growth or stress resistance.

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