A novel gene, LCS7, that regulates plant calcium sensitivity and its applications.

Abstract

This invention belongs to the field of plant genetic engineering and discloses... LCS7 The application of genes or their encoded proteins in regulating plant sensitivity to low calcium levels, wherein the plants are Arabidopsis thaliana and tomato. LCS7 The nucleotide sequences of the gene are shown in Arabidopsis thaliana SEQ.ID.NO.1 and tomato SEQ.ID.NO.4, respectively. This gene encodes a pyrophosphate hydrolase that regulates cytoplasmic pyrophosphate homeostasis, thereby controlling leaf growth under low calcium conditions. This invention provides a gene resource with potential for genetic improvement, laying the foundation for breeding low-calcium tolerant crop varieties and providing important evidence for further research on the molecular mechanism of low-calcium-induced blossom-end rot in tomatoes.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, specifically to a novel gene regulating plant sensitivity to low calcium levels. LCS7 And its applications. Background Technology

[0002] Calcium is an essential macronutrient for plant growth and development, playing a vital role. It is not only a key component of cell walls and cell membranes but also acts as a core signaling molecule in the cytoplasm, participating in various physiological processes. Calcium deficiency is widespread in agricultural production, especially in vegetables and fruit trees. In leafy vegetables, calcium deficiency often manifests as heart rot, yellowing edges, and leaf tip necrosis, typical examples being heart rot in Chinese cabbage and black rot in celery. In solanaceous crops, calcium deficiency leads to browning and sunken spots at the fruit tip, most commonly blossom-end rot in tomatoes and peppers. When the calcium concentration is below 0.29 mM, approximately 53% of tomato fruits develop blossom-end rot. Furthermore, calcium deficiency in fruit trees can cause fruit deformities, cracking, and sunken spots on the peel, such as bitter pit in apples and fruit cracking in citrus.

[0003] Whether a plant is calcium deficient depends on both the available calcium content in the soil and the plant's own absorption and translocation capacity. According to the median calcium concentration in the topsoil layer (0-25 cm) nationwide, only 1.96% of the soil contains available calcium. In arid regions, although the total amount of calcium in the soil is relatively high, the available calcium content is low due to the large proportion of mineral calcium. In humid regions, available calcium is easily lost due to leaching from rainfall and acidic ion exchange, and plants in these areas often face the risk of calcium deficiency. Unlike other nutrients, plant calcium deficiency is not solely caused by insufficient soil calcium content but is also influenced by various environmental factors. Due to the difficult redistribution of calcium, plant tissues can only obtain calcium through transpiration; therefore, tissues with weaker transpiration are more prone to calcium deficiency symptoms. Any factor affecting transpiration, such as rainy weather or high temperatures and drought, can trigger calcium deficiency in plants. Furthermore, potassium (K)... + Mg 2+ and NH4 + isocations and Ca 2+ Antagonistic effects can also affect plant calcium absorption. Therefore, even if the soil is not deficient in calcium, plants may still exhibit calcium deficiency symptoms. Because the causes of calcium deficiency in plants are complex, fertilization alone is often insufficient to completely solve the problem. Therefore, breeding low-calcium-tolerant crop varieties has become a feasible and promising strategy for preventing calcium deficiency in plants. In the efficient molecular breeding of low-calcium-tolerant crops, identifying and analyzing the genes regulating plant sensitivity to low calcium levels is particularly important.

[0004] Currently, studies have reported some genes associated with plant growth in low-calcium environments. These genes are mainly involved in calcium absorption and transport, as well as cell wall synthesis. For example, vacuolar calcium... 2+ / H + The retrotransporter (CAX1) gene plays a crucial role in calcium transport between the cytoplasm and vacuoles, and overexpression of this gene exacerbates calcium deficiency symptoms in plants (de Freitas S, Padda M, Wu Q, et al. Dynamic alternations in cellular and molecular components during blossom-end rot development in tomatoes expressing sCAX1, a consistently active calcium transporter). 2+ / H + Antiporter from arabidopsis[J]. Plant physiology, 2011, 156(2): 844-855), while its mutants showed stronger tolerance to low calcium. In addition, genes related to cell wall synthesis, such as the GSL family of genes related to callose synthesis, are also involved. gsl1 , gsl8 and gsl10The mutant exhibits defective new leaf growth under low calcium conditions, accompanied by severe cell death (e.g., Shikanai Y, Yoshida R, Hirano T, et al. Callose Synthesis Suppresses Cell Death Induced by Low-Calcium Conditions in Leaves[J]. Plant Physiology, 2020, 182(4): 2199-2212; and Shikanai Y, Takahashi S, Enomoto Y, et al. Arabidopsis Glucan Synthase-Like1 (GSL1) Is Required for Tolerance to Low-Calcium Conditions and Exhibits a Function Comparable to GSL10[J]. Plant and Cell Physiology, 2022, 63(10): 1474-1484; and Shikanai Y, Asada M, Sato T, et al. Role of GSL8 in low calcium tolerancein Arabidopsis thaliana[J]. Plant Biotechnology, 2022, 39(3): 221-227). However, although these genes play an important role in the low calcium sensitivity phenotype of plants, their specific mechanisms of action are not fully understood, and their potential in genetic improvement has not been fully explored and applied. Therefore, their application in actual production is relatively limited in the near future. Summary of the Invention

[0005] The purpose of this invention is to provide LCS7 The application of genes or their encoded proteins in regulating plant calcium sensitivity, by providing a novel gene with genetic improvement potential and capable of regulating plant calcium sensitivity. LCS7 It and its encoded protein enable its application in regulating low calcium sensitivity in Arabidopsis and tomato.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] LCS7 Application of genes or their encoded proteins in regulating plant sensitivity to low calcium levels.

[0008] In this invention, low calcium means a calcium concentration of less than 0.3 mM.

[0009] Furthermore, Arabidopsis thaliana LCS7 The application of genes or their encoded proteins in regulating plant sensitivity to low calcium levels, in the Arabidopsis thaliana. LCS7 The coding region nucleotide sequence of the gene is shown in SEQ.ID.NO.2, referring to the Arabidopsis thaliana. LCS7 The amino acid sequence of the protein encoded by the gene is shown in SEQ.ID.NO.3.

[0010] This invention discovered a low-calcium hypersensitive mutant through forward genetic screening in Arabidopsis thaliana. lcs7 ( low- Calcium sensitive 7 This mutant exhibits cessation of new leaf growth under low calcium (0.2 mM Ca) conditions, but normal calcium absorption and transport capabilities. This mutant provides a novel strategy and genetic resource for improving plant sensitivity to low calcium. Through BSA (Bulked Segregant Analysis) resequencing localization, gene complementation, and gene knockout verification, the key genes controlling plant sensitivity to low calcium were identified. LCS7 The gene ID is AT1G15690, and it is annotated as vacuolar inorganic pyrophosphatase AVP1 (Arabidopsis vacuolar H + -pyrophosphatase 1).

[0011] Furthermore, tomatoes SlLCS7 The application of genes or their encoded proteins in regulating plant sensitivity to low calcium levels, in the tomato variety. SlLCS7 The coding region nucleotide sequence of the gene is shown in SEQ.ID.NO.5, referring to the tomato. SlLCS7 The amino acid sequence of the gene-encoded protein is shown in SEQ.ID.NO.6.

[0012] This invention identifies a gene that regulates calcium sensitivity in tomatoes. SlLCS7 The gene ID is Sloyc07G007600. Furthermore, the gene in tomatoes was knocked out using CRISPR / Cas9 technology. SlLCS7 The gene was used to verify its role in regulating the low calcium sensitivity of tomatoes.

[0013] Furthermore, through overexpression LCS7 Genes or their encoded proteins reduce the plant's sensitivity to low calcium stress, that is, increase tolerance to low calcium stress or resistance to calcium stress.

[0014] LCS7 Genes or their encoded proteins mediate pyrophosphate homeostasis, regulating leaf growth in plants under low calcium conditions. This invention, through... lcs7Low-calcium phenotype and pyrophosphate detection in mutants and transgenic lines that specifically clear cytoplasmic pyrophosphate revealed that LCS7-mediated pyrophosphate homeostasis regulates leaf growth under low-calcium conditions.

[0015] The plant in question is either Arabidopsis thaliana or tomato.

[0016] The present invention also provides LCS7 The application of genes or their encoded proteins in plant breeding, wherein, when applying, screening for overexpression of the genes or proteins is performed. LCS7 Plants containing genes or their encoded proteins can be used to obtain low-calcium tolerant crops.

[0017] Furthermore, Arabidopsis thaliana expressing the gene were screened. LCS7 The plant containing the gene or the protein it encodes, said Arabidopsis thaliana LCS7 The coding region nucleotide sequence of the gene is shown in SEQ.ID.NO.2, referring to the Arabidopsis thaliana. LCS7 The amino acid sequence of the protein encoded by the gene is shown in SEQ.ID.NO.3.

[0018] Furthermore, screening for expression-enhancing tomatoes SlLCS7 The tomato plant containing the gene or the protein it encodes. SlLCS7 The coding region nucleotide sequence of the gene is shown in SEQ.ID.NO.5, referring to the tomato. SlLCS7 The amino acid sequence of the gene-encoded protein is shown in SEQ.ID.NO.6.

[0019] Furthermore, the plant is Arabidopsis thaliana or tomato.

[0020] Compared with existing technologies, this invention is the first to discover, locate, and clone a novel gene that regulates the low calcium sensitivity of plants. LCS7 Functional studies were conducted. This invention relates to Arabidopsis thaliana. LCS7 The low calcium phenotype and pyrophosphate accumulation in overexpressed transgenic lines were verified; the results showed that overexpression LCS7 It can significantly restore the growth of new leaves under extremely low calcium (0.1 mM Ca) conditions and reduce low-calcium-induced pyrophosphate accumulation, indicating that LCS7-mediated pyrophosphate homeostasis has the potential for genetic improvement in regulating calcium sensitivity. Furthermore, this invention utilizes CRISPR / Cas9 technology to knock out tomatoes... SlLCS7 Genetic verification revealed that the absence of SlLCS7 leads to the death of new leaves and the outbreak of blossom-end rot in fruits under low calcium conditions.

[0021] Therefore, this invention regulates the cytoplasmic pyrophosphate level by genetically manipulating the expression of LCS7, thereby controlling the plant's low calcium sensitivity, and has the potential for genetic improvement. Attached Figure Description

[0022] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0023] Figure 1 Wild-type Col-0 and point mutant from Example 1 lcs7 Statistical results of aboveground phenotype and biomass after 14 days of low calcium treatment, scale bar 1 cm;

[0024] Figure 2 Wild-type Col-0 and point mutant from Example 1 lcs7 Phenotypic and quantitative analysis of new leaf death after 14 days of low calcium treatment, scale bar 1 mm;

[0025] Figure 3 Wild-type Col-0 and point mutant from Example 1 lcs7 Comparison of calcium content in the aboveground parts after 14 days of low calcium treatment;

[0026] Figure 4 As in Example 2, based on lcs7 Distribution of single nucleotide polymorphisms (SNPs) in ×Col-0 F2 population cluster segregation analysis (BSA);

[0027] Figure 5 Arabidopsis thaliana in Example 2 LCS7 A schematic diagram of the (AT1G15690) gene structure, and lcs7 (L753F) lcs7-6 CR and lcs7-33 CR The specific mutation type and location of the mutant;

[0028] Figure 6 Wild-type Col-0 and knockout mutant in Example 3 lcs7-6 CR , lcs7-33 CR Statistical results of aboveground phenotype and biomass after 14 days of low calcium treatment, scale bar 1 cm;

[0029] Figure 7 The wild-type Col-0 and knockout mutants in Example 4 lcs7-6 CR The aboveground phenotypic and biomass statistics of its complementary transgenic lines after 14 days of low calcium treatment, scale bar 1 cm;

[0030] Figure 8 Example 5: Wild-type Col-0 and point mutant lcs7 Pyrophosphate content in the aboveground parts after 14 days of low calcium treatment;

[0031] Figure 9The transgenic line expressing yeast pyrophosphate hydrolase IPP1 in Example 5 recovered after 14 days of low calcium treatment. lcs7 Growth of new leaves and pyrophosphate content in the aboveground parts of the mutant;

[0032] Figure 10 As a control in Example 6 EV Knockout mutants lcs7-6 CR and overexpression LCS7 Western blot results of LCS7 protein detection in transgenic lines;

[0033] Figure 11 As a control in Example 6 EV With overexpression LCS7 Statistical results of aboveground phenotype and biomass of transgenic lines after 14 days of low calcium treatment, scale bar 1 cm;

[0034] Figure 12 Tomato in Example 7 SlLCS7 (Solyc07g007600) Gene Structure Diagram sllcs7-8 CR , sllcs7-14 CR The specific mutation type and location of the knockout mutant, and sllcs7-8 CR , sllcs7-14 CR In knockout mutants SlLCS7 The level of expression;

[0035] Figure 13 The wild-type WT and tomato knockout mutants in Example 7 sllcs7-8 CR , sllcs7-14 CR Phenotypic analysis of necrotic spots on new leaves, statistical analysis of dead area, and pyrophosphate content after 21 days of low calcium treatment, scale bar 1 cm;

[0036] Figure 14 The wild-type WT and tomato knockout mutants in Example 7 sllcs7-8 CR , sllcs7-14 CR Phenotypic and frequency of blossom-end rot in fruit after 45 days of low calcium treatment, scale bar 1 cm.

[0037] Figure 15 For the purpose of constructing complementary lines pLCS7:LCS7 , pLCS7:IPP1 Vectors and overexpression lines p35s: LCS7 and pUBQ10:LCS7A schematic diagram of the EcoRI restriction site of the pGreen0229 binary vector. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0039] Example 1: Arabidopsis thaliana low-calcium hypersensitive mutant lcs7 Acquisition and aboveground phenotypic analysis

[0040] In this embodiment, Arabidopsis thaliana Col-0 was used as the material. The seeds of Arabidopsis thaliana Col-0 were treated with the chemical mutagen ethyl methanesulfonate (EMS). Through its alkylation effect on DNA bases, point mutations were randomly induced throughout the genome, thereby obtaining a population with extensive genetic variation. The EMS-mutated population was then screened for low-calcium-sensitive mutants under 0.2 mM Ca conditions, resulting in a low-calcium-sensitive hypersensitive mutant. lcs7 ( low- calcium sensitive 7 ).

[0041] The low-calcium hypersensitive mutant obtained above lcs7 Wild-type Col-0 seeds were treated for 14 days in Yamagami medium containing normal calcium (2 mM Ca) and low calcium (0.2 mM Ca). Observations using a camera or stereomicroscope, along with statistical analysis of aboveground biomass and new leaf mortality, revealed that under low calcium conditions... lcs7 The aboveground growth of the mutant was significantly inhibited. Figure 1 Furthermore, the new leaves showed obvious necrosis and wilting, and were unable to unfold normally. Figure 2 ).

[0042] To investigate the association between phenotype and calcium transport, another experiment was conducted: the low-calcium hypersensitive mutants obtained above were... lcs7 Wild-type Col-0 was sown on Yamagami medium containing normal calcium (2 mM Ca) and low calcium (0.3 mM Ca). After 14 days of treatment, 0.2 g of aerial samples were collected, dried, and then digested with concentrated nitric acid at high temperature to prepare a clear digestion solution. The calcium content was determined by MP-AES. The results showed that, regardless of whether the calcium conditions were normal or low, lcs7 The calcium content in the aboveground parts of the mutant was not significantly different from that of the wild-type Col-0. Figure 3 This indicates that the stunted growth of its new leaves was not caused by abnormal calcium transport.

[0043] Example 2: Genes regulating plant low calcium sensitivity LCS7 Positioning

[0044] The one obtained in Example 1 lcs7 The F2 population, derived from the self-pollination of the F1 generation obtained by crossing the mutant with the Col-0 wild-type, was used as the material. Under 0.2 mM Ca conditions, resistant individuals (35 plants) with fully expanded true leaves and sensitive individuals (34 plants) with severely inhibited true leaf growth were screened based on the true leaf growth phenotype of individuals in the F2 population. Genomic DNA was extracted from the aboveground parts of each individual plant using the FastPure Plant DNA Isolation Mini Kit. The DNA from the 35 resistant individuals was then mixed in equal amounts to construct a resistance pool, and the DNA from the 34 sensitive individuals was mixed in equal amounts to construct a sensitivity pool for subsequent sequencing analysis. DNA library construction and high-throughput sequencing were performed by Shanghai Ouyi Biotechnology Co., Ltd., using the Illumina NovaSeq 6000 sequencing platform. Library construction was performed using the Illumina TruSeq DNA LT Sample Prep kit. The raw sequencing data were first subjected to a quality control process to remove low-quality reads, and the remaining clean reads were aligned with the Arabidopsis thaliana reference genome TAIR10. Based on the alignment results, the proportion of reads containing mutant alleles at each locus was calculated and defined as the SNP index. The difference in SNP index (ΔSNPindex) between the sensitive and resistant pools was further calculated to reflect the allele frequency difference at this locus between the two extreme phenotype populations. Finally, based on a sliding window analysis method (window size 1 Mb, step size 100 kb), a sliding average of the ΔSNP index across the entire genome was calculated, and the results were presented in the form of a Manhattan plot. Figure 4 At the same time, show the most likely ones. LCS7 The gene structure of candidate gene AT1G15690, and lcs7 Point mutation type, where the amino acid at position 753 changes from leucine to phenylalanine (L753F) Figure 5 ).

[0045] Example 3: Obtaining the Arabidopsis LCS7 knockout mutant and analyzing its low-calcium phenotype in its aboveground parts

[0046] Using the "target Design" function of the online website CRISPR-GE (http: / / skl.scau.edu.cn / ), a study was conducted targeting Arabidopsis thaliana. LCS7Candidate gene AT1G15690 was used to design specific targets, and targets located in exon regions with GC content of 45-70% and predicted low off-target rates were screened. Using the "primer design" function on the website, appropriate AtU3 / U6 promoters and primer design methods were selected, primers were automatically generated and synthesized by Shanghai Sangon Biotech. The primer sequences used are shown in SEQ.ID.NO.7 and SEQ.ID.NO.8. Overlapping PCR was used to ligate the U3 / U6 promoter and the target. Then, using Golden Gate technology, the fragment containing the AtU3 / U6 promoter driving the target was inserted into the pYLCRISPR / Cas9-H linear vector digested with Bsa I-HF. After sequencing confirmed, Arabidopsis thaliana was analyzed. LCS7 Knockout vector construction. The vector was transformed into Agrobacterium GV3101 and wild-type Col-0 was transformed using the Arabidopsis thaliana flower-dipping method to obtain T0 generation plants. After harvesting T0 generation seeds, positive seedlings (T1 generation) were obtained through hygromycin resistance selection. Self-pollination of positive T1 generation plants produced T2 generation plants, which were then verified by DNA sequencing. LCS7 Gene editing of the target region was assessed. Two homozygous mutants with different editing types were screened from the T2 generation and named... lcs7-6 CR and lcs7-33 CR ( Figure 5 The above-mentioned knockout mutant and wild-type Col-0 were sown on Yamagami medium containing normal calcium (2 mM Ca) and low calcium (0.2 mM Ca). After 14 days of culture, photographic observation and aboveground biomass statistics showed that the knockout mutant in wild-type Col-0... LCS7 Genes can produce and lcs7 The point mutant exhibits a similar low-calcium-sensitive phenotype, with new leaf growth ceasing under low-calcium conditions. Figure 6 ).

[0047] Example 4: Arabidopsis thaliana LCS7 Obtaining complementary transgenic lines and analyzing their low-calcium phenotype in aboveground parts

[0048] Primers were designed from the wild-type Arabidopsis thaliana Col-0 genomic DNA, with primer sequences shown in SEQ.ID.NO.9 and SEQ.ID.NO.10, to amplify a fragment from 4400 bp upstream of the start codon to the stop codon of the LCS7 gene (i.e., the pLCS7:LCS7 fragment). Using a one-step cloning method, this fragment was inserted into the pGreen0229 linear vector digested with EcoRI-HF. Verification was achieved through enzyme digestion and sequencing. pGreen0229-pLCS7:LCS7 The vector was constructed correctly. The vector was then transformed into the Agrobacterium GV3101 strain, and infected using the Arabidopsis thaliana flower-dipping method. lcs7-6 CRKnockout mutants were used to obtain T0 generation plants. After harvesting seeds from the T0 generation, seeds containing red fluorescence were screened using the RFP channel of a stereomicroscope; these were designated as the T1 generation. After self-pollination of the T1 generation to produce the T2 generation, the seed fluorescence segregation ratio was calculated. Single-copy insertion complementary transgenic lines with a segregation ratio of 3:1 were selected and planted individually. The obtained complementary transgenic lines ( pLCS7:LCS7 / lcs7-6 CR #1 and pLCS7:LCS7 / lcs7-6 CR #3 ), lcs7-6 CR Knockout mutants and Col-0 wild-type were simultaneously sown on Yamagami medium containing normal calcium (2 mMCa) and low calcium (0.2 mMCa). After 14 days of culture, photographic observation and aboveground biomass statistics revealed that: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] pLCS7:LCS7 The fragment can be fully recovered. lcs7- 6 CR New leaf growth of mutants under low calcium conditions ( Figure 7 Combining the results of Example 3 with those of this example, it is shown that... LCS7 Genes are crucial for the growth of new leaves in Arabidopsis thaliana under low calcium conditions.

[0049] Example 5: Arabidopsis thaliana LCS7 Functional analysis of regulation of low calcium sensitivity

[0050] To verify the gene function of LCS7 in regulating the growth of low-calcium leaves, this example used an ultrasensitive pyrophosphate assay kit and a microplate reader to detect the levels of Col-0 wild-type and low-calcium leaf growth. lcs7 The pyrophosphate content in the aboveground parts of the mutant under low calcium conditions was analyzed, and the results showed that, compared with the wild type, it was lower under normal calcium conditions. lcs7 Pyrophosphate accumulates in the mutant, while low calcium leads to... lcs7 The accumulation of pyrophosphate is more severe in the middle. Figure 8 Furthermore, in this embodiment, primers were designed to amplify genomic DNA from wild-type Arabidopsis thaliana Col-0. LCS7 The IPP1 coding region sequence was amplified from the 4400 bp upstream of the start codon and from a yeast cDNA library. Primer sequences used are shown in SEQ.ID.NO.9, SEQ.ID.NO.11, SEQ.ID.NO.12, and SEQ.ID.NO.13. A one-step cloning method was used to ligate the above two fragments to the pGreen0229 linear vector digested with EcoRI-HF. Enzyme digestion verification and sequencing confirmed the sequence. pGreen0229-pLCS7:IPP1. The vector was constructed correctly. The above vector was transformed into Agrobacterium GV3101, and wild-type Col-0 was transformed using the Arabidopsis thaliana flower-dipping method to obtain T0 generation plants. After the T0 generation seeds were harvested, seeds containing red fluorescence were screened using the RFP channel of a stereomicroscope (T1 generation); after the T1 generation self-pollinated to produce the T2 generation, the fluorescence segregation ratio was calculated, and single-copy insertion overexpression transgenic lines meeting the 3:1 ratio were selected and individual plants were harvested for seed. The pGreen0229-pLCS7:IPP1 transgenic line (…) was obtained. pLCS7: IPP1 / lcs7 #1 and pLCS7: IPP1 / lcs7 #2 Yeast seeds were sown on Yamagami medium with normal calcium (2 mMCa) and low calcium (0.2 mMCa). After 14 days of culture, the aboveground parts were photographed and the pyrophosphate content was measured. The results showed that the expression of yeast pyrophosphate hydrolase IPP1 was significantly restored. lcs7 The mutant showed increased new leaf growth and reduced pyrophosphate accumulation after 14 days of low calcium treatment. Figure 9 In summary, this embodiment demonstrates the main function of LCS7 as a pyrophosphate hydrolase in promoting new leaf growth under low calcium conditions.

[0051] Example 6: Arabidopsis thaliana LCS7 Obtaining transgenic lines with overexpression and analyzing their low-calcium phenotype in aboveground parts

[0052] Primers were designed to amplify from a wild-type Arabidopsis thaliana cDNA library. LCS7 The nucleotide sequence of the gene coding region, and the primer sequences used are shown in SEQ.ID.NO.11, SEQ.ID.NO.14, and SEQ.ID.NO.15. A one-step cloning method was used to insert this fragment, along with a 35S or UBQ10 promoter fragment, into the pGreen0229 linear vector digested with EcoRI-HF. Enzyme digestion verification and sequencing confirmed the correct construction of the pGreen0229-p35S / UBQ10:LCS7 vector. This vector was transformed into the Agrobacterium GV3101 line, and wild-type Col-0 was transformed using the Arabidopsis thaliana flower-dipping method to obtain T0 generation plants. After harvesting seeds from the T0 generation, seeds containing red fluorescence (T1 generation) were screened using the RFP channel of a stereomicroscope. After self-pollination of the T1 generation to produce the T2 generation, the fluorescence segregation ratio was calculated, and single-copy overexpression transgenic lines meeting a 3:1 ratio were selected and harvested individually. The obtained single-copy overexpression transgenic lines were subjected to Western blotting to determine the LCS7 protein level, in order to clarify the abundance of LCS7 protein in different overexpression transgenic lines. Figure 10 The obtained overexpression transgenic lines ( p35s:LCS7 / Col-0 #1 and #2 , pUBQ10:LCS7 / Col-0 #3 and #4The pGreen0229 empty transgenic line was simultaneously sown on Yamagami medium containing normal calcium (2 mMCa) and ultra-low calcium (0.1 mMCa). After 14 days of culture, photographs were taken, aboveground biomass was counted, and pyrophosphate content was measured. It was found that the overexpression transgenic line could significantly alleviate the growth inhibition of new leaves under low calcium conditions and reduce the accumulation of low calcium-induced pyrophosphate. Figure 11 This indicates that regulation is achieved through genetic manipulation. LCS7 Gene expression can improve a plant’s sensitivity to low calcium levels.

[0053] Among them, Examples 4-6 are used to construct complementary lines. pLCS7:LCS7 , pLCS7:IPP1 Vectors and overexpression lines p35s:LCS7 and pUBQ10:LCS7 The EcoRI restriction sites of the pGreen0229 binary vector are as follows: Figure 15 As shown.

[0054] Example 7: Tomato SlLCS7 Obtaining knockout mutants and analyzing their low-calcium phenotype in newly formed tissues

[0055] Using the CRISPR-GE online website (http: / / skl.scau.edu.cn / ) to target tomatoes SlLCS7 Two specific targets were designed using the primer sequences shown in SEQ.ID.NO.16 and SEQ.ID.NO.17. The synthesized target sequences were annealed and ligated into a vector containing the AtU3 / U6 promoter. The resulting AtU3 / AtU6-sgRNA fragment was then ligated into the BGK012 vector, and confirmed by sequencing. SlLCS7 The gene knockout vector was constructed correctly. The knockout vector was transformed into the Agrobacterium GV3101 strain using the tomato cotyledon infection method. Tissue culture seedlings (T0 generation) were obtained through callus induction, resistance induction differentiation, and rooting culture. After self-pollination of the T0 generation, T1 generation seeds were harvested. SlLCS7 DNA sequencing of the target region identified two different types of editing. sllcs7 Homozygous mutants were named respectively. sllcs7-8 CR and sllcs7-14 CR ( Figure 12 ).Will sllcs7-8 CR , sllcs7- 14 CR Knockout mutants and wild-type (WT) tomatoes were hydroponically cultured for 21 days in Hoagland nutrient solution with normal calcium (1 mMCa) and low calcium (0.03 mMCa), respectively. Statistical analysis of necrotic spots on new leaves and pyrophosphate content testing revealed that under low calcium conditions… SlLCS7Gene deletion causes numerous necrotic spots to appear on new tomato leaves, and compared to the wild type... sllcs7 More pyrophosphate accumulates under low calcium conditions. Figure 13 Furthermore, the three fruits were hydroponically cultured in Hoagland nutrient solutions with normal calcium (1 mMCa) and low calcium (0.1 mMCa) for 45 days, respectively. Observation through photography and frequency statistics of blossom-end rot on the fruit revealed that under low calcium conditions… SlLCS7 Gene deletion led to an outbreak of blossom-end rot in tomato fruits. Figure 14 The results of this embodiment show that... LCS7 The mechanism by which genes regulate plant low calcium sensitivity is conserved in plants. This mechanism can be effectively applied to tomatoes, providing a clear target and feasible pathway for improving the low calcium tolerance of tomatoes.

[0056] The foregoing illustrative description illustrates the invention and its embodiments. Obviously, the invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of this invention should be considered within the scope of protection of this invention.

Claims

1. The use of Arabidopsis LCS7 gene or its encoded protein in regulating the low calcium sensitivity of plants, characterized in that, The nucleotide sequence of the coding region of the Arabidopsis LCS7 gene is shown as SEQ. ID. NO. 2, and the amino acid sequence of the protein encoded by the Arabidopsis LCS7 gene is shown as SEQ. ID. NO. 3; and the plant is Arabidopsis.

2. Use of the tomato SlLCS7 gene or the protein encoded by the same for modulating the sensitivity of a plant to low calcium, characterized in that, The nucleotide sequence of the coding region of the tomato SlLCS7 gene is shown as SEQ. ID. NO. 5, and the amino acid sequence of the protein encoded by the tomato SlLCS7 gene is shown as SEQ. ID. NO. 6; and the plant is tomato.

3. Use according to claim 1, characterized in that, The low calcium sensitivity of Arabidopsis is reduced by overexpressing the Arabidopsis LCS7 gene or the protein encoded thereby.

4. Use according to claim 2, characterized in that, The low calcium sensitivity of tomato is reduced by overexpressing the tomato SlLCS7 gene or the protein encoded thereby.

5. Use according to claim 3, characterized in that, The pyrophosphate homeostasis mediated by the Arabidopsis LCS7 gene or the protein encoded thereby regulates the leaf growth of Arabidopsis under low calcium condition.

6. Use according to claim 4, characterized in that, The pyrophosphate homeostasis mediated by the tomato SlLCS7 gene or the protein encoded thereby regulates the leaf growth of tomato under low calcium condition.

7. Use of the Arabidopsis LCS7 gene or the protein encoded by the same in plant breeding, characterized in that, The low calcium tolerant plant is obtained by screening the plant overexpressing the Arabidopsis LCS7 gene or the protein encoded thereby; the nucleotide sequence of the coding region of the Arabidopsis LCS7 gene is shown as SEQ. ID. NO. 2, and the amino acid sequence of the protein encoded by the Arabidopsis LCS7 gene is shown as SEQ. ID. NO. 3; and the plant is Arabidopsis.

8. Use of the tomato SILCS7 gene or the protein encoded by it in plant breeding, characterized in that, The low calcium tolerant plant is obtained by screening the plant overexpressing the tomato SlLCS7 gene or the protein encoded thereby; the nucleotide sequence of the coding region of the tomato SlLCS7 gene is shown as SEQ. ID. NO. 5, and the amino acid sequence of the protein encoded by the tomato SlLCS7 gene is shown as SEQ. ID. NO. 6; and the plant is tomato.

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