Novel gene LCS7 for regulating and controlling low-calcium sensitivity of plant and application thereof

Abstract

The invention belongs to the field of plant genetic engineering, and discloses application of an LCS7 gene or a protein coded by the LCS7 gene in regulation and control of low-calcium sensitivity of plants, the plants are arabidopsis thaliana and tomatoes, and nucleotide sequences of the LCS7 gene are respectively shown as SEQ ID NO.1 of arabidopsis thaliana and SEQ ID NO.4 of tomatoes. The gene encodes pyrophosphoric acid hydrolase, regulates the steady state of cytoplasm pyrophosphoric acid, and further controls the growth of leaves under a low-calcium condition. The invention provides a gene resource with genetic improvement potential, lays a foundation for cultivating low-calcium-resistant crop varieties, and provides an important basis for further researching a molecular mechanism of tomato low-calcium-induced blossom-end rot.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant genetic engineering, and in particular to a new gene for regulating plant low calcium sensitivity LCS 7 and its application. BACKGROUND

[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 signal transduction molecule in the cytoplasm, participating in various physiological processes. In agricultural production, calcium deficiency is widespread, especially in vegetables and fruit trees. When leafy vegetables are deficient in calcium, leaf symptoms such as dry burning heart, yellowing of the edges, and necrosis of the leaf tips often occur. Typical examples include dry burning heart in Chinese cabbage and black rot in celery. Calcium deficiency in solanaceous crops leads to brown and sunken spots on the top of the fruit, most commonly seen in tomato and pepper as gummy stem blight. When the calcium concentration is lower than 0.29 mM, about 53% of tomato fruits develop gummy stem blight. In addition, calcium deficiency in fruit crops leads to fruit malformation, split fruit, and sunken spots on the fruit skin, such as bitter canker in apples and split fruit in citrus.

[0003] Whether a plant is deficient in calcium is determined by the available calcium content in the soil and the plant's own absorption and transport capacity. According to the median value of calcium concentration in the surface layer (0-25 cm) of the soil nationwide, only 1.96% of the soil has available calcium. In arid regions, although the total amount of soil calcium is 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 precipitation leaching and acidic ion displacement, and plants in these regions often face the risk of calcium deficiency. Unlike other nutrients, plant calcium deficiency is not only caused by insufficient soil calcium content, but also influenced by various environmental factors. Due to the difficulty of redistributing calcium, different tissues of the plant can only obtain calcium supply through transpiration, so tissues with weaker transpiration are more likely to exhibit calcium deficiency symptoms. Any factor that affects transpiration, such as overcast and rainy weather or high temperature and drought, can trigger calcium deficiency in plants. In addition, K + , Mg 2+ , and NH4 + ions have antagonistic effects on Ca 2+ , which also affect the plant's absorption of calcium. Therefore, although the soil does not lack calcium, plants may still exhibit calcium deficiency symptoms. Due to the complexity of the reasons for plant calcium deficiency, it is often difficult to completely solve this problem by fertilization alone. Therefore, cultivating low-calcium-tolerant crop varieties has become a feasible and promising strategy to prevent plant calcium deficiency. In the high-efficiency molecular breeding of low-calcium-tolerant crops, it is particularly important to identify and analyze genes that regulate plant low calcium sensitivity.

[0004] Currently, some genes related to plant growth in low-calcium environment have been reported, which are mainly involved in calcium absorption and transport and cell wall synthesis. For example, vacuolar Ca 2+ / H + The reverse transporter (CAX1) gene plays an important role in the transport of calcium between cytoplasm and vacuole, and overexpression of this gene will exacerbate the calcium deficiency symptoms of 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 constitutively active Ca 2+ / H + antiporter from arabidopsis[J]. Plant physiology,2011, 156(2): 844-855), and its mutant shows stronger low-calcium tolerance. In addition, genes related to cell wall synthesis, such as the GSL family of callose synthesis-related genes gsl1 、 gsl8 and gsl10Mutants, new leaf growth appears defects under low calcium conditions, and accompanied by severe cell death (such as 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 tolerance in 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, the specific mechanism of action is not fully understood, and its potential in genetic improvement has not been fully tapped and applied, so it is relatively limited in practical production in the near future. SUMMARY

[0005] The purpose of the present application is to provide LCS 7 The application of the gene or its encoded protein in regulating the low calcium sensitivity of plants, by providing a new gene with genetic improvement potential and regulating the low calcium sensitivity of plants LCS 7 And its encoded protein, realize in the application of regulating the low calcium sensitivity of Arabidopsis and tomato.

[0006] In order to achieve the above-mentioned purpose of the present application, the technical scheme adopted by the present application is as follows: LCS 7 The application of the gene or its encoded protein in regulating the low calcium sensitivity of plants.

[0007] In the present application, low calcium refers to calcium concentration less than 0.3 mM.

[0008] Further, ArabidopsisLCS 7 application of the gene or the encoded protein in regulating plant low calcium sensitivity, the Arabidopsis thaliana LCS 7 The coding region nucleotide sequence of the gene is shown as SEQ.ID.NO.2, the Arabidopsis thaliana LCS 7 The amino acid sequence of the protein encoded by the gene is shown as SEQ.ID.NO.3.

[0009] The present application finds a low calcium hypersensitive mutant through Arabidopsis thaliana forward genetics screening lcs7 ( low- Calcium sensitive 7 ). The mutant shows that the new leaves stop growing under low calcium (0.2 mM Ca) conditions, but the calcium absorption and transport capacity is normal. The mutant provides a new strategy and genetic resources for improving plant low calcium sensitivity. Through BSA (Bulked Segregant Analysis) resequencing positioning, gene complementation and gene knockout verification, the key gene controlling plant low calcium sensitivity is determined LCS 7 . The ID of the gene is AT1G15690, which is annotated as vacuolar inorganic pyrophosphatase AVP1 (Arabidopsis vacuolar H + -pyrophosphatase 1).

[0010] Further, the tomato SlLCS 7 application of the gene or the encoded protein in regulating plant low calcium sensitivity, the tomato SlLCS 7 The coding region nucleotide sequence of the gene is shown as SEQ.ID.NO.5, the tomato SlLCS 7 The amino acid sequence of the protein encoded by the gene is shown as SEQ.ID.NO.6.

[0011] The present application determines a gene regulating tomato low calcium sensitivity SlLCS 7 , the gene ID is Sloyc07G007600. Further, through CRISPR / Cas9 technology to knock out the SlLCS 7 gene in tomato, the role of the gene in regulating tomato low calcium sensitivity is verified.

[0012] Further, by overexpressing LCS 7 the gene or the encoded protein, the low calcium sensitivity of the plant is reduced, that is, the low calcium stress tolerance or calcium stress resistance is improved.

[0013] LCS 7 The gene or the encoded protein mediates pyrophosphate homeostasis to regulate leaf growth of the plant under low calcium conditions. The present application finds that LCS7 mediates pyrophosphate homeostasis to regulate leaf growth under low calcium conditions through the low calcium phenotype and pyrophosphate detection of the mutant and the cytosolic pyrophosphate transgenic strain. lcs7 ​

[0014] The plant is Arabidopsis or tomato.

[0015] The application also provides LCS 7 application of the gene or the protein coded by the gene in plant breeding, wherein overexpression of the gene or the protein coded by the gene is screened to obtain a low-calcium tolerant crop. LCS 7 application of the gene or the protein coded by the gene in plant breeding, wherein overexpression of the gene or the protein coded by the gene is screened to obtain a low-calcium tolerant crop.

[0016] Further, overexpression of the Arabidopsis LCS 7 gene or the protein coded by the gene is screened, wherein the coding region nucleotide sequence of the Arabidopsis LCS 7 gene is shown as SEQ.ID.NO.2, and the amino acid sequence of the protein coded by the Arabidopsis LCS 7 gene is shown as SEQ.ID.NO.3.

[0017] Further, overexpression of the tomato SlLCS 7 gene or the protein coded by the gene is screened, wherein the coding region nucleotide sequence of the tomato SlLCS 7 gene is shown as SEQ.ID.NO.5, and the amino acid sequence of the protein coded by the tomato SlLCS 7 gene is shown as SEQ.ID.NO.6.

[0018] Further, the plant is Arabidopsis or tomato.

[0019] Compared with the prior art, the application first discovers, locates and clones a new gene regulating plant low-calcium sensitivity LCS 7 , and performs function research. LCS 7 The low-calcium phenotype and pyrophosphate accumulation of the Arabidopsis LCS 7 overexpression transgenic line are verified; the results show that overexpression of the Arabidopsis SlLCS 7 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 the LCS7 mediated pyrophosphate homeostasis has genetic improvement potential in regulating calcium sensitivity.

[0020] Therefore, the application regulates cytoplasmic pyrophosphate level by genetically manipulating the expression of LCS7, thereby regulating plant low-calcium sensitivity, and has genetic improvement potential. BRIEF DESCRIPTION OF DRAWINGS

[0021] The specific embodiments of the application are further described in detail below with reference to the accompanying drawings.

[0022] Figure 1Wild type Col-0 and point mutants in Example 1 lcs7 Phenotype of aerial part and biomass after 14 days of low calcium treatment, scale bar 1 cm; Figure 2 Wild type Col-0 and point mutants in Example 1 lcs7 Phenotype of new leaves and quantification of dead leaves after 14 days of low calcium treatment, scale bar 1 mm; Figure 3 Wild type Col-0 and point mutants in Example 1 lcs7 Calcium content of aerial part after 14 days of low calcium treatment; Figure 4 SNP distribution map based on BSA of F2 population of Col-0 x Col-0 in Example 2 lcs7 Figure 5 Arabidopsis thaliana (AT1G15690) gene structure in Example 2, and LCS 7 Specific mutation type and location of mutants lcs7 (L753F), lcs7-6 CR and lcs7-33 CR Specific mutation type and location of mutants Figure 6 Wild type Col-0 and knock-out mutants in Example 3 lcs7-6 CR , lcs7-33 CR Phenotype of aerial part and biomass after 14 days of low calcium treatment, scale bar 1 cm; Figure 7 Wild type Col-0, knock-out mutants and transgenic lines in Example 4 lcs7-6 CR Phenotype of aerial part and biomass after 14 days of low calcium treatment, scale bar 1 cm; Figure 8 Wild type Col-0 and point mutants in Example 5 lcs7 Pyrophosphate content of aerial part after 14 days of low calcium treatment; Figure 9 Transgenic lines expressing yeast pyrophosphatase IPP1 in Example 5 restore lcs7 growth of new leaves and pyrophosphate content of aerial part of mutants after 14 days of low calcium treatment; Figure 10 Control, knock-out mutants and overexpression EV in Example 6 lcs7-6 CR and overexpression LCS 7 ​Western Blot detection results of LCS7 protein in transgenic lines; Figure 11 Phenotype of overexpression of EV and overexpression of LCS 7 Phenotype of overexpression of Figure 12 Phenotype of overexpression of SlLCS 7 Gene structure of Solyc07g007600, sllcs7-8 CR 、 sllcs7-14 CR Specific mutation type and position of knockout mutants, and sllcs7-8 CR 、 sllcs7-14 CR Expression level of SlLCS 7 in knockout mutants; Figure 13 Phenotype of overexpression of sllcs7-8 CR 、 sllcs7-14 CR Phenotype of new leaf necrotic spots, dead area statistics and pyrophosphate content after 21 days of low calcium treatment, scale 1 cm; Figure 14 Phenotype of overexpression of sllcs7-8 CR 、 sllcs7-14 CR Phenotype of fruit stem-end rot and its frequency after 45 days of low calcium treatment, scale 1 cm; Figure 15 Vector for constructing complementary lines pLCS7:LCS7 、 pLCS7:IPP1 and overexpression lines p35s: LCS 7 and pUBQ10:LCS 7 EcoR I restriction site map of pGreen0229 binary vector. DETAILED DESCRIPTION

[0023] The application will be further described below in conjunction with specific examples, but the scope of protection of the application is not limited to this.

[0024] Example 1: Arabidopsis low calcium hypersensitive mutants lcs7 obtained and analysis of their aerial phenotype In the present embodiment, Arabidopsis Col-0 is used as the material, and the seeds of Arabidopsis Col-0 are treated with a chemical mutagen ethyl methanesulfonate (EMS) to randomly induce point mutations in the whole genome and thus obtain a population with extensive genetic variation, through the alkylation of DNA bases. The EMS mutagenized population is screened for low-calcium-sensitive mutants under the condition of 0.2 mM Ca, and a low-calcium hypersensitive mutant lcs7 low- calcium sensitive 7

[0025] The low-calcium hypersensitive mutant obtained above lcs7 is sowed in Yamagami medium containing normal calcium (2 mM Ca) and low calcium (0.2 mM Ca) for 14 days, and it is observed by a camera or a stereo microscope and the aboveground biomass and the death of new leaves are counted, and it is found that the aboveground growth of the mutant is significantly inhibited lcs7 under low-calcium conditions Figure 1 , and the new leaves show obvious necrosis and wilting and cannot be normally unfolded Figure 2 .

[0026] To explore the correlation between the phenotype and calcium transport, another experiment is set up: the low-calcium hypersensitive mutant obtained above lcs7 is sowed in Yamagami medium containing normal calcium (2 mM Ca) and low calcium (0.3 mM Ca), and 0.2 g of the aboveground sample is taken after 14 days, dried, and then digested by concentrated nitric acid at high temperature to prepare a clear digestion solution, and the calcium content is detected by MP-AES. The results show that, under normal calcium or low calcium conditions, lcs7 there is no significant difference between the calcium content in the aboveground part of the mutant and that in the wild type Col-0 Figure 3 , indicating that the growth stagnation of the new leaves is not caused by abnormal calcium transport.

[0027] Example 2: Gene for regulating the low-calcium sensitivity of plants LCS 7 Location of the gene The low-calcium hypersensitive mutant obtained in Example 1 lcs7 ​​The F2 population was obtained by selfing the F1 generation from the cross between the mutant and the Col-0 wild type. Under the condition of 0.2 mM Ca, the resistant individuals (35) with fully expanded true leaves and the sensitive individuals (34) with severely inhibited true leaf growth were screened according to the true leaf growth phenotype of individuals in the F2 population. The genomic DNA was extracted from the aboveground part of each individual using the FastPure Plant DNA Isolation Mini Kit, and then the DNA of the 35 resistant individuals was mixed in equal amounts to construct a resistant pool, and the DNA of the 34 sensitive individuals was mixed in equal amounts to construct a sensitive pool for subsequent sequencing analysis. The construction of the DNA library and high-throughput sequencing were completed by Shanghai Ouhui Biological Technology Co., Ltd., and the sequencing platform was Illumina NovaSeq 6000. The library was constructed using the Illumina TruSeq DNA LT Sample Prep kit. The raw data obtained by sequencing were first subjected to a quality control process to remove low-quality reads, and the clean reads were aligned to the Arabidopsis reference genome TAIR10. According to the alignment results, the proportion of reads occupied by the mutant allele of each site was calculated and defined as the SNP index. Further, the difference in SNP index (ΔSNP index) between the sensitive pool and the resistant pool was calculated to reflect the difference in allele frequency at this site between the two extreme phenotype populations. Finally, based on the sliding window analysis method (window size 1 Mb, step size 100 kb), the sliding average calculation of ΔSNP index was carried out in the whole genome range, and the results were presented in the form of a Manhattan plot (Fig. 2). Figure 4 ). At the same time, the gene structure of the most likely candidate gene AT1G15690 was shown, and the mutation type of the point mutation, i.e., the mutation of the 753th amino acid from leucine to phenylalanine (L753F) was shown. LCS 7 lcs7 Figure 5

[0028] Example 3: Obtaining of Arabidopsis LCS7 knockout mutant and analysis of low calcium phenotype in the aboveground part The "target Design" function of the online website CRISPR-GE (http: / / skl.scau.edu.cn / ) was used to design the guide RNA (gRNA) targeting the Arabidopsis LCS7 gene. The gRNA sequence was synthesized by Shanghai Generay Biotech Co., Ltd., and the sequence was as follows: 5'-TTGCGGCGGTTTTCACCGTT-3'. The gRNA was cloned into the pCR-Blunt II-TOPO vector to construct the gRNA expression vector pCR-Blunt II-TOPO-gRNA. The gRNA expression vector pCR-Blunt II-TOPO-gRNA was transformed into the Agrobacterium tumefaciens strain EHA105 by freeze-thaw method. The transformed Agrobacterium tumefaciens strain EHA105 was used to transform Arabidopsis thaliana Col-0 wild type by floral dip method. The T1 generation was obtained by selfing the F1 generation from the cross between the mutant and the Col-0 wild type. Under the condition of 0.2 mM Ca, the resistant individuals (35) with fully expanded true leaves and the sensitive individuals (34) with severely inhibited true leaf growth were screened according to the true leaf growth phenotype of individuals in the F2 population. The genomic DNA was extracted from the aboveground part of each individual using the FastPure Plant DNA Isolation Mini Kit, and then the DNA of the 35 resistant individuals was mixed in equal amounts to construct a resistant pool, and the DNA of the 34 sensitive individuals was mixed in equal amounts to construct a sensitive pool for subsequent sequencing analysis. The construction of the DNA library and high-throughput sequencing were completed by Shanghai Ouhui Biological Technology Co., Ltd., and the sequencing platform was Illumina NovaSeq 6000. The library was constructed using the Illumina TruSeq DNA LT Sample Prep kit. The raw data obtained by sequencing were first subjected to a quality control process to remove low-quality reads, and the clean reads were aligned to the Arabidopsis reference genome TAIR10. According to the alignment results, the proportion of reads occupied by the mutant allele of each site was calculated and defined as the SNP index. Further, the difference in SNP index (ΔSNP index) between the sensitive pool and the resistant pool was calculated to reflect the difference in allele frequency at this site between the two extreme phenotype populations. Finally, based on the sliding window analysis method (window size 1 Mb, step size 100 kb), the sliding average calculation of ΔSNP index was carried out in the whole genome range, and the results were presented in the form of a Manhattan plot (Fig. 2). LCS 7 ​​​Candidate 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. LCS 7 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. LCS 7 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... LCS 7 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 ).

[0029] Example 4: Arabidopsis thaliana LCS 7 Obtaining complementary transgenic lines and analyzing their low-calcium phenotype in aboveground parts 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... LCS 7 Genes are crucial for the growth of new leaves in Arabidopsis thaliana under low calcium conditions.

[0030] Example 5: Arabidopsis thaliana LCS 7 Functional analysis of regulation of low calcium sensitivity 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. LCS 7 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.

[0031] Example 6: Arabidopsis thaliana LCS 7 Obtaining transgenic lines with overexpression and analyzing their low-calcium phenotype in aboveground parts Primers were designed to amplify from a wild-type Arabidopsis thaliana cDNA library. LCS 7 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. LCS 7 Gene expression can improve a plant’s sensitivity to low calcium levels.

[0032] 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.

[0033] Example 7: Tomato SlLCS 7 Obtaining knockout mutants and analyzing their low-calcium phenotype in newly formed tissues Using the CRISPR-GE online website (http: / / skl.scau.edu.cn / ) to target tomatoes SlLCS 7 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. SlLCS 7 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. SlLCS 7 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… SlLCS 7Gene deletion leads to a large number of necrotic spots on the new leaves of tomato, and the number of necrotic spots is more than that of the wild type sllcs7 More pyrophosphate accumulation is produced under low calcium condition Figure 13 In addition, the three are respectively watered in normal calcium (1 mM Ca) and low calcium (0.1 mM Ca) Hoagland nutrient solution for 45 days, and it is found through photographing and observing and counting the frequency of fruit stem rot that under low calcium condition, SlLCS 7 Gene deletion leads to the outbreak of tomato fruit stem rot Figure 14 The results of the embodiment show that, LCS 7 The mechanism of gene regulating the low calcium sensitivity of plants is conservative in plants, and the mechanism can be effectively applied to tomato, thereby providing a clear target and a feasible path for improving the low calcium tolerance of tomato.

[0034] The above is described schematically for the present application and the embodiments thereof, and obviously, the present application is not limited to the above embodiments, and there can be many variations. All the variations directly derived or thought by those skilled in the art from the disclosure of the present application should be considered as the protection scope of the present application.

Claims

1. Application of the LCS7 gene or its encoded protein in regulating plant low calcium sensitivity.

2. The application according to claim 1, characterized in that, The application of the Arabidopsis LCS7 gene or its encoded protein in regulating plant low calcium sensitivity, wherein the nucleotide sequence of the coding region of the Arabidopsis LCS7 gene is shown in SEQ.ID.NO.2, and the amino acid sequence of the protein encoded by the Arabidopsis LCS7 gene is shown in SEQ.ID.NO.

3.

3. The application according to claim 1, characterized in that, The application of the tomato SlLCS7 gene or its encoded protein in regulating plant low calcium sensitivity, wherein the nucleotide sequence of the coding region of the tomato SlLCS7 gene is shown in SEQ.ID.NO.5, and the amino acid sequence of the protein encoded by the tomato SlLCS7 gene is shown in SEQ.ID.NO.

6.

4. The application according to any one of claims 1-3, characterized in that, Overexpression of the LCS7 gene or its encoded protein reduces the plant's susceptibility to low calcium levels.

5. The application according to claim 4, characterized in that, The LCS7 gene or its encoded protein mediates pyrophosphate homeostasis to regulate plant leaf growth under low calcium conditions.

6. The application according to any one of claims 1-3, characterized in that, The plant in question is either Arabidopsis thaliana or tomato.

7. Application of the LCS7 gene or its encoded protein in plant breeding, wherein plants expressing the LCS7 gene or its encoded protein are screened to obtain low-calcium tolerant crops.

8. The application according to claim 7, screening plants that overexpress the Arabidopsis LCS7 gene or the protein encoded therein, wherein the nucleotide sequence of the coding region of the Arabidopsis LCS7 gene is shown in SEQ.ID.NO.2, and the amino acid sequence of the protein encoded by the Arabidopsis LCS7 gene is shown in SEQ.ID.NO.

3.

9. The application according to claim 8, screening plants that overexpress the tomato SlLCS7 gene or its encoded protein, wherein the nucleotide sequence of the coding region of the tomato SlLCS7 gene is shown in SEQ.ID.NO.5, and the amino acid sequence of the protein encoded by the tomato SlLCS7 gene is shown in SEQ.ID.NO.

6.

10. The application according to any one of claims 7-9, characterized in that, The plant in question is either Arabidopsis thaliana or tomato.

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