Chimonanthus praecox calmodulin protein gene CpCML25 and application thereof

Abstract

The invention relates to a chimonanthus praecox calmodulin protein CpCML25 and an application thereof. The CpCML25 gene is cloned from chimonanthus praecox, the CDS region of the CpCML25 gene is 570 bp, and 189 amino acids are encoded. Under drought, salt, high temperature and low temperature stress and induction of hormones ABA and MeJA, CpCML25 is up-regulated to different degrees. And performing agrobacterium tumefaciens-mediated transformation on the overexpression vector to obtain the 35S:: CpCML25 / arabidopsis thaliana strain. Compared with a wild type, the overexpressed strain shows the phenomena that the flowering time is advanced, the plant height is lower, the rosette leaf length is smaller, the number of stem leaves and primary lateral branches is increased and the like. The overexpression CpCML25 improves the drought resistance of the arabidopsis thaliana, and reduces the salt tolerance and cold tolerance of the arabidopsis thaliana.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology, and specifically relates to a wintersweet. CpCML25 Genes, the proteins they encode, and their applications. Background Technology

[0002] Winter plum ( Chimonanthus praecox (L.) is a deciduous shrub belonging to the Calycanthaceae family and the Chimonanthus genus. It is endemic to China, with a rich variety of species and a wide distribution in central and southwestern my country. The flowers of the Chimonanthus are waxy yellow and have a strong fragrance. It is a well-known flowering plant in landscaping, and its unique flower color, fragrance, and cultural significance have made it popular in plant landscaping, bonsai, and cut flower markets, giving it high ornamental, economic, and cultural value.

[0003] With the development of molecular biology and high-throughput sequencing technology, molecular research on wintersweet has gradually become a hot topic, mainly focusing on two aspects: flower development and response to abiotic stress. Sui Shunzhao constructed the first wintersweet cDNA library at Southwest University, screening out 114 functional genes, including 19 genes related to wintersweet flowering. Based on this, Liu et al. constructed a genome database of wintersweet at different stages of flower development; Li et al. constructed a database on how low temperatures break the dormancy of wintersweet flower buds and induce their expansion and opening. The establishment of these databases has accelerated the development of research on the function of wintersweet-related genes. Currently, research on wintersweet flower development mainly focuses on flowering induction and flower morphogenesis. Huang et al. discovered that wintersweet... CpWRKY71, CpWRKY75 Heterologous overexpression of Arabidopsis thaliana genes resulted in a significant upregulation of flowering-related endogenous genes and a markedly earlier flowering period. Additionally, CpWRKY71 The gene also accelerated the senescence rate of Arabidopsis leaves, indicating that CpWRKY71 Genes are involved in flower morphogenesis and leaf growth and development. Xu and Liu et al. discovered... CpVIL2-As2i Genes cause early flowering in Arabidopsis thaliana. CpVIL2-As2i pro::GUS The promoter has strong activity in flowers, and the levels of exogenous hormones ABA and IAA are related to... CpVIL2-As2i pro::GUS Promoter activity is negatively correlated. Liu et al. identified miRNAs and their target genes in wintersweet flower buds using high-throughput sequencing technology, and found that miRNAs are involved in the regulation of the dormant release process in wintersweet flower buds. Cao et al. found... CpSRG1 Heterologous overexpression promoted growth and flowering in Arabidopsis thaliana and delayed senescence. Hou et al. found that superleaf members... CpFUL , CpSEPs and CpAGL6s It is related to breaking dormancy and bud differentiation and development, and CpFUL Genes significantly promote earlier flowering in Arabidopsis thaliana. Hou et al. identified changes in the protein level of candidate genes associated with autonomous pathways and found... CpFPA1 Heterologous overexpression advanced the flowering period of Arabidopsis thaliana. Tian Mingkang et al. discovered that wintersweet...CpAP2-L11 The gene is involved in the induction of flowering in wintersweet, and its overexpression promotes flowering in Arabidopsis thaliana. Li et al. explored the minimum chilling requirement and regulatory mechanism for breaking the dormancy of wintersweet flower buds under natural and artificial conditions, and found that... CpFT1 Overexpression of the gene significantly promoted flowering in Arabidopsis thaliana. Li studied the distribution characteristics of nectaries in the perianth segments of Chimonanthus praecox, finding that nectaries were concentrated in the adaxial region of the inner and middle perianth segments. He also discovered... CpCRC ( CRABS CLAW ) and four YABBY The expression levels of family genes are highly correlated with the distribution of nectar glands, suggesting that these genes may be involved in the development of nectar glands and the formation of fragrance in wintersweet flowers; Kamran et al. constructed a transcription factor for wintersweet. CpbHLH Whole-genome databases have identified transcription factors related to floral fragrance and color. CpbHLH25 and CpbHLH59 , CpbHLH112 and CpMYB2 They can interact; Aslam et al. discovered CpMYC2 and CpbHLH13 The expression levels were highest during the peak flowering period, and overexpression of both promoted the synthesis of fragrance-related compounds (monoterpenes and sesquiterpenes) in Arabidopsis thaliana; Shang Junzhong constructed a genome-wide database of fragrance-related genes in wintersweet and discovered 99 fragrance-related genes; Liu et al. discovered, through bimolecular fluorescence complementation experiments and yeast double hybridization technology, that... CpCZF1 and CpCZF2 The gene causes the absence of stamens and partial petaloidization in Arabidopsis thaliana. In addition, they also found... CpC3H3 Overexpression of the gene led to earlier flowering and increased drought tolerance in Arabidopsis thaliana. Liu Daofeng discovered... CpMADS1 Genes cause petaloidization of stamens in some petunia lines. Lei Xinghua, Zhang et al. discovered this. CpAP3 Heterologous overexpression of genes can induce petaloidization of stamens in tobacco and petunia. These studies on the development of wintersweet flowers provide data resources for the ornamental traits, flowering period regulation, and cultivation of wintersweet.

[0004] Regarding responses to abiotic stresses, Liu et al. found CpLEA5 Genes enhance the cold and drought resistance of Arabidopsis thaliana; Lv et al. discovered heterologous overexpression CpCAF1 Genes that cause Arabidopsis to flower earlier and enhance its cold resistance; Wu et al. found CpBBX19 Heterologous overexpression enhanced the salt and drought tolerance of Arabidopsis thaliana; Lin et al. found CpNAC68 It can improve the tolerance of Arabidopsis thaliana to low temperature, drought, salt and high temperature stress; Li et al. found that heterologous overexpression can enhance the tolerance of Arabidopsis thaliana to low temperature, drought, salt and high temperature stress. CpSIZ1 The gene improved the cold resistance and vegetative growth of Arabidopsis thaliana, but delayed flowering and advanced leaf senescence. Liu et al. found through prokaryotic expression and heterologous overexpression of Arabidopsis thaliana that...CpLEA5 It can enhance the low-temperature and drought resistance of Arabidopsis thaliana. Similarly, Ren Fei et al. found that... CpLEA Expression levels were upregulated at low temperatures, and heterologous overexpression enhanced the cold and drought resistance of Arabidopsis thaliana; Tian Mingkang discovered that wintersweet... CpCBL8 and CpCIPK9 The signaling system positively regulates the drought and salt tolerance of Arabidopsis thaliana, but negatively regulates its cold tolerance and flowering time. In addition, there are some molecular studies on wintersweet related to plant growth, development, and metabolites. For example, Wang and Zhang et al. discovered molecules involved in the synthesis of strigolactones. CpCCD7 and CpMAX1a Heterologous overexpression of these genes reduced the number of branches in Arabidopsis thaliana. Jiang et al. identified genes responsible for the synthesis of the aromatic compounds linalool and benzyl acetate by combining metabolomics, genomics, transcriptomics, and enzyme assays. CpTPS3 (CpLIS) and CpBAHD2 (CpBEAT) .

[0005] With the development of high-throughput sequencing technology, several members of the CML family have been identified in multiple plant species. Among them, Arabidopsis thaliana (… Arabidopsis 50, corn Zea mays 46, barley ( Hordeum vulgare 80, tomatoes Solanum lycopersicum 52 cucumbers Cucumber 44, soybeans Glycine soja 144, green beans ( Phaseolus vulgaris 111 tea trees Camellia sinensis 60, Paulownia ( Paulownia fortune 58, Phoebe zhennan ( Phoebe bournei 60, alfalfa Medicago truncatula 53, apples Malus domestica 83, grapes Vitis vinifera 62, strawberries Fragaria vesca 36, grapefruit Vitis amurensis Rupr 54, cabbage ( Brassica napus 79, Ginkgo ( Ginkgo biloba 21, chrysanthemums Chrysanthemum seticuspe 82. Studies have shown that the expression pattern of CML proteins is specific, and CML genes with high homology may perform drastically different physiological functions. As an important calcium ion signal transduction and sensing protein, CML proteins participate in the plant growth and development cycle, biotic and abiotic stresses, enabling plants to make corresponding physiological responses when facing abiotic stresses. In recent years, research on the function of CML proteins has developed rapidly. CML25 As a member of the calmodulin-like protein family, it has also yielded some research results. For example, Wang et al. discovered that overexpression... AtCML25The mutant promoted pollen germination and pollen tube elongation in Arabidopsis thaliana, while the mutant reduced pollen germination rate, pollen tube elongation, and Ca2+ resistance. 2+ Sensitivity, indicating AtCML25 Positive regulation of Ca in Arabidopsis pollen germination 2+ Signal regulation network; Yan et al., through omics analysis of the transcriptome of birch trees in response to low temperature, discovered... BpCML25 The gene is Ca upregulated under low temperature stress 2+ It is one of the sensing proteins; however, the main species studied for CML function are crops and economic crops, with very few reported in ornamental plants, especially flowering plants, and none in wintersweet.

[0006] Wintersweet is a precious winter-flowering plant in landscaping. Researching the function of key genes involved in its low-temperature-induced flowering process can enrich the reserve of wintersweet flowering-related genes, potentially leading to the cultivation of wintersweet varieties with stronger resistance, higher economic value, and greater ornamental value. Furthermore, through research on wintersweet... CpCML25 Conducting functional studies is of great theoretical and practical significance for the research on the stress resistance and flowering period regulation of wintersweet. Summary of the Invention

[0007] The purpose of this invention is to provide calmodulin protein CpCML25 from wintersweet and its applications.

[0008] First, the present invention provides a calmodulin protein CpCML25 of the wintersweet family, the amino acid sequence of which is shown in SEQ ID No. 2.

[0009] The present invention also provides a gene encoding the calmodulin protein CpCML25 of the aforementioned wintersweet.

[0010] In a specific embodiment of the present invention, the nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0011] The present invention also provides a vector containing the gene. Preferably, the vector may be an overexpression vector or a silencing vector.

[0012] The present invention also provides engineered bacteria containing the aforementioned gene or vector. Preferably, the engineered bacteria may be *Escherichia coli* or *Agrobacterium tumefaciens*.

[0013] This invention also provides the use of the gene in promoting early flowering in plants. Specifically, a vector containing the gene is transferred into the plant genome and overexpressed in transgenic plants.

[0014] This invention also provides the use of the gene in promoting early germination of plant seeds. Specifically, a vector containing the gene is transferred into the plant genome and overexpressed in transgenic plants.

[0015] The present invention also provides the use of the gene in improving the tolerance of plants to drought stress. Specifically, a vector containing the gene is transferred into the plant genome and overexpressed in transgenic plants.

[0016] The present invention also provides the use of the gene in increasing the sensitivity of plants to cold and / or salt stress. Specifically, a vector containing the gene is transferred into the plant genome and overexpressed in transgenic plants.

[0017] This invention also provides the ability of the gene to promote hypocotyl elongation, inhibit root elongation, and / or promote lateral root development in plants. Specifically, a vector containing the gene is transferred into the plant genome and overexpressed in transgenic plants.

[0018] The beneficial effects of this invention are as follows: This study focuses on wintersweet. CpCML25 The genes underwent bioinformatics analysis and subcellular localization analysis; qRT-PCR analysis was performed. CpCML25 Expression characteristics of the gene in different tissues, different flower development stages, and under different abiotic stresses and exogenous hormones in *Chimonanthus praecox*; heterologous overexpression in wild-type *Arabidopsis thaliana* Columbia-0 (Col-0). CpCML25 Genes, on 35S:: CpCML25 / Phenotypic observations, physiological index measurements after abiotic stress treatment, and expression analysis of related endogenous genes were performed on Arabidopsis thaliana lines. The main results are as follows: (1) Gene structure analysis results show that wintersweet CpCML25 The gene has only one exon and no introns. It contains a 570 bp CDS region encoding 189 amino acids; phylogenetic relationships indicate that *Chimonanthus praecox*... CpCML25 The gene is most closely related to the submerged camphor tree; amino acid structure analysis results show that the CpCML25 protein possesses Motif 1-6 and conserved domains EF-hand7 and EF-hand8; promoter cis-acting element prediction results indicate that... CpCML25 The gene promoter contains a light-responsive core element, as well as hormone and abiotic stress-responsive elements; subcellular localization results in tobacco indicate that wintersweet... CpCML25 It is located in the cell membrane and cell nucleus.

[0019] (2) The results of the analysis of expression characteristics in different tissues showed that, CpCML25 The gene was expressed at the highest level in wintersweet flowers. It was also found in different parts of the floral organs. CpCML25 The gene was expressed at its highest level in the stamens, followed by the median perianth, inner perianth, and outer perianth, while its expression was lowest in the pistil. Analysis of the gene's expression characteristics at different stages of flower development showed that... CpCML25The gene showed a gradual increase followed by a decrease during the chilling accumulation period and the flowering process, reaching its highest value at the initial flowering stage. It is speculated that... CpCML25 The gene is induced by low temperature and positively regulates the winter flowering of wintersweet. Analysis of expression characteristics under different abiotic stresses and exogenous hormone treatments showed that under drought, salinity, high temperature, low temperature stress, and induction by the hormones ABA and MeJA, CpCML25 All showed varying degrees of upward adjustment. (Inference / Speculation) ​ Genes have various functions in responding to abiotic stresses and hormones.

[0020] (3) Obtaining expression vectors through Agrobacterium-mediated transformation ​ Arabidopsis thaliana strains. Phenotypic observations were conducted on the overexpression lines (OEx / y / z): compared with Col-0, the overexpression lines exhibited earlier flowering time, lower plant height, smaller rosette leaves, and an increased number of stem leaves and primary lateral branches.

[0021] (4) Using WT as a control, the overexpressing lines were subjected to drought, salt, and low-temperature stress treatments, and physiological measurements were performed after the stresses. The results showed that under drought stress, the overexpressing lines had higher proportions of normal plants, higher chlorophyll fluorescence index, higher chlorophyll content, higher superoxide dismutase activity, higher relative water content, and higher proline content than the WT lines, while lower malondialdehyde content, lower electrolyte permeability, and lower stomatal opening. However, under salt and low-temperature stress, the results were completely opposite. This indicates... ​ It improved the drought resistance of Arabidopsis thaliana, but reduced its salt tolerance and cold resistance.

[0022] In summary, this application addresses... ​ The function of the gene in the development and abiotic stress response of wintersweet was explored. Heterologous overexpression ​ The gene positively regulates the flowering time of Arabidopsis thaliana and inhibits vegetative growth; at the same time, it improves the drought tolerance of Arabidopsis thaliana, but reduces its salt tolerance and cold tolerance. This application enriches the molecular regulatory mechanism of wintersweet flower development and provides a theoretical basis for the regulation of wintersweet flowering period. Attached Figure Description

[0023] ​ As shown ​ PCR products of gene amplification. M (marker): DNA molecular weight standard (DL2000), AL2000, 1 is... ​ 2 is a blank control.

[0024] ​ As shown ​ Gene structure diagram.

[0025] ​ As shown ​Nucleotide and amino acid sequences. Note: Start codons are marked in bold red and underlined; stop codons are marked with a red asterisk; EF-hand domains are marked in blue.

[0026] ​ The diagram shows the phylogenetic tree and domain structure of the CpCML25 system.

[0027] ​ The image shows the subcellular localization analysis of CpCML25 in tobacco.

[0028] ​ As shown ​ Gene expression characteristics in different tissues.

[0029] ​ As shown ​ Gene expression characteristics at different stages of flower development. Note: BS: bud stage; DPE: early petal emergence stage; DPL: late petal emergence stage; IBE: early initial opening stage; IBL: late initial opening stage; OF: full bloom stage; WP: decline stage.

[0030] ​ As shown ​ Gene expression analysis under different treatments.

[0031] ​ The image shown is pCAMBIA-1300- ​ Double enzyme digestion verification of the overexpression vector. Note: M is DNA maker (DL2000); lane 1 shows the double enzyme digestion result of the overexpression vector; lane 2 shows the vector plasmid control.

[0032] ​ As shown ​ Screening of T1 generation transgenic Arabidopsis thaliana lines.

[0033] ​ As shown ​ Transgenic Arabidopsis T1 generation PCR verification (M is DNA molecular weight standard AL2000; 1-11 are transgenic lines; PK is positive control; CK is blank control).

[0034] ​ As shown ​ Relative expression levels of transgenic Arabidopsis thaliana T2 generation.

[0035] ​ As shown ​ Phenotypic observation of strains (scale bar: 2 cm).

[0036] ​ As shown ​ Statistical observation of germination rate, hypocotyl and root length of the strain (scale bar is 1 cm).

[0037] ​ The image shows the effect of cold stress. ​ Phenotype of the strain (scale bar is 2 cm).

[0038] ​ The image shows the effect of cold stress. ​ Phenotypic statistics of strains and chlorophyll fluorescence parameters (scale bar is 2 cm).

[0039] ​ The image shows the effects of salt stress. ​ Phenotypic of transgenic Arabidopsis thaliana strains (scale bar 2 cm).

[0040] ​ The image shows the effects of salt stress. ​ Phenotypic and chlorophyll fluorescence index of transgenic Arabidopsis thaliana lines (scale bar: 2 cm).

[0041] ​ The image shows the effects of drought stress. ​ Phenotypic of transgenic Arabidopsis thaliana lines (scale bar 2 cm).

[0042] ​ The image shows the effects of drought stress. 35::CpCML25 Phenotypic statistics of strains and chlorophyll fluorescence parameters (scale bar is 2 cm).

[0043] Figure 21 The figures show the effects of salt, drought, and low temperature stress. 35::CpCML25 Stomatal conductance of the strain (scale bar: 20 micrometers).

[0044] Figure 22 The figures show the effects of salt, drought, and low temperature stress. 35::CpCML25 Physiological indicators of the strain (scale bar is 1 cm). Detailed Implementation

[0045] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0046] Example 1: Wintersweet CpCML25 Cloning of genes Winter plum CpCML25 The cDNA sequence of the gene was obtained from the transcriptome database of wintersweet flower buds that had undergone low-temperature dormancy breaking and bud enlargement in a flower laboratory. Specific amplification primers were designed across the ORF using Primer 5.0 software, and their specificity was verified on NCBI. The primer sequences are shown in Table 1.

[0047] Table 1. Gene amplification primers

[0048] Using cDNA from leaves of the 'Suxin' wintersweet variety as a template, the target gene was... CpCML25Perform PCR amplification. Detect the obtained PCR products by electrophoresis (1% agarose gel), and the results are as follows: Figure 1 As shown. The target gene fragment was recovered using a gel extraction kit and then ligated into the pMD19-T vector to obtain pT. -CpCML25 . pT -CpCML25 Transformed *E. coli* competent cells DH5α, and single colonies with clear boundaries were picked and placed in LB broth. After shaking on a constant temperature shaker (37℃, 200 rpm) until the bacterial suspension became turbid, PCR detection was performed, and bacterial suspensions with correct bands were sequenced to obtain the final results. CpCML25 The full-length gene sequence is 679 bp, as shown in SEQ ID No. 1. Gene structure analysis results indicate that *Chimonanthus praecox*... CpCML25 A gene has only one exon and no introns, such as Figure 2 As shown. Analysis using DNAMAN software. CpCML25 Gene sequencing results. For example... Figure 3 As shown, CpCML25 The gene contains a 570 bp CDS region, a 52 bp 5' UTR, and a 57 bp 3' UTR, encoding 189 amino acids (SEQ ID No. 2). Transmembrane activity analysis of the CpCML25 protein was performed using the TMHMMServer online database. The results showed that CpCML25 does not possess a transmembrane region; SignalP-5.0 prediction confirmed that the CpCML25 protein does not contain a signal peptide, indicating that this protein is not a secretory protein.

[0049] like Figure 4 As shown, a phylogenetic tree was constructed using model plants. The results showed that wintersweet... CpCML25 The gene clusters with those of *Cinnamomum camphora*, maize, lotus, grape, sunflower, tomato, and tobacco, and is most closely related to *Cinnamomum camphora*. Motif structure analysis of the CpCML25 protein shows that, like CML25 in most species, it possesses Motif 1 / 2 / 3 / 4 / 5 / 6, and the order of these six Motifs is essentially the same. Domain analysis of the CpCML25 protein shows that, like all CML25 proteins, it possesses conserved domains EF-hand7 and EF-hand8, and their order is identical. This demonstrates the conservation of the CpCML25 protein domains.

[0050] The transient expression vector pCAMBIA-1300GFP- fusion protein was constructed using homologous recombination. CpCML25 PCR and electrophoresis were performed on the bacterial culture, and the results showed that the product length was the same as the sequence alignment result, and there were no mutations in the bacterial culture samples, proving that this vector can be transiently expressed in tobacco cells.

[0051] Tobacco leaves infected with bacterial solution for 36 hours were prepared and observed under a laser confocal microscope. The results showed that the GFP fluorescence signal of the unloaded 1300-GFP was distributed throughout the entire cell structure. Under red light, the cell nucleus was excited to emit red light, and the two merged to present a yellow color. pCAMBIA-1300GFP- CpCML25 The GFP fluorescence signal of the vector accumulates in the cell nucleus and cell membrane. The cell nucleus emits red light under the red light channel, and the two are fused in the cell nucleus and emit yellow light, indicating that the wintersweet... CpCML25 Located in the cell membrane and cell nucleus ( Figure 5 ). Example 2: Analysis of the expression characteristics of the CpCML25 gene in wintersweet

[0052] Leaves, floral organs (inner / middle / outer petals, pistils / stamens), young fruits, and roots and stems of wintersweet seedlings were collected from well-grown, healthy, and pest-free adult wintersweet plants for analysis. CpCML25 The expression characteristics of genes in different tissues and the primers for qRT-PCR are shown in the table below.

[0053] Table 2 Primers for Quantitative Fluorescence

[0054] qRT-PCR was used to detect the effects of qRT-PCR on wintersweet. CpCML25 Analysis of gene expression characteristics in different tissues of wintersweet revealed that ( Figure 6 ), CpCML25 The gene is expressed at the highest level in the flowers of wintersweet, while it is expressed in small amounts in the stems, leaves, and fruits. In the floral organs, CpCML25 The gene was expressed at the highest level in the stamens, followed by the middle / inner / outer perianth segments. CpCML25 The gene expression level was lowest in the pistil, and the expression level in the stamen was approximately 12.74 times that in the pistil. Therefore, it is speculated that the wintersweet... CpCML25 Genes may be related to the flowering process.

[0055] To further clarify the meaning of wintersweet CpCML25 The expression characteristics of genes during flower development were analyzed using qRT-PCR at different stages of wintersweet flower development. The results are as follows: Figure 7 As shown, CpCML25 The expression level of the gene first increased and then decreased during the chilling accumulation phase (November 2nd to December 10th), reaching its highest value on November 28th and its lowest value on December 10th; from the bud swelling stage to the late early flowering stage, CpCML25 The expression level gradually increased, reaching its peak in the late early blooming stage (December 28th), and then began to decline from the full bloom stage. Meanwhile, the expression level in the late early blooming stage was approximately three times that during the chilling accumulation period (November 1st). Therefore, it is speculated that the expression level of *Chimonanthus praecox* is related to the wintersweet. CpCML25 Genes may have the function of promoting early flowering.

[0056] To further analyze wintersweet CpCML25 The expression characteristics of genes under abiotic stress and hormone treatment were investigated. CpCML25 Gene function. Healthy, uniformly growing six-leafed wintersweet seedlings were selected and subjected to abiotic stress treatments (300mM NaCl, 50% PEG, 42℃ and 4℃) and exogenous hormone treatments (50μM ABA, 100μM MeJA). Before treatment, the seedlings were thoroughly watered for three days to allow them to recover. Seedlings in the abiotic stress treatment group were immersed in the same tray, while the leaves of each seedling in the exogenous hormone treatment group were sprayed with 200mL of water. The untreated control group was treated with the same distilled water. At 0h, 2h, 6h, 12h, 24h, and 36h for each treatment, leaves from the same location on the seedlings were harvested. The leaves were wrapped in sterilized aluminum foil, quickly placed in liquid nitrogen for freezing, and stored at -80℃.

[0057] The leaves of six-leaf stage wintersweet under different treatments were analyzed by qRT-PCR. The results are as follows: Figure 8 As shown, in the exogenous hormone treatment group, compared with 0h before treatment, after 2h of treatment with 50μM ABA... CpCML25 Gene expression levels decreased to a minimum at 2 hours, then rapidly increased at 6 hours, decreased again at 12 hours, and then gradually increased. Similarly, in the 100 μM MeJA treatment group, expression levels decreased to a minimum at 2 hours, then rapidly increased to a maximum at 6 hours, and then gradually decreased. In the 50% PEG treatment group, CpCML25 Gene expression levels increased immediately after 50% PEG treatment, reached their peak at 6 hours, and then gradually decreased; after treatment with 300 mM NaCl, CpCML25 Gene expression levels rose rapidly, gradually decreased after 2 hours, dropped rapidly after 6 hours to reach their lowest point at 12 hours, then rose rapidly again after 24 hours before declining once more. In the 42℃ and 4℃ treatment groups, CpCML25 Gene expression levels increased after treatment at 42℃, peaked at 6 hours, then decreased, before starting to rise again at 12 hours. After treatment at 4℃, CpCML25 Gene expression levels were slowly upregulated to 6 hours and then gradually decreased, with the expression level reaching its maximum at 6 hours.

[0058] Example 3: Winter Plum CpCML25 Gene function verification Comparative analysis using Snapgene software CpCML25 The gene sequence and the sequence of the plant binary expression vector pCAMBIA-1300 were compared, and restriction endonuclease sites were finally selected. Kpn 1 and Sal 1. Design specific upstream and downstream primers respectively. Use restriction endonucleases. Kpn 1 and Sal1. For pT- CpCML25 The recombinant plasmid and the plant overexpression vector pCAMBIA-1300 were double-digested and ligated overnight to obtain the desired result. CpCML25 Gene plant overexpression vector pCAMBIA-1300- CpCML25 After enzyme digestion, the cells were transformed into competent *E. coli* cells. The bacterial suspension was then spread onto LB agar plates containing Kan-resistant bacteria using a sterile spreader. Single colonies were picked and shaken until the suspension became turbid before bacterial testing. PCR-positive suspensions were sent for further testing. After confirming the absence of base mutations in the sequencing results, plasmids were extracted using a kit. The double enzyme digestion verification results are shown below. Figure 9 As shown. The plant overexpression vector pCAMBIA-1300- was verified to be correct. CpCML25 Agrobacterium competent cells GV3101 were transformed into Arabidopsis thaliana using the inflorescence inoculation method. Mature Arabidopsis seeds obtained after inoculation were sown on MS solid medium (containing 50 mg / L Hyg, pH=5.8). Arabidopsis successfully transformed with the overexpression vector exhibited normal leaf and root growth and dark green leaves. Conversely, Arabidopsis that failed to transform showed no resistance, could not grow normally, and died at the two-leaf stage. Figure 10 ).

[0059] Arabidopsis thaliana lines that had undergone resistance selection for two weeks and were growing normally were marked and transplanted. Genomic DNA extracted from healthy young leaf materials was used as a template, and the overexpression vector pCAMBIA-1300- was used. CpCML25 The plasmid served as a positive control, wild-type Arabidopsis thaliana (WT) as a negative control, and an equal volume of deionized water as a blank control. The expression vector pCAMBIA-1300- was used. CpCML25 The transgenic lines were verified by PCR using upstream primers and downstream universal primers for the vector. Electrophoresis showed that the bands of the transgenic lines were the same length as the positive control, while the WT lines showed no bands. Seeds were collected from individual plants of the PCR-verified positive transgenic Arabidopsis lines for further resistance screening until homozygous transgenic lines were obtained. Results of some lines are shown below. Figure 11 As shown.

[0060] To select high-expression and moderate-expression lines, homozygous T2 generation was used. 35S::CpCML25 Total RNA was extracted from whole plant samples of Arabidopsis thaliana and WT at the 6-leaf stage. Arabidopsis thaliana was used as the primary source of RNA. AtActin The gene was used as an internal reference gene, and reverse transcription was performed for qRT-PCR. The results showed that the lines with the highest expression levels were OE13-2, OE-8, and OE13-1, with OE13-2 and OE-8 lines significantly higher than OE14-1, OE-7, and OE-12 lines. Figure 12 Meanwhile, in the OE14-1, OE-7, and OE-12 strains... CpCML25The expression level was also significantly higher than that of WT. Therefore, based on the comprehensive analysis of expression levels and the health status of the lines, the high-expression line OE13-2 and the medium-expression line OE14-1 were finally selected for phenotypic observation and subsequent experiments.

[0061] Example 4 CpCML25 Phenotypic observation and statistics of transgenic Arabidopsis thaliana Using wild-type Arabidopsis thaliana WT as a control, homozygous... 35::CpCML25 / Phenotypic observation of Arabidopsis T2 generation lines revealed that the bolting time and the number of rosette leaves at bolting were significantly different from those of the WT line in the high-expression line OE13-2 and the medium-expression line OE14-1. Figure 13 (Table 3). Specifically, the bolting time of overexpression plants was significantly earlier, and when transitioning from the vegetative growth stage to the reproductive growth stage, the average number of rosette leaves in the overexpression lines was 6.81–7.28, while that in the wild-type line (WT) was 12.28. Simultaneously, the rosette leaf length and plant height of the overexpression lines OE13-2 and OE14-1 were significantly different from the wild-type line WT. At five weeks of age, the rosette leaf length of the overexpression lines was smaller than that of WT, with an average leaf length of 1.79–1.81 cm, compared to 2.22 cm in WT. The rosette leaf width of the medium-expression line OE14-1 did not differ significantly from that of WT, but the rosette leaf width of the high-expression line OE13-2 did differ significantly from that of WT. Compared to WT, at eight weeks of age, the overexpression lines were shorter in height and had shorter siliques. Furthermore, the overexpression lines had more primary lateral branches than WT, but fewer secondary lateral branches. The above results indicate that heterologous overexpression CpCML25 The gene promotes flowering in Arabidopsis thaliana, while at the same time inhibiting its vegetative growth to some extent.

[0062] Table 3. Phenotypic statistics of the 35::CpCML25 / Col-0 transgenic Arabidopsis T2 generation lines

[0063] Note: The data algorithm is "mean ± standard deviation", one-way ANOVA and Duncan's multiple comparisons; a, b, and c indicate significant differences at the level (P<0.05).

[0064] To investigate CpCML25 The role of genes in seed germination was investigated through seed germination experiments and statistical observations of hypocotyl and root length. The results are as follows: Figure 14 As shown. Regarding the final germination rate, there was no significant difference among the lines, with 2-3 seeds failing to germinate in each line. 35::CpCML25 / Arabidopsis thaliana lines exhibited earlier germination times. Figure 14C). Overexpression lines began germinating 15 hours after vernalization, while WT lines began germinating around 30 hours later. The overexpression lines germinated earlier than WT lines, and the earlier germination occurred with increasing expression levels, indicating that... CpCML25 Heterologous gene expression promoted earlier germination in Arabidopsis thaliana, suggesting it may break dormancy or be involved in calcium metabolism. 2+ Regulatory networks that influence plant growth and development.

[0065] Hypocotyl elongation is the first stage in which plants break dormancy and enter the photosynthetic stage. Its elongation time and length are influenced by various factors, internal hormones, and regulatory networks. Hypocotyl lengths were measured after 7 days of dark culture in overexpression lines and WT. The results showed that the hypocotyl lengths of the overexpression lines OE14-1 and OE13-2 were significantly longer than those of WT. Figure 14 A, B), which indicates that CpCML25 Heterologous gene expression promoted hypocotyl elongation in Arabidopsis, suggesting it may be involved in calcium metabolism. 2+ Regulatory networks that influence plant growth and development.

[0066] Plant roots play a crucial role in nutrient transport and resistance to abiotic stress. Root lengths were measured after 7 and 14 days of light exposure in overexpression lines and root-weighted (WT) plants. Results showed that at 7 days of light exposure, the root lengths of the overexpression lines were shorter than those of WT plants, and the high-expression line OE13-2 showed a highly significant difference compared to WT. Figure 14 (A, B). After 14 days of light cultivation, the root length of the overexpression lines was shorter than that of the WT lines, and the differences were highly significant. However, at this time, the root apex of the overexpression lines had more branches. These results indicate that... CpCML25 Heterologous gene expression inhibited root elongation in Arabidopsis thaliana but promoted lateral root development. Furthermore, during the experiment, it was observed that the overexpression lines had more and longer root hairs than the WT lines, which may be related to cell elongation and stress resistance in plants. Example 5: Stress resistance analysis of CpCML25 transgenic Arabidopsis thaliana

[0067] 1. Cold tolerance of CpCML25 transgenic Arabidopsis thaliana Using wild-type Arabidopsis thaliana WT as a control, the T2 generation, which was conventionally cultured for approximately 14 days, was compared with that of wild-type Arabidopsis thaliana WT. 35S::CpCML25 / Low-temperature stress and observation of Arabidopsis thaliana and WT revealed the following: After 12 h of treatment at 4℃, all overexpressing lines and WT showed normal growth with no significant changes compared to before treatment; after 12 h of dark treatment at -4℃, all lines showed obvious frost damage and wilting, with the frost damage phenotype of the overexpressing lines being more pronounced than that of WT. The frost damage area of ​​the medium-expressing line OE14-1 was approximately 50%, the frost damage area of ​​the high-expressing line OE13-2 exceeded 50%, while the frost damage area of ​​WT was less than 30%; after 12 h of recovery at 4℃, all lines and WT showed some recovery, but the degree of recovery decreased with increasing expression levels; after 7 days of conventional culture, all overexpressing lines and WT recovered to normal growth, but most leaves of the high-expressing line OE13-2 withered and some plants died without recovery, while only a small number of leaves of the medium-expressing lines OE14-1 and WT withered and almost no plant deaths. Figure 15 The above results indicate that heterologous overexpression CpCML25 Genes make Arabidopsis thaliana more sensitive to low temperatures.

[0068] To further validate the overexpression lines 35S::CpCML25 / To assess the cold tolerance of Arabidopsis thaliana lines and WT plants after transplanting and conventional culture for approximately 14 days, untreated overexpressing lines and WT plants were subjected to -4℃ low-temperature stress treatment, followed by chlorophyll fluorescence imaging and index measurement. The results showed that before cold stress treatment, all lines grew normally. There were no significant differences in steady-state maximum photodynamic efficiency QY_max (Fv / Fm), steady-state photochemical quenching parameter qL-Lss, and steady-state fluorescence decay rate Rfd_Lss among all lines and WT plants, and Fv / Fm-Lss was around 0.79, indicating healthy plant growth. After 1 hour of -4℃ stress, the proportion of normal plants in all lines decreased, and this decrease further decreased with increasing expression levels. Figure 16 A, B(a)). The values ​​of QY_max, qL-Lss, and Rfd_Lss in all lines decreased after cold stress, and the decrease in overexpressing plants was significantly greater than that in WT ( ). Figure 16 B(b)-(d)) indicates that the PSII of the transgenic plants suffered greater damage after cold stress, further proving that CpCML25 Heterologous overexpression of genes reduces the cold resistance of Arabidopsis thaliana, making it more sensitive to low temperatures.

[0069] 2. CpCML25 Salt tolerance of Arabidopsis thaliana overexpression Using wild-type Arabidopsis thaliana WT as a control, the homozygous T2 generation was compared with 35S::CpCML25 / Arabidopsis thaliana were subjected to salt stress, and phenotypes were observed. Seedlings were watered for 3 days before treatment to allow them to recover, and photographs were taken. Three days later, overexpressing lines and the control group (WT) were irrigated with a 200 mM NaCl solution. Results are as follows: Figure 17As shown, after 4 days of salt stress, all Arabidopsis strains maintained normal growth, except for the high-expression strain OE13-2, which showed some leaf wilting. After 7 days of salt stress, WT leaves exhibited yellowing and purple phenotypes, the medium-expression strains showed only a small number of yellowing leaves, while the high-expression strain OE13-2 showed some leaf death and large-area leaf yellowing. After 14 days of salt stress, a small number of WT leaves died, while the overexpression strains showed large-area leaf death. By 18 days of salt stress, about 50% of the leaves in the WT strain had died but new leaves were still growing, while the overexpression strains died from the leaf surface to the base of the leaf stalk. Among them, the survival rate of the high-expression strain OE13-2 was less than 20%. These results indicate that heterologous overexpression... CpCML25 The gene enhances Arabidopsis's sensitivity to salt.

[0070] To further validate the overexpression lines 35S::CpCML25 / To assess salt tolerance, Arabidopsis thaliana lines and WT plants that had been conventionally cultured for approximately 14 days after transplanting were subjected to 200 mM NaCl salt stress treatment, and chlorophyll fluorescence imaging and index measurements were performed. The results showed that before salt stress treatment, all lines grew normally. There were no significant differences in steady-state maximum photodynamic efficiency QY_max (Fv / Fm), steady-state photochemical quenching parameter qL-Lss, and steady-state fluorescence decay rate Rfd_Lss among all lines and WT plants, and Fv / Fm-Lss was around 0.79, indicating healthy plant growth. After 4 days of salt stress, the proportion of normal plants in all lines decreased, and this decrease further decreased with increasing expression levels. Figure 18 A, B(a)). The values ​​of QY_max, qL-Lss, and Rfd_Lss of all lines decreased after salt stress, and the decrease in the overexpression lines was significantly greater than that in WT ( Figure 18 B) indicates that PSII in transgenic plants is more severely damaged after salt stress, further proving that CpCML25 Heterologous overexpression of genes reduces the salt tolerance of Arabidopsis thaliana.

[0071] 3. CpCML25 Overexpression of drought tolerance in Arabidopsis thaliana Using wild-type Arabidopsis thaliana WT as a control, the T2 generation homozygous strain was compared with the control group. 35S::CpCML25 Arabidopsis thaliana were subjected to drought stress, and phenotypes were observed. Seedlings were watered for 3 days before treatment to allow them to recover, and photographs were taken. Three days later, overexpressing lines and whole wheat plants were irrigated with a 30% PEG solution. Results are as follows: Figure 19As shown, after 6 days of drought stress, all Arabidopsis lines maintained normal growth, with only the WT and high-expression lines OE13-2 showing yellowing at the leaf tips. After 10 days of drought stress, WT leaves showed partial yellowing and wilting, while the overexpression lines showed only a small number of yellowing leaves. After 14 days of drought stress, a small number of WT leaves withered, some yellowed, and large areas wilted, while the overexpression lines showed only a small area of ​​wilting leaves. By the 18th day of drought stress, all WT lines had withered, while the overexpression lines showed less wilting than WT and remained green. The medium-expression line OE14-1 showed yellowing and purple stress phenotypes, but still did not show wilting. These results indicate that heterologous overexpression... CpCML25 Genes that enhance the drought resistance of Arabidopsis thaliana.

[0072] To further validate the overexpression lines 35S::CpCML25 To assess drought tolerance, Arabidopsis thaliana lines and WT plants that had been conventionally cultured for approximately 14 days after transplanting were subjected to 30% PEG stress treatment, and chlorophyll fluorescence imaging and index measurements were performed. Results showed that before salt stress treatment, all lines grew normally. There were no significant differences in steady-state maximum photodynamic efficiency (QY_max) (Fv / Fm), steady-state photochemical quenching parameter (qL-Lss), and steady-state fluorescence decay rate (Rfd_Lss) among the lines and WT, and Fv / Fm-Lss was around 0.79, indicating healthy plant growth. After 4 days of PEG stress, the QY_max (Fv / Fm), qL-Lss, and Rfd_Lss values ​​of the overexpressing lines were significantly higher than those of WT. Figure 20 B). This indicates that the PSII in transgenic plants suffered less damage after cold stress, further proving... CpCML25 Heterologous overexpression of genes enhances the drought resistance of Arabidopsis thaliana.

[0073] 4. Stomatal conductance and physiological parameters after overexpression in Arabidopsis thaliana under stress. Under abiotic stress, plants reduce transpiration rates by decreasing stomatal conductance to cope with different stresses. Therefore, to further validate the overexpression lines... 35S::CpCML25 To assess the salt, drought, and cold tolerance of the overexpressing strains and WT detached leaves, simulated salt, drought, and low-temperature stresses were applied. Stomatal conductance and opening levels were observed and statistically analyzed for each strain. Three healthy Arabidopsis thaliana plants with consistent growth were selected from each strain. Three fields of view were observed under a microscope for each plant, and three stomata were counted in each field of view. The results showed that ( Figure 21In the untreated control group, there were no significant differences in stomatal opening among the different lines. After stress treatment, stomatal conductance decreased in all lines. Under salt stress, the stomatal conductance of the overexpressing lines was greater than that of the WT line, with OE14-1 showing a highly significant difference from WT. Although the high-expression line OE13-2 did not differ significantly from WT, the average value of the overexpressing lines was higher than that of WT. Under drought stress, the stomatal conductance of the overexpressing lines was less than that of WT, with OE14-1 showing a highly significant difference from WT. Under low temperature stress, the stomatal conductance of the overexpressing lines was less than that of WT, and the difference was highly significant. These results further demonstrate that... CpCML25 Heterologous gene expression improves the drought tolerance of Arabidopsis thaliana, but reduces its salt tolerance and cold tolerance.

[0074] like Figure 22 As shown, the overexpression lines, including untreated and normally growing strains and the WT line, were used as controls. The effects on the overexpression lines were investigated. 35S:: CpCML25 Salt, drought, and low temperature stress treatments were applied, and physiological indicators were measured. The results showed that in the untreated control group, except for the chlorophyll content, which showed a significant difference between the OE14-1 and WT lines, the contents of other indicators did not differ significantly among the lines, indicating that the growth status of the lines was similar and the physiological measurement results were reliable.

[0075] Under salt stress (200 mM NaCl), the chlorophyll content of all lines decreased, and the chlorophyll content of the overexpression lines OE14-1 and OE13-2 was significantly lower than that of the WT lines. Figure 5-14 A); the superoxide dismutase (SOD) activity of the overexpression lines was lower than that of the WT lines, and the high-expression line OE13-2 showed a highly significant difference from the WT line. Figure 22 B); After salt stress, the proline content of all lines increased, but the proline content of the overexpressing lines was lower than that of the WT line, and the proline content of the medium-expressing line OE14-1 was significantly lower than that of the WT line. Figure 22 C); meanwhile, the relative water content of the overexpression lines was lower than that of WT. Among them, OE13-2 was not significantly different from WT, while OE14-1 showed a significant difference from WT. Figure 22 D); In addition, the malondialdehyde (MDA) content of the overexpression lines was higher than that of the WT lines. Among them, the MDA content of OE14-1 was significantly higher than that of WT (D). Figure 22 E); similarly, the malondialdehyde content of the overexpression lines was also higher than that of WT, and the difference was extremely significant (E). Figure 22 F); NBT and DAB staining results indicate that hydrogen peroxide (H2O2) and oxygen ions (O2) are present in the overexpression lines. 2- The content of ) is higher than that of WT ( Figure 22 The above results indicate that the salt tolerance of the overexpression lines is lower than that of the WT lines.

[0076] Under drought stress (30% PEG), the chlorophyll content of all lines increased, with no significant difference compared to WT. However, the average chlorophyll content of the overexpression lines OE14-1 and OE13-2 was higher than that of the WT lines. Figure 22 A); the superoxide dismutase (SOD) activity of the overexpression lines was significantly higher than that of the WT lines. Figure 22 B); Under drought stress, the proline content of the overexpressing lines was higher than that of the WT, and the difference was highly significant. Figure 22 C); meanwhile, the overexpression lines had a higher relative water content than the WT lines, and the difference was highly significant ( Figure 22 D); In addition, the malondialdehyde content and electrolyte permeability of the overexpression lines were lower than those of WT, and the differences were significant and extremely significant, respectively. Figure 22 E, F); NBT and DAB staining results indicate that hydrogen peroxide (H2O2) and oxygen ions (O2) are present in the overexpression lines. 2- The content of all of them was lower than that of WT ( Figure 22 The above results indicate that the overexpression lines have stronger drought resistance than the WT lines.

[0077] Under low temperature stress (4℃), the chlorophyll content of all lines decreased, and the chlorophyll content of the overexpression lines was significantly lower than that of the WT lines. Figure 22 A); the superoxide dismutase (SOD) activity of the overexpression lines was lower than that of the WT lines, and the difference was extremely significant. Figure 22 B); After low temperature stress, the proline content of all strains increased, but the proline content of the overexpression strains was significantly lower than that of WT (B). Figure 22 C); meanwhile, the relative water content of the overexpression lines was lower than that of WT. Among them, OE14-1 was not significantly different from WT, while OE13-2 showed a significant difference from WT. Figure 22 D); In addition, the malondialdehyde (MDA) content and electrolyte permeability of the overexpression lines were higher than those of WT. Although the MDA content of OE13-2 was not significantly different from that of WT, the average value of the overexpression lines was higher than that of WT. Figure 22 E). The electrolyte permeability of OE13-2 and OE14-1 was significantly higher than that of WT (E). Figure 22 F); NBT and DAB staining results indicate that hydrogen peroxide (H2O2) and oxygen ions (O2) are present in the overexpression lines. 2- The content of ) is higher than that of WT ( Figure 22 The above results indicate that the overexpression lines have lower cold tolerance than the WT lines.

[0078] In summary, heterologous overexpression CpCML25 The gene improved the drought resistance of Arabidopsis thaliana, but reduced its cold and salt tolerance.

[0079] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The calmodulin protein CpCML25 of wintersweet, characterized in that, The amino acid sequence is shown in SEQ ID No.

2.

2. The gene encoding the calmodulin protein CpCML25 of the wintersweet as described in claim 1.

3. The gene as described in claim 2, characterized in that, The nucleotide sequence is shown in SEQ ID No.

1.

4. A vector containing the gene of claim 2 or 3.

5. Engineered bacteria containing the gene described in claim 2 or 3.

6. The use of the gene according to claim 2 or 3 in promoting early flowering in plants, characterized in that, The vector containing the gene was transferred into the plant genome and overexpressed in the transgenic plants.

7. The use of the gene according to claim 2 or 3 in promoting early germination of plant seeds, characterized in that, The vector containing the gene was transferred into the plant genome and overexpressed in the transgenic plants.

8. Use of the gene of claim 2 or 3 in improving plant tolerance to drought stress, wherein a vector containing the gene is transferred into the plant genome and overexpressed in transgenic plants.

9. Use of the gene of claim 2 or 3 in increasing the sensitivity of plants to cold and / or salt stress, wherein a vector containing the gene is transferred into the plant genome and overexpressed in transgenic plants.

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