Chimonanthus praecox calmodulin protein gene CpCML45 and application thereof

Abstract

The invention relates to a chimonanthus praecox calmodulin protein CpCML45 and an application thereof. The CpCML45 gene is cloned from chimonanthus praecox, the maximum ORF frame of the CpCML45 gene is 627 bp, and 208 amino acids are encoded. According to the invention, a pCAMBIA-2301G-CpCML45 overexpression vector is constructed and is transferred into arabidopsis thaliana Col-0. Through overexpression of CpCML45, the leaf area of transgenic arabidopsis thaliana is significantly increased, the inflorescence is significantly increased, and the leaf area and the inflorescence are in direct proportion to the expression quantity. Through overexpression of CpCML45, drought tolerance and salt tolerance of transgenic arabidopsis thaliana are enhanced, but low temperature tolerance of the transgenic arabidopsis thaliana is reduced.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology, and specifically relates to a wintersweet. CpCML45 Genes, the proteins they encode, and their applications. Background Technology

[0002] Winter plum ( Chimonanthus praecox Chimonanthus praecox, also known as yellow plum blossom or waxwood, is a perennial deciduous shrub or small tree belonging to the genus Chimonanthus in the family Calycanthaceae. It is a winter-flowering tree species unique to my country, mainly produced in central and southern my country, with large-scale distribution in Sichuan, Yunnan, and Guizhou provinces, possessing abundant wild resources. It was artificially introduced and cultivated during the Tang Dynasty, boasting a history of over a thousand years. Due to its unique ornamental and economic value, it is deeply loved by people.

[0003] Research on genes related to stress and adverse conditions in wintersweet is also a key focus of molecular biology research on wintersweet. Liu et al. cloned a gene containing abundant late-embryonic proteins from wintersweet. CpLEA5 ( Late embryogenesis abundant protein 5 ), and successfully transformed into E. coli to induce its expression, the results showed CpLEA5 It can improve the cold resistance of strains, and after heterologous transformation of Arabidopsis thaliana, it enhances the cold and drought resistance of Arabidopsis thaliana; Liu Daofeng et al. found that overexpression CpTAF10 ( TATA box- binding protein associated factors Genes that significantly improve the germination rate of transgenic Arabidopsis thaliana under salt stress; Wu Huafeng et al. used the transcription factor of wintersweet. CpBBX19 ( B-box-containing protein 19 Successfully transferred to Arabidopsis thaliana, it improved the drought and salt tolerance of the overexpressed Arabidopsis; Lin et al. discovered that wintersweet CpNAC68 ( NAM, ATAF1 / 2, CUC2 The gene can be induced by different hormones (GA, JA, SA) and abiotic stresses (drought, salt, high temperature, low temperature). After heterologous transformation of Arabidopsis thaliana, it will improve the survival rate of transgenic Arabidopsis thaliana under various abiotic stresses. Liu Huamin et al. cloned a CCCH-type zinc finger protein gene from wintersweet. CpC3H3 Discover heterogeneous transformation CpC3H3 It can induce early flowering in overexpressing Arabidopsis thaliana and also enhance the drought tolerance of transgenic Arabidopsis thaliana.

[0004] So far, it has been used in Arabidopsis thaliana ( Arabidopsis thaliana 50 were identified in the study. AtCMLs In rice ( Oryza sativa 32 were identified in the study. OsCMLs In tea trees ( Camellia sinensis 60 were identified in the study. CsCMLs In cucumbers ( Cucumis sativus 44 were identified in the study. CsaCMLs In alfalfa (tribulus terrestris) Medicago truncatula 50 were identified in the study. MtCMLsIn the root of all veins ( Lotus japonicas 19 were identified in the study. LjCMLs Previous generations, through their analysis of some of these... CMLs Cloning and functional analysis of family members revealed that CMLs play important regulatory roles in plant growth and development (such as root development, pollen germination, and flowering regulation), biotic stress (such as pathogens and herbivore attacks), and abiotic stress (such as drought, salt damage, low temperature, ultraviolet light, and mechanical damage). Moreover, these proteins do not appear to have functional redundancy, but rather play a core and highly specific role in coordinating plant adaptation to the environment.

[0005] Currently regarding CMLs Most research focuses on model plants, crops, or fruit trees, while research on ornamental flowering trees is less common. CMLs Studies on wintersweet are relatively rare. CMLs No research on this topic has been reported. Wintersweet, with its unique tree shape, abundant blossoms, and rich fragrance, is widely used in landscaping. It is not only a delicious food but also a traditional Chinese medicine used to treat various chronic diseases, possessing high ornamental and economic value. Research on its functional genes related to abiotic stress can provide important theoretical and practical value for improving the stress resistance of wintersweet cultivated in open fields. Summary of the Invention

[0006] The purpose of this invention is to provide calmodulin protein CpCML45 from wintersweet and its applications.

[0007] First, this invention provides a calmodulin protein CpCML45 from wintersweet, the amino acid sequence of which is shown in SEQ ID No. 2: The present invention also provides a gene encoding the calmodulin protein CpCML45 of the wintersweet as described in claim 1.

[0008] In a specific embodiment of the present invention, the nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0009] The present invention also provides a vector containing the gene. Preferably, the vector may be an overexpression vector or a silencing vector.

[0010] The present invention also provides engineered bacteria containing the aforementioned gene or vector. Preferably, the engineered bacteria may be *Escherichia coli* or *Agrobacterium tumefaciens*.

[0011] The present invention also provides the use of the gene in increasing plant leaf area and / or increasing inflorescence height.

[0012] Specifically, a vector containing the gene is transferred into the plant genome and overexpressed in transgenic plants.

[0013] The present invention also provides the use of the gene in improving the tolerance of plants to drought and / or salt stress.

[0014] 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 increasing the plant’s sensitivity to cold stress.

[0016] Specifically, a vector containing the gene is transferred into the plant genome and overexpressed in transgenic plants.

[0017] The beneficial effects of this invention are as follows: This invention is cloned from 'Suxin' wintersweet. CpCML45 The gene has a maximum ORF frame size of 627 bp, encodes 208 amino acids, and has only one exon; CpCML45 The promoter contains multiple light, hormone, and stress response elements; CpCML45 The encoded protein contains two conserved EF-hand domains; multiple sequence alignment and phylogenetic analysis showed that CpCML45 is not highly similar to CML45 proteins in other species, and is most closely related to CmCML45 of Camphor Tree; subcellular localization results showed that CpCML45 is located in the nucleus and plasma membrane.

[0018] right CpCML45 Analysis of expression characteristics in different tissues and organs revealed that... CpCML45 The relative expression level was highest in the roots of *Chimonanthus praecox*, followed by the stems, leaves, and flowers. Among the floral organs, the inner perianth segments showed the highest relative expression level, while the stamens showed the lowest. This expression occurred during the chilling accumulation and bud opening stages. CpCML45 Expression levels were highest in early November when cold storage was needed to accumulate, but decreased as low temperatures continued to accumulate. CpCML45 Expression levels gradually decreased, reaching a significant decrease at the early bud opening stage, and then remained at low to medium levels throughout the bud opening period. Treatment of six-leaf stage seedlings of *Chimonanthus praecox* with exogenous hormones ABA and MeJa induced expression of [a specific hormone] in the leaves of *Chimonanthus praecox* seedlings. CpCML45 Significant upregulation; abiotic stress (drought, salt, 4℃) treatment of six-leaf stage seedlings can also induce the upregulation of [a specific substance] in wintersweet leaves. CpCML45 The expression pattern under 42°C treatment differs from the above treatments: under drought, salinity, and low temperature stress, CpCML45 The expression level showed a trend of first increasing and then decreasing, while the expression level under high temperature stimulation was... CpCML45 Expression levels were significantly downregulated at 2 hours, and then gradually recovered.

[0019] This invention successfully constructed pCAMBIA-2301G- CpCML45The expression vector was overexpressed and transformed into Arabidopsis thaliana Col-0. Twenty-two transgenic lines were screened using resistance medium. After PCR identification, they were screened up to the T3 generation. qRT-PCR was then used to screen for high, medium, and low expression levels in lines OE5-17, OE13-14, and OE5-13 for validation. CpCML45 Gene function.

[0020] a. Phenotypic observation of the overexpression lines and WT lines revealed that... 35S::CpCML45 There was no significant difference in the number of rosette leaves between the Arabidopsis thaliana and WT lines at bolting time (1 cm) before bolting, indicating that... CpCML45 It does not participate in the regulation of flowering time, but the leaf area and inflorescence height of transgenic Arabidopsis are significantly increased, and these increases are proportional to the expression level.

[0021] b. 35S::CpCML45 Seeds of different Arabidopsis thaliana T3 generations and WT lines were sown in MS medium simulating drought and salt stress. Results showed that the transgenic lines had superior seed germination rate and taproot growth compared to the WT lines. After drought and salt stress, the survival rate of the transgenic lines was higher than that of the WT lines, and the contents of chlorophyll, proline, soluble protein, and SOD were also higher. Meanwhile, the relative conductivity and malondialdehyde content were lower in the transgenic lines than in the WT lines, indicating that the transgenic lines had stronger drought and salt tolerance than the WT lines. Measurements of reactive oxygen species accumulation and stomatal conductance further corroborated these results. After cold stress, the phenotypic and physiological changes of the overexpressing lines were the opposite of those under drought and salt stress.

[0022] c. Through the 35S::CpCML45 After different Arabidopsis thaliana T3 generation lines and WT lines were subjected to different stress treatments (drought, salt, -4℃), chlorophyll fluorescence parameters of each line were measured. The results showed that after drought and salt stress, the QY_max, QY_lss, and NPQ_lss of the overexpressing lines were all higher than those of the WT lines, indicating that their photosystem II was less damaged; while after low temperature stimulation, the QY_max, QY_lss, and NPQ_lss of the overexpressing lines were all lower than those of the WT lines, indicating that their photosystem II was more severely damaged.

[0023] d. Select Arabidopsis thaliana CBF1 / 3 , 14-3-3λ , CRPK1 , COR15B qRT-PCR will be performed to detect... 35S:: CpCML45 The expression levels of low-temperature stress response genes in Arabidopsis thaliana and WT lines were analyzed, and the results showed that the transgenic lines... CBF1 / 3 and COR15B The expression levels were significantly lower than those of the WT strain, and at the same time, 14-3-3λ and CRPK1 The expression level of was significantly higher than that of the WT strain. Attached Figure Description

[0024] Figure 1 The image shown is of wintersweet. CpCML45 Gel electrophoresis image of the largest ORF box sequence clone.

[0025] Figure 2 The image shows multiple sequence alignment (A) and phylogenetic analysis (B) of CpCML45 with other homologous proteins.

[0026] Figure 3 As shown CpCML45 Prediction of promoter cis-acting elements.

[0027] Figure 4 The diagram shows the construction of the subcellular localization vector (A), single-colony PCR identification (B), and sequencing results (C). Lane M represents the DNA molecular weight standard DL2000, lanes 1 to 7 represent different single clones, and CK is the negative control.

[0028] Figure 5 The image shows the subcellular localization analysis of CpCML45 (scale bar: 50 micrometers).

[0029] Figure 6 As shown CpCML45 Expression characteristic analysis. A: CpCML45 A: Relative expression levels in different tissues and organs; B: Chill accumulation during flower development and the flower bud opening stage (flower bud breaking dormancy). CpCML45 Expression patterns; C~H: under different hormone treatments and abiotic stresses CpCML45 Presentation pattern. Data are expressed as mean ± standard error; different letters represent... P The difference was significant at the <0.05 level.

[0030] Figure 7 As shown 35S::CpCML45 Overexpression vector construction (A), single colony PCR identification (B), and sequencing results (C). Lane M represents the DNA molecular weight standard DL2000, lanes 1 to 6 represent different single clones, and CK is the negative control.

[0031] Figure 8 As shown 35S:: CpCML45 / Resistance screening of Arabidopsis thaliana T1 (A) and T3 (B) lines (scale bar: 2.5 cm).

[0032] Figure 9 As shown 35S:: CpCML45 / PCR identification of Arabidopsis thaliana T1 generation lines. Lane M: DNA molecular weight standard DL2000; Lanes 1 to 21: CpCML45 Transgenic lines; WT: wild-type Arabidopsis thaliana; CK: negative control; PK: positive control.

[0033] Figure 10 As shown 35S:: CpCML45 Screening of Arabidopsis thaliana T2 generation lines. Data are mean ± standard error; different letters indicate different values. P The difference was significant at the <0.05 level.

[0034] Figure 11 As shown 35S:: CpCML45 Phenotypic analysis of Arabidopsis thaliana T3 generation lines (scale bar: 2 cm). Data in Figures E-H are mean ± standard error; different letters represent different values. P The difference was significant at the <0.05 level.

[0035] Figure 12 As shown 35S:: CpCML45 Germination rate (A) and rooting status (B, C) of Arabidopsis thaliana strains at different mannitol concentrations (scale bar: 1 cm).

[0036] Figure 13 As shown 35S:: CpCML45 Phenotypes of Arabidopsis thaliana strains under drought stress (scale bar: 2 cm).

[0037] Figure 14 As shown 35S:: CpCML45 Phenotypes of Arabidopsis thaliana strains under low temperature stress (scale bar: 2 cm).

[0038] Figure 15 As shown 35S:: CpCML45 / Stomatal aperture (A, B) and water loss rate of detached leaves (C) of Arabidopsis thaliana strains under stress (scale bar: 50 micrometers).

[0039] Figure 16 As shown 35S:: CpCML45 Physiological indicators of Arabidopsis thaliana strains under stress (scale bar: 1 cm).

[0040] Figure 17 As shown 35S:: CpCML45 / Chlorophyll fluorescence imaging (A) and parameters (B~D) of Arabidopsis thaliana strains under stress (scale bar: 1 cm). Detailed Implementation

[0041] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0042] Example 1: Wintersweet CpCML45 Cloning of genes The experiment used CpCML45 The gene sequence was obtained from a transcriptome database of wintersweet flower buds that have undergone dormancy and bud opening due to low-temperature processing. SnapGene 4.1.8 was used in... CpCML45Specific primers were designed flanking the largest ORF (Open reading frame) of the gene. The primer sequences are shown in Table 1.

[0043] Table 1 Primer List

[0044] cDNA extracted from the leaves of the 'Suxin' wintersweet was used as a template to amplify a sample containing... CpCML45 full-length coding sequence cDNA ( Figure 1 The sequence is shown in SEQ ID No. 1, which contains a 627bp CDS region and a 40bp / 115bp 5' / 3'-UTR region, consistent with those in transcriptome databases.

[0045] Using online software CpCML45 Protein-encoded features were predicted, and the results showed: CpCML45 The encoded protein contains 208 amino acids (SEQ ID No. 2) and its molecular formula is C2. 1059 H 1668 N 272 O 330 S 15 CpCML45 is a hydrophilic protein. Its secondary structure consists of 59.62% α-helices, 6.73% extended strands, 6.73% β-turns, and 26.92% random coils. Analysis of its spatial structure reveals that CpCML45 is a multi-stranded folded protein, predominantly α-helices, containing two distinct α-helix-loop-α-helix structures (i.e., EF-hand structures).

[0046] Using MEGA-X software, the wintersweet CpCML45 and lotus ( Nelumbo nucifera ), upland cotton ( Gossypium hirsutum ), submerged camphor ( Cinnamomum micranthum Multiple sequence alignment of CML45 homologous proteins from multiple species, including [list of species], was performed. The results are as follows: Figure 2 Like most CML45 proteins, A, CpCML45 contains two EF-hand structures, but its similarity to CML45 proteins in other species is not high. It has the highest homology with CmCML45 of Camphor Tree (52.28%), followed by MpCML45 of Apple (42.93%).

[0047] To further analyze the evolutionary relationships of CpCML45, a phylogenetic tree was constructed using MEGA-X software for CpCML45 in wintersweet and CML45 homologs in multiple species. The results are as follows: Figure 2 B: CpCML45 is the same as upland cotton, lotus, submerged camphor, and grape ( Vitis vinifera They cluster together and are most closely related to the submerged camphor tree.

[0048] Through online software analysis, it was discovered that CpCML45 The promoter contains multiple TATA-box and CAAT-box core promoter elements, photoresponsive elements such as Box4, TCT-motif, and chs-CMA1a, and also possesses the hormone ABA-responsive element ABRE and multiple abiotic stress-responsive elements, including the low-temperature-responsive element LTR, the drought-responsive element MBS, and the drought, high-salt, and low-temperature-responsive element MYB. Figure 3 ).

[0049] pCAMBIA1300- was constructed using homologous recombination. CpCML45 -mCherry subcellular localization vector ( Figure 4 A) Colony PCR identification results are as follows Figure 4 B, the PCR amplification band is between 500bp and 750bp, the length is correct. The corresponding bacterial culture sample was sent for sequencing. (The sequencing results are...) Figure 4 C) No abnormalities were found, and the material can be used for subsequent experiments.

[0050] Subcellular localization results showed that tobacco cells injected with Agrobacterium carrying the empty vector pCAMBIA-1300mCherry exhibited RFP (Red fluorescent protein) fluorescence signals in both the nucleus and cell membrane. In contrast, the negative control group (perhaps a control without any bacterial culture) only showed blue light emission from the nucleus under DAPI channel stimulation, indicating that the experimental vector and strain were normal. Transient expression of pCAMBIA-1300mCherry-CpCML45 resulted in blue light emission from the nucleus under DAPI channel stimulation, indicating good cell condition and reliable experimental results. Furthermore, RFP fluorescence signals were detected in both the nucleus and plasma membrane. Therefore, it is speculated that CpCML45 is localized in the nucleus and plasma membrane. Example 2: Expression characteristics of the CpCML45 gene in wintersweet

[0051] Using roots, stems, leaves, fruits, whole flowers, and early-opening floral organs (outer perianth segments, median perianth segments, inner perianth segments, stamens, and pistils) of mature 'Suxin' wintersweet as materials, RT-PCR was performed. CpCML45 Tissue-specific analysis was performed, and the primers are shown in Table 1.

[0052] CpCML45 Tissue-specific analysis results showed that ( Figure 6 A), CpCML45 It is expressed in different tissues and organs, but the expression levels vary significantly. CpCML45The expression level was highest in the roots of wintersweet, followed by the stems, and decreased sequentially in the leaves, whole flowers, and young fruits, with the weakest expression in different floral organs. The expression level in the stamens was only 0.04, less than 1 / 100 of the expression level in the roots, showing a significant difference. Therefore, it is speculated that this gene may be related to root development and stress resistance.

[0053] Flower buds from mature 'pure-heart' wintersweet plants during the chilling accumulation and bud opening stages were used as samples for analysis. CpCML45 Expression patterns in these two stages.

[0054] CpCML45 The results of the expression patterns of chilling accumulation and bud opening stages in wintersweet flower development showed that ( Figure 6 B) CpCML45 Expression levels were highest in early November when cold storage was needed to accumulate, but decreased as low temperatures continued to accumulate. CpCML45 The expression level continuously decreased, and it decreased significantly at the early stage of flower bud opening, then stabilized until the decay period (1.2).

[0055] Using the 'Plain Heart' wintersweet at the six-leaf stage as material, nine wintersweet seedlings of uniform growth were selected for each treatment. The substrate was thoroughly watered before treatment, and the seedlings were allowed to recover for 3 days before treatment. The specific treatments included: (1) Hormone treatment: Spray the seedling leaves with 50μM ABA and 100μM MeJA respectively until the leaves are wet and dripping water.

[0056] (2) Drought and salt treatment: 300mM NaCl and 50% (m / v) PEG8000 (Polyethyleneglycol, PEG) were used for root irrigation, and the irrigation volume depended on the volume of the flower pot.

[0057] (3) 4℃ and 42℃ treatment: Place them in artificial climate chambers at 4℃ and 42℃ respectively (only adjust the temperature conditions, and set the other conditions according to the artificial climate chamber conditions).

[0058] For further analysis CpCML45 Gene function, according to CpCML45 Promoter cis-acting element analysis results were obtained, and the effects of different exogenous hormones (100 μM MeJA and 50 μM ABA) and abiotic stresses (4℃, 42℃, 50% PEG8000 and 300 mM NaCl) on treatment were determined. CpCML45 The expression pattern, the result is as follows Figure 6 CH. CpCML45 It could be induced to express under all treatments, but the changes in expression were not entirely the same. After low-temperature treatment, CpCML45 The relative expression level gradually increased, reaching its peak at 6 hours, which was 4.6 times the pre-treatment relative expression level. Subsequently... CpCML45 The relative expression level gradually decreased to a stable level; after high-temperature treatment CpCML45 The expression pattern is the opposite of that of low-temperature treatment. CpCML45 The relative expression level was significantly downregulated after treatment, reaching its lowest value at 6 h and then gradually upregulated; after PEG simulated drought stress, CpCML45 The relative expression level generally showed a trend of first increasing and then decreasing, reaching its highest value at 6 hours, which was 3 times that before treatment; after NaCl stress, CpCML45 The relative expression level first gradually increased, reaching a peak at 6 hours, and then gradually decreased back to the pre-treatment level; after treatment with two exogenous hormones, MeJA and ABA, CpCML45 Expression levels were significantly upregulated, reaching their highest values ​​at 12h and 24h, respectively, which were 20.1 times and 2.9 times that at 0h.

[0059] Example 3: Wintersweet CpCML45 Construction and functional validation of plant gene overexpression vectors Based on the MCS region of the plant expression vector pCAMIA-2301G and CpCML45 Gene sequence characteristics, ultimately leading to the selection of appropriate restriction enzyme sites. Xba I and EcoR Ⅰ, with cn- CpCML45 Using a template, primers were designed to add the restriction enzyme site sequence. CpCML45 Protective bases were added to both ends of the largest ORF; primer sequences are shown in Table 1. After PCR product recovery, it was ligated into the pMD19-T cloning vector, and the resulting positive clone plasmid was named pT. -CpCML45 .

[0060] Select restriction endonucleases Xbal and EcoR I. The pCAMIA-2301G plant expression vector and pT -CpCML45 Linearization. Recovery of the vector fragment and pT from the pCAMIA-2301G plant expression vector. -CpCML45 The target gene fragment was used to construct pCAMBIA-2301G- using the T4 ligation method. CpCML45 Overexpression vector ( Figure 7 A) Colony PCR identification results are as follows Figure 7 B, the PCR amplification band length is correct. The corresponding bacterial sample was sent for testing, and the sequencing results were obtained. Figure 7 C) No mutations, deletions, or insertion sites were observed. The correctly sequenced bacterial cultures were then amplified and extracted using plasmids, transformed into Agrobacterium competent cells, and then transformed into Arabidopsis thaliana using the flower-dipping method.

[0061] The T1 generation seeds collected after infection were sown in MS medium containing 50 mg / L Kan and 100 mg / L Cb. The experimental results after 14 days are as follows: Figure 8A. It can be clearly observed that Arabidopsis seedlings successfully transformed into the overexpression vector developed 6 true leaves with tender green leaves, while seedlings that were not transformed stopped growing after developing two true leaves, and the plants were thin, weak, and yellowed. Ultimately, 22 single plants were selected on the resistance medium and transplanted into an artificial greenhouse for cultivation. Screening continued until the T2 generation, and lines with a positive to negative seedling segregation ratio of 3:1 were selected for transplantation, ultimately obtaining homozygous T3 generation lines (…). Figure 8 B).

[0062] To further identify pCAMBIA-2301G- CpCML45 To determine whether the overexpression vector was successfully transferred into Arabidopsis thaliana, gDNA was extracted from the transgenic and WT lines using the CTAB method and then used as templates for PCR amplification. The PCR results are as follows: Figure 9 A total of 22 transgenic lines were screened on the resistant medium. However, some lines were not used for gDNA extraction due to poor growth. Of the remaining 16 lines, except for lanes 1 and 3 where no bands were amplified, and lanes 9, 18, and 21 where the bands were faint, all other lanes amplified bright bands with the same length as the positive control bands, indicating successful transformation of pCAMBIA-2301G- CpCML45 These lines were further screened to homozygosity using overexpression vectors for subsequent experiments.

[0063] by 35S::CpCML45 Using different individual plants from Arabidopsis thaliana T2 generation and WT lines as materials, identification was performed by qPCR. CpCML45 Gene expression levels in overexpression lines. Experimental results showed that, except for the WT line, CpCML45 It was expressed in all transgenic lines, and showed significant differences. Figure 10 The three strains with the highest expression levels were OE5-17, OE13-14, and OE13-18, with OE5-17 showing the highest expression level. CpCML45 The expression level was significantly higher than that of OE13-14 and OE13-18. The lowest expression level was observed in line OE20-10, but it was not significantly different from the WT line. Therefore, OE5-13, which had a lower expression level and was significantly different from the WT line, was selected for subsequent experiments. Finally, lines OE5-17, OE13-14, and OE5-13 were selected for subsequent experiments. Example 4 Phenotypic Observation of Transgenic Arabidopsis

[0064] To explore CpCML45 The function of genes, CpCML45 Phenotypic observations were conducted on OE5-17 / OE13-14 / OE5-13 and WT lines with high / medium / low gene expression levels. The results showed that the bolting time (based on a bolting length of 1 cm) and the number of rosette leaves in the OE5-17, OE13-14, and OE5-13 lines were not significantly different from those in the WT line. Figure 11 (A, B, E, and F) The number of rosette leaves is one of the important indicators reflecting the transformation of plant vegetative growth to reproductive growth. However, the number of rosette leaves in the overexpression lines and WT lines was mostly concentrated between 10 and 11, showing no significant difference, indicating that... CpCML45 Gene overexpression did not affect the flowering period of Arabidopsis thaliana. However, the rosette leaf area of ​​the overexpressing lines at 35 days was significantly reduced. Figure 11 C, H) and inflorescence height at 55 days ( Figure 11 Both D and G showed significant differences from WT and were directly proportional to the expression level, suggesting that this was due to... CpCML45 The phenomenon caused by gene overexpression. In existing... CMLs In the study of family member functions, no results similar to those in this experiment were found. It is speculated that the increased leaf size and inflorescence height in the transgenic lines are due to differences between different species.

[0065] Will 35S:: CpCML45 Seeds of different Arabidopsis thaliana T3 generation lines and WT lines were sown on MS medium containing different concentrations of mannitol. Germination and rooting were observed after 12 days. In MS medium without mannitol, there was no significant difference in germination rate among the lines. When the mannitol stress concentration reached 150 mM, the germination rates of lines OE5-17 and OE13-14 were significantly higher than those of the WT line, and the germination rate of line OE5-13 was also slightly higher than that of the WT line. When the mannitol stress concentration reached 200 mM, the germination rate of the WT line was only 8.55%, while the germination rates of lines OE5-17 / OE13-14 / OE5-13 were 44.40% / 27.95% / 13.52% (…). Figure 12 A). The taproot length of each line was measured. The results showed that under normal growth conditions, except for the OE5-17 line, whose taproot length was significantly greater than that of the WT line, the root lengths of the other transgenic lines were not significantly different from those of the WT line. However, with the increase of Mannitol concentration, the transgenic lines showed stronger growth advantages. Under Mannitol stress of 100mM, 150mM, and 200mM, the taproot lengths of the OE5-17 and OE13-14 lines were significantly greater than those of the WT line. The root length of the OE5-13 line under Mannitol stress of 100mM and 200mM was also significantly greater than that of the WT line. Figure 12 (B and C). In conclusion, CpCML45 Overexpression can improve the drought tolerance of Arabidopsis thaliana.

[0066] Arabidopsis thaliana transplanted into square pots were subjected to natural drought treatment, and phenotypes were observed. The substrate was thoroughly watered before treatment to ensure normal growth of each plant line. Photographs were taken and recorded two days after the seedlings had recovered. The results showed that on the sixth day of natural drought, all plant lines exhibited stress phenotypes such as wilting and drooping rosette leaves, pale leaf gloss, and purplish discoloration. Figure 13By the 8th day of natural drought, nearly 50% of the WT series plants had shrunk in size and their leaves had drooped due to water loss, but most of the OE13-14 and OE5-17 series plants were still growing normally. By the 10th day of natural drought, the substrate in the square pots had become significantly lighter and the surface had dried into clumps. Almost no green leaves were visible in the WT and OE5-13 series, but nearly 50% of the OE13-14 series still had green leaves, and over 80% of the OE5-17 series remained healthy. After resuming watering for all series after 10 days of drought, by the 3rd day after rehydration, nearly 20% of the WT and OE5-13 series plants had not fully recovered, while almost all plants in the OE13-14 and OE5-17 series had fully recovered. Therefore, it is further speculated that… CpCML45 Overexpression can improve the drought resistance of Arabidopsis thaliana.

[0067] Arabidopsis thaliana transplanted into square pots were subjected to cold stress, and phenotypes were observed. Before treatment, all lines were ensured to be growing normally. They were placed in a 4℃ climate chamber (temperature and light conditions remained the same as before treatment) for 24 hours of cold acclimatization. Then, they were placed in a low-temperature climate chamber, and the temperature was adjusted to -4℃ for freezing treatment. After 8 hours, different degrees of frost damage were observed in each line. Approximately 60% of the plants in the OE5-17 line showed severe wilting, and approximately 50% of the plants in the OE13-14 and OE5-13 lines also showed severe wilting. However, less than 15% of the plants in the WT line showed the frost damage phenotype. Figure 14 After transferring each strain to a 4℃ climate chamber for 24 hours, some plants with less severe frost damage gradually recovered. After transferring the strains to a greenhouse for one week, some plants with severe frost damage completely died; nearly 40% of the plants in the OE5-17, OE13-14, and OE5-13 strains died completely. The WT strains had almost no dead plants, with only a small number showing yellowing and wilting leaves. In conclusion, overexpression... CpCML45 It enhanced the sensitivity of Arabidopsis thaliana to low temperatures.

[0068] Stomatal movement can reflect a plant's stress resistance. To further verify the stress resistance of Arabidopsis thaliana overexpressing this gene, stomatal conductance was observed in different lines after stress. The results showed that there was no significant difference in stomatal conductance among the control groups. Figure 15 (A and B) After stress treatment, the guard cells of all lines contracted to some extent, promoting stomatal closure and resisting external stress. After treatment with 300 mM mannitol, the stomatal conductance of OE5-17, OE13-14, and OE5-13 lines was significantly lower than that of the WT line. After treatment with 50 mM NaCl, the stomatal conductance of OE5-17 and OE13-14 lines was significantly lower than that of the WT line. However, after 4℃ stress, the stomatal conductance of OE5-17, OE13-14, and OE5-13 lines was significantly higher than that of the WT line. In conclusion, overexpression... CpCML45It can enhance the drought and salt tolerance of transgenic Arabidopsis by affecting stomatal movement, but reduces its tolerance to low temperatures.

[0069] Meanwhile, the water loss of detached leaves from different Arabidopsis strains was recorded continuously for 7 hours. The results are as follows: Figure 15 C. The WT strain showed a water loss rate of 36.29% one hour after in vitro in vitro, significantly higher than the overexpression strains OE5-17, OE13-14, and OE5-13. Seven hours after in vitro in vitro in vitro, the WT strain's water loss rate reached 87.91%, significantly higher than the OE5-17 strain. Apart from these, no significant differences were observed at other time points. However, it is noteworthy that throughout the entire water loss measurement period, the mean water loss rates of the OE5-17 and OE13-14 strains were lower than those of the WT strain. Therefore, it is speculated that the overexpression strain... CpCML45 To some extent, it can improve the water retention capacity of transgenic plants.

[0070] Under drought and salt stress, the electrolyte permeability and malondialdehyde content of the OE5-17 strain were significantly lower than those of the WT strain, while the activities of soluble protein, proline, and superoxide dismutase were significantly higher than those of the WT strain. Figure 16 A and C~F), while O 2- The accumulation of H2O2 also corresponds to other physiological indicators ( Figure 16 G and H); It is worth noting that the chlorophyll content of the OE5-17 line was significantly higher than that of the WT line under salt stress, but there was no significant difference between the two lines under drought stress, although its average value was higher than that of the WT line (G and H). Figure 16 B). Under drought stress simulated by 15% PEG, the electrolyte permeability, malondialdehyde (MDA) content, proline content, and superoxide dismutase (SOD) content of the OE13-14 strain were significantly different from those of the WT strain. Simultaneously, the average chlorophyll and soluble protein contents were higher in the OE13-14 strain than in the WT strain, indicating that it has a greater drought tolerance than the WT strain. Under salt stress, the physiological indicators of the OE13-14 strain, such as electrolyte permeability, chlorophyll, and proline content, were significantly different from those of the WT strain. Furthermore, the average MDA content was lower in the OE13-14 strain than in the WT strain, while the average soluble protein and SOD activities were higher, all indicating that its salt tolerance is stronger than that of the WT strain.

[0071] The physiological changes of the OE5-17 and OE13-14 lines under cold stress were exactly the opposite of those under drought and salt stress. Specifically, the chlorophyll content, soluble protein accumulation, proline content, and superoxide dismutase activity of the OE5-17 and OE13-14 lines were significantly lower than those of the WT lines, while their electrolyte permeability and malondialdehyde content were significantly higher than those of the WT lines. 2- The H2O2 accumulation was also greater than that of the WT strain, indicating overexpression. CpCML45 It reduced Arabidopsis' tolerance to low temperatures.

[0072] Chlorophyll fluorescence imaging was performed on OE5-17, OE13-14 and WT lines after different stress treatments. Figure 17 A), and chlorophyll fluorescence parameters were statistically analyzed. The experimental results showed that in the control group, there were no significant differences in the maximum quantum efficiency QY_max, actual quantum efficiency QY_lss, and non-chemical fluorescence quenching coefficient NPQ_lss among the various lines. Figure 17 (B~D) After different stress treatments, the QY_max and QY_lss of all lines showed a decreasing trend, indicating damage to Photosystem II (PSII). Among them, the mean QY_max of OE5-17 and OE13-14 lines under drought stress was higher than that of WT lines, and their QY_lss was significantly higher, indicating that the PSII damage degree of WT lines was higher than that of transgenic lines. Under salt stress, the QY_max and QY_lss of OE5-17 and OE13-14 lines were significantly greater than those of WT lines, indicating that the PSII damage degree of transgenic lines was lower than that of WT lines. However, under cold stress, the QY_max and QY_lss of OE5-17 line were significantly lower than those of WT lines, indicating that its PSII damage degree was lower. The overexpression rate was higher than that of the WT lines. NPQ_lss reflects the plant's photosensitivity. Plants typically activate photosensitivity mechanisms under stress, resulting in an increase in NPQ_lss. However, this mechanism may gradually become inactive with increasing stress intensity or duration, leading to a decrease in NPQ_lss. Under drought and salt stress, NPQ_lss in all lines showed a decreasing trend, indicating a tendency for photosensitivity mechanism inactivation and damage to photosynthetic structures. The degree of photosynthetic structure damage was higher in the WT lines than in the transgenic lines. After cold stress, NPQ_lss in all lines showed an increasing trend, indicating activation of the photosensitivity mechanism, which mitigates damage to photosynthetic structures by consuming excess light energy. The increase in NPQ_lss in the WT lines was significantly higher than that in the OE5-17 and OE13-14 lines. These results further demonstrate the effectiveness of overexpression. CpCML45 This enhanced the drought and salt tolerance of transgenic Arabidopsis thaliana, but reduced its tolerance to low temperatures.

[0073] 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 CpCML45 of wintersweet, characterized in that, The amino acid sequence is shown in SEQ ID No.

2.

2. The gene encoding the calmodulin protein CpCML45 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 described in claim 2 or 3 in increasing plant leaf area and / or inflorescence height, characterized in that, The vector containing the gene was transferred into the plant genome and overexpressed in the transgenic plants.

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

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

9. A transgenic plant with increased tolerance to drought and / or salt stress and decreased tolerance to low temperatures, characterized in that, The plant heterologously expresses the gene described in claim 2 or 3.

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