CN115490762B

CN115490762B - Chimonanthus praecox CpCBL8 gene, protein encoded by same and application thereof

Info

Publication number: CN115490762B
Application number: CN202210511305.3A
Authority: CN
Inventors: 李志能; 田明康; 眭顺照; 李先源; 李俊彩
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2023-06-27
Anticipated expiration: 2042-05-11
Also published as: CN115490762A

Abstract

The invention relates to the field of plant molecular biology, in particular to a Chimonanthus praecox CpCBL8 gene, a coded protein and application thereof. Designing a specific primer for the total length of a CPCBL8 gene CDS sequence obtained from transcriptome sequencing, taking a wax plum leaf cDNA as a template, and obtaining the target fragment by utilizing a PCR amplification technology, wherein the nucleotide sequence of the target fragment is shown as SEQ ID NO.1, and the amino acid of the coded protein is shown as SEQ ID NO. 2. CpCBL8 is a positive regulator of salt and drought stress response and is a potential gene for improving the salt and drought tolerance of Chimonanthus praecox. The heterologous expression of the Chimonanthus praecox CpCBL8 can enhance the salt tolerance and drought tolerance of the overexpression Arabidopsis thaliana, but can make the Arabidopsis thaliana intolerant to low temperature stress and have the effect of inhibiting flower development.

Description

Chimonanthus praecox CpCBL8 gene, protein encoded by same and application thereof

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to a Chimonanthus praecox CpCBL8 gene, a protein encoded by the Chimonanthus praecox CpCBL8 gene and application of the Chimonanthus praecox CpCBL8 gene.

Background

Chimonanthus praecox (Chimonanthus praecox) is a fallen leaf shrub of Chimonanthus genus of Chimonanthaceae family, is named as beeswax due to its fragrant smell and color, and is called "four friends in snow" together with camellia japonica, narcissus and white plum. The Chimonanthus praecox is a traditional rare flower and tree in China, has a cultivation history of over one thousand years, is also a few garden plants flowering in winter, and has extremely high ornamental value and economic value.

The Chimonanthus praecox has the effects of coloring and scenting environment. The unique physical period enables the wintersweet to bloom in cold winter, adds color and fragrance for winter, and is an indispensable landscape plant for building a landscape. The Chimonanthus praecox has good fragrance and unique flowering time, is widely used as a landscaping plant in Japan, europe and America and the like, has high ornamental value, has strong adsorption effect on various toxic and harmful gases, particularly chlorine and sulfur dioxide, and is an important tree species for beautifying and purifying urban environment.

Volatile terpenoid and benzene compounds can be emitted from the honeyglands near the ends of the festive sheets in the wax Mei Hualei, and the compounds have rich fragrance, so that the essential oil extracted from the flowers of the Chimonanthus praecox is widely used as the main component of perfumes and various seasonings, and meanwhile, the essential oil of the Chimonanthus praecox is also rich in various bioactive components such as phenols and flavonoid compounds, and has obvious functions of diminishing inflammation, resisting oxidation, resisting aging and the like, so that the essential oil of the Chimonanthus praecox is widely used as a raw material of cosmetics; the Chimonanthus flower also has important medicinal value, and the extract is a potential source of natural bactericides, and is also applied to pharmaceutical engineering, and is mainly used for treating cough, rheumatism and measles; it is worth mentioning that the sesquiterpenoids separated and extracted from the fruits and leaves of the Chimonanthus praecox have cytotoxicity to cancer cells, thereby playing a role in inhibiting the growth of the cancer cells and providing a new direction and thought for the research and development of medicines.

In 1998, chen Longqing and the like firstly adopt RAPD (Random amplified polymorphic DNA, RAPD) molecular marker technology, analyze the genetic diversity and genetic differentiation of natural living groups of the Chimonanthus praecox from the DNA level, and provide important scientific basis for germplasm resource preservation, introduction and domestication, genetic improvement and new variety cultivation of the Chimonanthus praecox; in 2007, zhao Kaige reports the division of the cultivation area of the Chimonanthus praecox and the genetic variation of the Chimonanthus praecox in different cultivation areas by adopting ISSR (Inter-simple sequence repeat, ISSR) and RAPD molecular marker technologies for the first time, provides important data support for optimizing a breeding sampling strategy, and also provides important theoretical basis for improving, identifying and preserving germplasm resources of the Chimonanthus praecox; in 2009, the university of agricultural in China utilizes AFLP (Amplified fragment length polymorphism, AFLP) and ISSR molecular marker technology to construct a first genetic linkage map in the Chimonanthus praecox seed, and the genetic map is used as a QTL (Quantitative trait locus, QTL) positioning framework, so that important genetic information is provided for Chimonanthus praecox molecular breeding; in recent years, the rapid development of high-throughput sequencing technology and gene mining technology has promoted the progress of analyzing gene expression differences, new gene discovery and gene function research, and simultaneously has promoted the development of molecular breeding technology. In 2012, headland, etc., a first Chimonanthus praecox cDNA library of southwest university is constructed, 95 genes related to environmental stress and plant defense are identified by using EST (Expressed sequence tag, EST) technology, 19 genes related to flower development are identified, and important gene resources are provided for researching Chimonanthus praecox functional genes; subsequently, liu Daofeng et al constructed transcriptome databases of different stages of the development of Chimonanthus praecox, li Zhineng et al constructed transcriptome databases that broken dormancy of Chimonanthus praecox buds at low temperature and made them expanded, and these databases provided important data resources for the study of the molecular mechanism of Chimonanthus praecox development. In 2019 and 2020, the university of Nanjing forestry and the college of lotus successively complete sequencing of chloroplast genome of Chimonanthus praecox (C.praecox var. Concolor) and Chimonanthus praecox (C.praecox var. Grandiforus), and provide important data reference and selection for genetic breeding, population genetics and DNA bar code research of Chimonanthus praecox; in 2020 and 2021, shang Junzhong and Shen Zhiguo, the whole genome sequencing and chromosome level assembly of the Chimonanthus praecox 'H29' (C.praecox 'H29') and the Chimonanthus praecox 'Hongyun') are finished respectively, the deficiency of lack of genome sequence in the research of Chimonanthus praecox molecular biology is complemented, and a brand-new gene reserve and data resource are provided for analyzing the genetic background of the Chimonanthus praecox, improving the ornamental property of the Chimonanthus praecox and the stress resistance of the Chimonanthus praecox, and a new chapter of the research of Chimonanthus praecox molecular biology is turned over.

Most of the existing researches on the CBL family are related to adversity stress, and researches on the flower development regulation of CBL family members are rarely reported. The transcriptome sequencing is carried out on the Chimonanthus praecox in the earlier stage of the subject group, and a transcriptome database which breaks dormancy of the Chimonanthus praecox buds at low temperature and enables the Chimonanthus praecox buds to expand is constructed. When the expression patterns of all CBL family members in the database are analyzed in different stages of the development of the Chimonanthus praecox, 1 key gene is found in all transcripts of the CBL, and the expression quantity of the gene is obviously different in different stages of the development of the Chimonanthus praecox, so that the gene is suspected to be possibly involved in the flowering phase regulation, and meanwhile, the expression quantity of the gene is obviously changed in different stages of the accumulation of the cold quantity required by the Chimonanthus praecox bud, and the gene is presumed to be possibly induced by low temperature and to be involved in responding to the low temperature stress.

In order to verify the function of the gene, the CBL family members of the Chimonanthus praecox are firstly screened and identified on the genome level to obtain all CBL family member sequences of the Chimonanthus praecox, then the transcript ID of the CBL in a transcriptome database is renamed according to the gene identification result, namely CpCBL8, and finally the CpCBL8 is subjected to key research so as to analyze the action and molecular mechanism of the Chimonanthus praecox in the growth and development process, thereby providing an important method and strategy for genetic improvement, new variety cultivation and flowering phase regulation of the Chimonanthus praecox and being beneficial to better applying the Chimonanthus praecox resources to garden landscaping.

Disclosure of Invention

The invention aims to provide wintersweet CpCBL8 and a coded protein and application thereof.

First, the present invention provides a Chimonanthus praecox CpCBL8 protein, which is:

1) A protein consisting of the amino acids shown in SEQ ID No. 2; or (b)

2) A protein derived from 1) which has equivalent activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.

The invention also provides a gene for encoding the Chimonanthus praecox CpCBL8 protein.

Preferably, the sequence of the gene is shown as SEQ ID No. 1.

The invention also provides a vector, a host cell and engineering bacteria containing the gene.

The invention also provides the application of the gene in delaying plant flowering, improving plant drought tolerance, improving plant salt tolerance and/or improving plant sensitivity to low temperature.

In one embodiment of the invention, the gene is transferred into the genome of a plant and overexpressed in a transgenic plant, causing the plant to delay flowering, improve drought tolerance, improve salt tolerance and/or improve sensitivity to low temperatures.

The present invention also provides a method for delaying flowering in plants by transferring a vector containing the gene into the genome of the plant and overexpressing the gene in the transgenic plant.

The present invention also provides a method for improving drought tolerance, salt tolerance and/or sensitivity to low temperatures in plants by transferring a vector containing said gene into the genome of said plant and overexpressing in transgenic plants.

The beneficial effects of the invention are as follows:

the present invention clones the full length of CpCBL8 from Chimonanthus praecox. Expression characteristic analysis shows that CpCBL8 has the highest expression level in leaves of Chimonanthus praecox, and secondly buds have the lowest expression level in flowers; the expression level of CpCBL8 in the perianth/perianth tablets was significantly higher than that in the perianth tablets, stamens and pistils at different sites of flowers. Subcellular localization experiments showed that CpCBL8 is localized to the plasma membrane and nucleus. Compared with WT, 35S is that CpCBL 8/Arabidopsis flowering phase is remarkably delayed, rosette leaf number is remarkably increased, leaf area is remarkably increased, inflorescence is remarkably increased, and the higher CpCBL8 expression quantity is, the more obvious phenotype change is. The measurement of the endogenous genes related to floral development shows that the expression levels of the over-expression lines SOC1, AP1 and LFY are significantly reduced compared with the WT. Salt and drought stress experiments show that compared with WT, the over-expression strain has lower wilting degree, higher superoxide dismutase activity, relative water content, chlorophyll and proline content, smaller pore conductivity, smaller malondialdehyde content and active oxygen accumulation, lower electrolyte leakage rate, and completely opposite measurement results of various physiological indexes under low-temperature stress.

In summary, cpCBL8 is a positive regulator of salt and drought stress responses, a potential gene that enhances the salt and drought tolerance of prunes. The heterologous expression of the Chimonanthus praecox CpCBL8 can enhance the salt tolerance and drought tolerance of the overexpression Arabidopsis thaliana, but can make the Arabidopsis thaliana intolerant to low temperature stress and have the effect of inhibiting flower development.

Drawings

FIG. 1 shows a gel electrophoresis pattern of a CpCBL8 maximum ORF sequence clone. M is DNA molecular weight standard AL2000,1, 2, 3 and 4 are different monoclonal.

FIG. 2 is a diagram showing the double cleavage assay of pCAMBIA-1300mCherry-CpCBL 8. M is DNA molecular weight standard AL2000,1, 2 and 5 are respectively an uncleaved pCAMBIA-1300mCherry plasmid, an digested pCAMBIA-1300mCherry plasmid and an digested cl-CpCBL8 plasmid; and 3 and 4 are pCAMBIA-1300mCherry-CpCBL8 recombinant vectors after enzyme digestion.

FIG. 3 shows the results of pCBL8 subcellular localization experiments. A. B, C are positive control, negative control and CpCBL8 experimental groups respectively; merge, bright, NLS and mCherry are mixed field, bright field, nuclear localization signal and fluorescence signal, respectively. The scale bar is 50 μm.

FIG. 4 shows the relative expression levels of CpCBL8 in different organs and tissues of Chimonanthus praecox.

FIG. 5 shows the double cleavage verification of pCAMBIA-2301G-CpCBL8 expression vector. M is DNA molecular weight standard AL2000,1 is pCAMBIA-232301G vector without enzyme, 2, 3 and 4 are pCAMBIA-232301G, pCAMBIA-2301G-CpCBL8 and tr-CpCBL8 after enzyme cutting respectively.

FIG. 6 shows T ₁ Detection of the CpCBL 8-over-expressed strain. M is DNA molecular standard weight AL2000, N is negative control, T is positive control, 2-33 and WT are different Arabidopsis strains.

FIG. 7 shows the CpCBL8 expression levels of different Arabidopsis over-expression lines. Data are mean ± standard error, single factor analysis of variance and multiple comparison of danken, different letters represent significant differences and P <0.05.

FIG. 8 shows 35S CpCBL8/Col-0 and WT strain phenotypes. The scale bar is 3 cm.

FIG. 9 shows the germination percentage (A) and root length (B and C) of CpCBL 8/Arabidopsis lines at 35S under salt stress at various concentrations. A and B are mean ± standard error, multiplex T-test, WT is control, P <0.05, P < 0.01; the scale of the C chart is 0.5 cm.

FIG. 10 shows 35S:: cpCBL 8/Arabidopsis phenotype under salt stress. The scale bar is 3 cm.

FIG. 11 shows 35S::: cpCBL 8/Arabidopsis germination rate (A) and root length (B and C) under mannitol stress at various concentrations. A and B are mean ± standard error, multiplex T-test, WT is control, P <0.05, P < 0.01; the scale of the C chart is 0.5 cm.

FIG. 12 shows 35S CpCBL 8/Arabidopsis phenotype under drought stress. The scale bar is 3 cm.

FIG. 13 shows 35S:: cpCBL 8/Arabidopsis phenotype under cold stress. The scale bar is 3 cm.

FIG. 14 shows 35S after cold stress CpCBL 8/Arabidopsis phenotype chlorophyll fluorescence parameters. The scale of the A diagram is 3 cm, and the scale of the E diagram is 1 cm.

FIG. 15 shows 35S: cpCBL 8/Arabidopsis plant in vitro leaf natural water loss rate. Air pore conductance (B and C) after different stress treatments. Panels a and B data are mean ± standard error, multiplex T-test, WT is control, and the same asterisks and broken lines in panel a represent the same strain. * P <0.05, < p.ltoreq.0.01, scale bar C is 10 μm.

FIG. 16 shows physiological indicators of 35S CpCBL 8/Arabidopsis overexpressing strain after stress treatment. Data of graphs A-F are mean value.+ -. Standard error, multiplex T test, WT is control group, P <0.05, P.ltoreq.0.01; panels G and H scale 1 cm.

FIG. 17 shows the relative expression levels of 35S CpCBL 8/Arabidopsis thaliana lines endogenous genes. Data are mean ± standard error, multiplex T-test, WT is control, P <0.05, p.ltoreq.0.01.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

EXAMPLE 1 cloning of the Chimonanthus Nitidus CpCBL8 Gene

The method is characterized in that the Chimonanthus praecox leaves at the 'mouth' with good growth vigor and no plant diseases and insect pests in the campus of southwest university are used as materials to extract total RNA, and the extracted RNA is used as a template to synthesize the first strand of cDNA.

And (3) using Primer Premier 5 software, taking CpCBL8 sequences screened from a transcriptome database which breaks dormancy of the Chimonanthus praecox buds at low temperature and makes the Chimonanthus praecox buds expanded as templates, and designing specific amplification primers, wherein PCR products comprise the maximum ORF (Open reading frame, ORF) sequence of the target gene. The designed primers are synthesized by Hua big gene company, and the specific primer sequences are shown in Table 1.

TABLE 1 primer list

And (3) carrying out PCR amplification on the full length of CpCBL8 by taking CpCBL8-cn-F and CpCBL8-cn-R as primers, and carrying out gel electrophoresis detection on the PCR amplified products of CpCBL8, wherein the result shows that the lengths of the products are basically consistent with the predicted lengths of the PCR products. Ligation of the Gene of interest to pMD ^TM After 19-carrier, colony PCR and sequencing are carried out, the colony PCR result is consistent with the gene cloning PCR result, the combined sequencing result shows that CpCBL8 obtained by cloning is 893bp long (figure 1), the complete 663bp CDS is contained, the sequence is shown as SEQ ID NO.1, the sequence is consistent with the sequence in a transcriptome database, the amino acid sequence of the coded protein is shown as SEQ ID NO.2, and the recombinant plasmid in the step is named cn-CpCBL8.

The pCAMBIA-1300mCherry-CpCBL8 recombinant vector is obtained by using a T4 connection method and a homologous recombination method, and the PCR amplification detection is carried out on the recombinant vector, so that the strip length is correct; sequencing the corresponding sample bacterial liquid, and feeding back a sequencing result without mutation, deletion or insertion sites; double restriction verification of the recombinant vector showed that pCAMBIA-1300mCherry-CpCBL8 could be cut into double bands by restriction endonuclease and the band length was correct (FIG. 2).

The subcellular localization experiment result shows that after the control group is infected by the empty agrobacterium carrying pCAMBIA-1300mCherry, fluorescent signals can be found at each position of the onion epidermal cells (figure 3A), and after the control group is infected by the empty agrobacterium carrying no carrier, only nuclear localization signals emit light (figure 3B), so that the experimental materials, the carrier and the strain are normal, and the experimental result is reliable; the transient expression of pCAMBIA-1300mCherry-CpCBL8, a fluorescent signal was detected in the plasma membrane and nucleus (FIG. 3C), indicating that CpCBL8 is localized in the plasma membrane and nucleus.

EXAMPLE 2 characterization of CpCBL8 expression in different organs and tissues of Chimonanthus praecox

The root, stem, leaf, bud, flower, young fruit, outer/middle/inner flower quilt sheet, stamen and pistil of' chimonanthus praecox adult plant with good growth vigor and no plant diseases and insect pests in school of southwest university are used as materials, 3 biological repetition are set for each sample, after sampling, the horses are wrapped by tinfoil paper, immersed in liquid nitrogen for freezing, and then stored in an ultralow temperature refrigerator at-80 ℃ for total RNA extraction and synthesis of cDNA first chain. qRT-PCR was performed using the cDNA obtained by reverse transcription as a template, and the expression characteristics of CpCBL8 in different organs and tissues of Chimonanthus praecox were analyzed, with 3 technical replicates per sample. Primer sequences are shown in the following table.

TABLE 2 primer list

Primer name	Primer sequence (5 '-3')
		CpCBL8-qRT-F1	GCTTGATTGGTTCTCATGTTG
CpCBL8-qRT-R1	TTCACGAACCTCAGACATTG
		CpTublin-F	TAGTGACAAGACAGTAGGTGGAGGT
CpTublin-R	GTAGGTTCCAGTCCTCACTTCATC
		CpActin-F	GTTATGGTTGGGATGGGACAGAAAG
CpActin-R	GGGCTTCAGTAAGGAAACAGGA

In different organs of Chimonanthus praecox, the expression level of CpCBL8 in leaves was highest, next to buds, the expression level in flowers was lowest, the expression level of CpCBL8 in leaves was 5.69 times that in flowers, and the expression difference was very remarkable (FIG. 4).

EXAMPLE 3 acquisition and characterization of CpCBL8 overexpressed Arabidopsis

As genetically transformed plant material, columbia ecological arabidopsis thaliana (Wild-type/Columbia, WT/Col-0) was used, which was grown in a university of southwest floral engineering research center greenhouse (23 ℃,16h day/8 h night), with a relative humidity of 60% and a planting matrix formulated from turf and vermiculite (1:1, v/v).

BamHI and SacI are selected as the linearization and recombination connection sites of the vector according to the characteristics of pCAMBIA-2301G expression vector and CpCBL8 gene sequence. And adding the screened restriction enzyme site sequences to two ends of the CpCBL8 cloning primer, and simultaneously adding a protective base, and cloning the CpCBL8 maximum ORF sequence carrying the corresponding restriction enzyme site sequence by taking cn-CpCBL8 as a template. The primer sequences are shown in Table 3.

TABLE 3 primer list

Separating PCR amplified product from reaction product by agarose gel electrophoresis, cutting gel block with correct length, recovering gel, and connecting gel recovered product of target gene to pMD ^TM On the 19-T vector, a recombinant plasmid was obtained, and finally the recombinant plasmid was designated tr-CpCBL8.

And (3) carrying out double enzyme digestion on the plant binary expression vectors pCAMBIA-2301G and tr-CpCBL8 recombinant plasmid according to the enzyme digestion site designed in the last step to obtain the linearized pCAMBIA-2301G and the target gene with the enzyme digestion site. The corresponding fragments are obtained by agarose gel electrophoresis and gel recovery, wherein the pCAMBIA-2301G expression vector enzyme digestion product is used for recovering long fragments, and the tr-CpCBL8 recombinant plasmid enzyme digestion product is used for recovering short fragments. And (3) connecting the tr-CpCBL8 short fragment with the pCAMBIA-2301G long fragment by using a T4 connection method to obtain the recombinant vector. The ligation product was transformed into E.coli competent cells, and then the transformed product was plated on LB solid medium plates (containing 50mg/L Kan); placing the culture medium plate in a constant temperature incubator in an inverted mode, setting the condition to 37 ℃ and culturing for 11-12 h; picking a plurality of cultured monoclonal colonies into 1000 mu L of LB liquid medium (containing 50mg/L Kan), then propagating by using a constant-temperature oscillator, setting the conditions at 37 ℃ and 180rpm, and culturing for 11-12 h; after the culture is completed, the positive strain is detected by using a PCR amplification and agarose gel electrophoresis method. The positive strain with the correct band length detected by gel electrophoresis is sent to the engine family biology company (Chongqing) for sequencing, single colony with correct sequencing is subjected to propagation and recombinant plasmid extraction, and double enzyme digestion verification is carried out on recombinant plasmid (figure 5). The vector which is verified to be error-free is named as pCAMBIA-2301G-CpCBL8, and the CpCBL8 genetic transformation recombinant vector is obtained.

The recombinant vector pCAMBIA-2301G-CpCBL8 was transferred into Agrobacterium-competent cell GV3101 (pSoup) by electrotransformation, and Arabidopsis thaliana was transformed by floral dip. Screening the over-expression strain by using MS culture medium (containing Kan 50 mg/L) to obtain T ₁ Over-expressed strain with T ₁ The result of the positive plant detection is shown in FIG. 6, wherein the negative control is the same volume of ultrapure water, and the positive control is pCAMBIA-2301G-CpCBL8 recombinant vector. The electrophoresis diagram shows that the negative control and WT strains have no bands, the over-expression strain bands and the positive control bands have the same length, lanes where the No. 4, 10, 17, 26, 27 and 28 strains are located have no bands, and the PCR detection positive strains are continuously screened to obtain the homozygous over-expression strains.

To select strains with different CpCBL8 expression levels, T was used ₂ CpCBL 8/Arabidopsis seedlings are taken as samples, qRT-PCR is carried out, and according to the results, three strains with highest expression levels are respectively OE13-1, OE3-1 and OE19-2 (figure 7), and the 3 strains respectively correspond to 13, 3 and 19 with clear bands in an over-expression strain positive identification electrophoresis chart (figure 6). The CpCBL8 expression level of the OE13-1 strain is obviously higher than that of OE3-1 and OE19-2, and the CpCBL8 expression level of the OE3-1 strain is also obviously higher than that of OE19-2; the expression level of the OE31-11 strain is low, but the expression level of the OE31-11 strain and the WT strain are also obviously different; the strains of OE8-5, OE13-2, OE15-3, OE30-1 and OE31-5 are not substantially expressed, and the expression levels of the strains are not significantly different from the WT strain. Finally, OE13-1, OE3-1 and OE31-11 are selected as 35S (CpCBL 8/Col) respectively by a method combining DNA detection and expression level detection0 high, medium and low expression lines, these 3 lines will be used for subsequent verification of CpCBL8 gene function.

For the screened T ₂ CpCBL 8/Arabidopsis lines are subjected to phenotypic observation, the bolting time and rosette number of OE13-1, OE3-1 and OE31-11 plants are found to be significantly different from those of WT (FIG. 8, table 4), the bolting time of the over-expression plants is significantly delayed, the number of rosette leaves is significantly increased, simultaneously, the rosette leaf area and inflorescence height of OE13-1 and OE3-1 plants are significantly different from those of WT, the over-expression Arabidopsis rosette leaves are larger at five weeks of age, and the over-expression Arabidopsis inflorescence at eight weeks of age is higher.

TABLE 4 35S CpCBL8/Col-0 and WT strain phenotype statistics

Plant phenotype	WT	OE13-1	OE3-1	OE31-11
					Bolting time (d)	28.75±0.22 ^b	31.30±0.33 ^a	30.90±0.39 ^a	30.70±0.35 ^a
Rosette leaf number (piece/piece)	12.05±0.21 ^b	15.25±0.40 ^a	14.45±0.44 ^a	14.45±0.37 ^a
					Rosette leaf length (cm)	1.87±0.04 ^c	2.77±0.05 ^a	2.49±0.03 ^b	1.95±0.04 ^c
Rosette leaf width (cm)	0.99±0.02 ^c	1.31±0.03 ^a	1.2±0.02 ^b	0.92±0.03 ^c
					Inflorescence height (cm)	24.76±1.19 ^c	35.40±0.26 ^a	32.15±0.22 ^b	26.85±0.54 ^c

EXAMPLE 4 identification of salt tolerance of overexpressed Arabidopsis thaliana

T with obvious difference of CpCBL8 expression level ₂ CpCBL 8/Arabidopsis and WT lines were sown on MS medium simulating varying degrees of salt stress (0, 10, 20, 50, 100mM NaCl), where the expressed lines were sown in tissue culture flasks containing Kan 50mg/L in medium, allowing sufficient space for plant roots to grow vertically. After 12d, the germination rate of each strain at different NaCl concentrations was counted. Root scanner is used for scanning roots of each strain under different concentration of NaCl stressRoot length is then measured according to the ruler drawn at the time of scanning. As a result, it was found that the CpCBL 8-highly expressed strain was more salt tolerant. Under the stress of 10, 20, 50 and 100mM NaCl, the germination rate of the seeds of the OE13-1 strain and the OE3-1 strain is obviously larger than that of the seeds of the WT strain (figure 9A), and the germination rate of the seeds of the OE31-11 strain with the low CpCBL8 expression and the germination rate of the seeds of the WT strain are not obviously different. As a result of measurement of seedling root length, it was found that the root length of OE13-1 strain was significantly greater than that of WT strain under 10, 20, 50 and 100mM NaCl stress (FIGS. 9B and C), and that the root length of OE3-1 strain was also significantly greater than that of WT strain under 10mM NaCl stress. The results show that the salt tolerance of the heterologously expressed CpCBL8 and the overexpressed Arabidopsis is obviously enhanced.

Part of seedlings (14 d-old) of each strain sown on a plate of an MS medium containing no NaCl are transplanted into square pots filled with a matrix, each square pot is transplanted with about 25 strains, and after conventional culture for 3w, salt stress treatment is performed. Each pot was irrigated with 100mL of 150mM NaCl solution, and each treatment was repeated three times, and a record of photographs was taken during stress.

For T transplanted into square basin ₂ CpCBL 8/Arabidopsis thaliana is subjected to salt stress treatment and phenotypic observation, each strain is watered with sufficient water before treatment to ensure normal growth, and the seedlings are treated with 150mM NaCl solution after 3 days of seedling reviving. Experimental results show that on day 3 of NaCl irrigation, the stress phenotype of each strain of Arabidopsis begins to appear, and rosette leaves begin to wilt and wither (FIG. 10); on day six, the WT strain had died nearly half, whereas only individual plants of OE13-1, OE3-1 and OE31-11 began to die; at day 10, the WT strain had died substantially, whereas only part of the strain OE13-1 with high CpCBL8 expression died; on day 13, the survival rate of OE13-1 plants was greater than 50%, the survival rate of OE3-1 plants was approximately 40%, and the survival rate of OE31-11 was approximately 20%. The above results further demonstrate that overexpression of CpCBL8 may enhance the salt tolerance of overexpressed Arabidopsis.

EXAMPLE 5 identification of drought tolerance in overexpressed Arabidopsis

T with obvious difference of CpCBL8 expression level ₂ CpCBL 8/Arabidopsis and WT strain seeds were sown on MS medium simulating varying degrees of drought (0, 50, 100, 200, 300mM mannitol), where the tableThe plant line is sown in a tissue culture bottle with a culture medium containing Kan of 50mg/L, so that the plant root system has sufficient space for vertical growth. And counting germination rates of all strains under the stress of mannitol with different concentrations after 12d (the germination standard is that two complete green cotyledons grow out of seedlings), scanning roots of all strains under the mannitol concentration by using a root system scanner, and measuring the root length according to a ruler drawn during scanning. The result shows that the CpCBL8 high-expression strain has stronger drought tolerance. The germination rate of OE13-1 seeds was significantly greater than that of the WT strain at 50, 100, 200 and 300mM mannitol stress, and that of OE3-1 seeds was significantly greater than that of WT in addition to 200mM, while that of OE31-11 seeds was significantly greater than that of the WT strain at 50mM and 100mM mannitol stress (FIG. 11A). As a result of measurement of seedling root length, it was found that OE13-1 and OE3-1 were both significantly longer than the WT strain under 50, 100, 200 and 300mM mannitol stress, and OE31-1 was also significantly longer than the WT strain under 50mM mannitol stress (FIGS. 11B and C). The result shows that the drought tolerance of the over-expressed Arabidopsis thaliana is obviously enhanced by the heterologous expression of CpCBL8.

Seedlings of each line (14 d years old) sown on MS medium plates without mannitol were transplanted into square pots containing a matrix, 25 plants were transplanted into each square pot, and after conventional cultivation for 3w, drought treatment was performed. Before treatment, making each basin of soil matrix fully absorb water, after 24h of seedling-restoring, making drought stress treatment, adopting natural drought method, after about 2 weeks, making rehydration, every treatment can be repeated for three times, and making photographing record in the drought period.

For T transplanted into square basin ₂ CpCBL 8/Arabidopsis are subjected to natural drought treatment and phenotype observation, and soil matrixes are fully watered before treatment to ensure that each strain grows normally. Experimental results show that on day 6 when watering is stopped, the square basin is obviously lighter in quality, the water in the planting matrix is seriously insufficient, and at the moment, the arabidopsis of each strain starts to have a stress phenotype, such as leaf wilting, leaf edge withering and leaf basal purple (figure 12); on day 10 when watering was stopped, the plant volume of the WT strain had been significantly reduced, while the overexpressing strains OE13-1, OE3-1 and OE31-11 still had more leaves growing normally; when the watering is stopped on the 13 th day,WT strains have been substantially free of fresh green leaves, while over-expressed strains OE13-1, OE3-1 and OE31-11 still have a small fraction of fresh green leaves; on day 3 of rehydration, approximately 20% of the WT plants were found to die completely, the remaining 80% recovered to normal, while the overexpressing strains OE13-1, OE3-1 and OE31-11 recovered to normal substantially all of the plants. The above results further demonstrate that heterologous expression of CpCBL8 may enhance drought tolerance of overexpressed Arabidopsis.

EXAMPLE 6 overexpression of Arabidopsis Cold tolerance

T after conventional cultivation for 3w in square basin ₂ And the generation of homozygous 35S is that CpCBL 8/Arabidopsis and WT strains are used as materials, 3 pots are taken from each strain, about 25 plants are transplanted in each pot, and low-temperature stress treatment is carried out. Firstly, cold acclimating for 9 hours at 4 ℃ in a climatic chamber, then, freezing for 9 hours at-4 ℃ in dark condition, thawing for 9 hours at 4 ℃ in dark condition, finally, transferring to normal culture condition for 7d recovery, and taking photos and recording the plants in different treatment periods. Experimental results show that after cold acclimation at 4 ℃ for 9 hours, each strain still grows normally without obvious phenotypic changes (fig. 13); freezing each strain at-4deg.C for 9 hr, wherein OE13-1 and OE3-1 strains seriously wilt, and only part of WT and OE31-11 strains appear wilt; after the freezing treatment, each strain is restored under the dark condition of 4 ℃, after 9 hours, the OE13-1 strain and the OE3-1 strain have no obvious sign of restoration, and partial strains of the WT strain and the OE31-11 strain are restored to normal; after transferring each line to the greenhouse for 7 days of conventional culture, it was found that each line had recovered to normal, but the OE13-1 and OE3-1 lines died approximately 50%, whereas the WT and OE31-11 lines, except for some of the plants, most of the plants still grew normally, especially the WT lines, with substantially no death. The above results indicate that heterologous expression of CpCBL8, over-expression of Arabidopsis thaliana is more sensitive to low temperatures.

And (3) carrying out treatment at-5 ℃ for 1 hour on the over-expression strain, counting the frostbite degree of each strain, and measuring chlorophyll fluorescence parameters of each strain. After each strain after being treated for 1h at the temperature of minus 5 ℃ is dark-adapted for 30min, a modulated chlorophyll fluorescence imaging system is used for photographing and measuring the maximum photochemical efficiency Fv/Fm and the electron transfer quantum rate phi of each strain _PSⅡ At least 3 individual plants per line were tested to further determine the cold tolerance of transgenic arabidopsis. The results showed that in the untreated control group, each strain grew normally, and there was no significant difference between the maximum photochemical efficiency (Fv/Fm) of each strain (FIGS. 14A, B and E) and the PS II electron transfer quantum efficiency (ΦPSII) (FIG. 14C); after freezing treatment, frostbite occurs to each strain, but the frostbite degree of the WT strain is lower, and the normal plant ratio is obviously higher than that of the OE13-1 strain; meanwhile, fv/Fm and ΦPS II of each strain are reduced, which indicates that PS II is damaged, but Fv/Fm and ΦPS II of OE13-1 and OE-31 are obviously lower than WT, which indicates that over-expression strain PS II is damaged to a higher degree. The above results further indicate that heterologous expression of CpCBL8, over-expressed Arabidopsis thaliana is more susceptible to low temperatures.

Example 7 overexpression of Arabidopsis in vitro leaf Natural Water loss Rate and stomatal conductivity under different stresses

To investigate the role CpCBL8 plays in participating in plant water retention capacity, the water loss rate measurements were performed for 8 consecutive hours on the in vitro leaves of the over-expressed lines. As can be seen from the measurement results, the water loss rate of the WT strain is significantly higher than that of OE13-1 and OE3-1 in 1h of the vitro strain (FIG. 15A), and the water loss rate of the WT strain is significantly higher than that of OE3-1 after 2h of the vitro strain and significantly higher than that of OE13-1 after 3h of the vitro strain. In the later period, there was no significant difference in water loss rate between the lines, but after 8 hours, the water loss rates of the WT lines had reached 68%, while the water loss rates of the OE13-1 and OE3-1 lines were 52% and 61% respectively, with no significant difference in water loss rates of the CpCBL8 low expressing lines OE31-11 and the WT lines throughout the stage. From this, it is presumed that the heterologous expression of CpCBL8 enhances the water-retaining ability of the overexpressed Arabidopsis thaliana in the early stage of dehydration (0 to 3 hours).

Stomatal movement is of great importance to plants against adversity stress. In order to verify 35S, cpCBL 8/Arabidopsis stress resistance is strong and weak, stress treatment is carried out on isolated leaves of each strain, the change of air pore conductivity is observed, the aspect ratio of air pores is counted, and more than 30 air pores are observed for each strain. As can be seen from the experimental results, under normal growth conditions, there was no significant difference in stomatal conductance for each strain (fig. 15B and C); simulated salt (50 mM NaCl) and drought (300 mM mannitol) stress on leaves revealed that the stomatal conductance of OE13-1 and OE3-1 was significantly less than that of WT; under the stress of low temperature (4 ℃), the pore conductance of each of OE13-1, OE3-1 and OE31-11 is obviously higher than that of WT, which indicates that the heterologously expressed Chimonanthus CpCBL8 and the over-expressed Arabidopsis can reduce the transpiration effect through pore movement, thereby enhancing the salt tolerance and drought tolerance, but becomes more sensitive to low temperature.

Example 8 overexpression of physiological index after stress treatment of Arabidopsis thaliana

CpCBL 8/Arabidopsis were subjected to 300mM NaCl, 30% PEG6000 and 4℃low temperature stress treatment, respectively, and then a plurality of physiological indexes were measured. The results show that in the control experiments, there was no significant difference in other indices except that OE13-1 had a significantly higher chlorophyll content than WT and malondialdehyde content significantly lower than WT strain (fig. 16B and E). Under salt and drought stress, the relative water content, chlorophyll and proline content of OE13-1 strain was significantly higher than that of WT strain (FIGS. 16A-C), the electrolyte permeability and malondialdehyde content were also significantly lower than that of WT strain (FIGS. 16D and E), while the superoxide dismutase activity was significantly higher than that of WT strain (FIG. 16F) while H ₂ O ₂ (FIG. 16G) and O ^2- (FIG. 16H) also significantly less accumulation than the WT strain; under salt stress, other physiological index measurement results show that the OE3-1 strain has stronger salt tolerance than the WT except that the chlorophyll and proline content is not obviously different from that of the WT strain; under the condition of 30% PEG6000 simulated drought stress, except that the malondialdehyde content is not obviously different from that of the WT strain, other physiological indexes show that the strain has stronger drought resistance than that of the WT strain; notably, while the above three indicators of OE3-1 strain were not significantly different from WT strain under salt and drought stress, the mean chlorophyll and proline content was higher than WT and the mean malondialdehyde content was lower than WT; the above results indicate that OE13-1 and OE3-1 have greater salt and drought tolerance than WT. On the other hand, the physiological index of OE13-1 after low temperature stress is completely opposite to the measurement results after salt and drought stress, which shows that OE13-1 has higher damage degree under low temperature stress, meanwhile, the malondialdehyde content of OE3-1 after low temperature stress is obviously higher than that of WT, the proline content is obviously lower than that of WT, the average value of relative water content, chlorophyll content and SOD activity is also lower than that of WT, which shows that OE3-1 has higher damage degreeHigh. The above experimental results further demonstrate that heterologous expression of CpCBL8 enhances salt and drought tolerance of overexpressed arabidopsis thaliana, but makes it more sensitive to low temperature stress.

Example 9 molecular mechanisms for overexpression of Arabidopsis phenotype and Cold tolerance changes

In order to explore the role of CpCBL8 in plant flowering phase regulation and low temperature stress response molecular mechanisms, expression analysis is performed on Arabidopsis endogenous genes related to flowering phase regulation and low temperature stress response. Actin2 was selected as an internal gene, FT (Flowering locus T), SOC1 (Suppressor of overexpression of CO 1), LFY (leaf) and AP1 (Apetala 1) as floral development regulatory pathway-related genes, CBF1/2/3 (C-repeat binding factor 1/2/3), COR47/15A/15B (Cold regulated 47/15A/15B), CRPK1 (Cold-responsive protein kinase 1) and 14-3-3λ (also referred to as G-box regulating factor, GRF 6) as low temperature response pathway-related genes, and qRT-PCR experiments were performed to investigate the molecular mechanism by which CpCBL8 functions in overexpressing Arabidopsis. Primers required for qRT-PCR were designed by NCBI on-line primer design tool, and were synthesized by the same department of Kyoho, and specific primer sequences are shown in Table 5.

TABLE 5 primer sequences

Since the study is based on transcriptome data development by breaking dormancy of Chimonanthus praecox buds at low temperature and expanding and opening the Chimonanthus praecox buds, and mainly relates to low-temperature induction and flower development, the related molecular mechanisms of flower development and low-temperature stress response are studied with great importance. Determining the relative expression quantity of the overexpression Arabidopsis thaliana flower development and cold resistance regulation pathway related genes, wherein the qRT-PCR result shows that under the normal growth condition, the relative expression quantity of the overexpression strain SOC1, LFY and AP1 is obviously lower than that of the WT, and the relative expression quantity of the FT is not obviously different from that of the WT (figure 17A); in the control group without the low temperature treatment, CPRK1 of OE13-1 strain, CPRK1 of 14-3-3λ and CPRK1 of OE3-1 strain were significantly higher than that of WT (FIG. 17B), CBF1/2/3 of OE13-1 strain, COR15A/15B/47 and COR15A/47 of OE3-1 strain were significantly lower than that of WT, while the relative expression of CBF3 of OE3-1 strain was significantly lower than that of WT (FIG. 17C); after low temperature treatment, the relative expression levels of CBF1/3 of the over-expressed strain OE13-1, CBF1 of OE3-1, COR15A/47 of OE13-1/3-1 and COR15B of OE13-1 are all significantly down-regulated, the expression level of COR15B in OE3-1 is significantly down-regulated, and simultaneously, the 14-3-3λ of the above two over-expressed strains is significantly up-regulated, while the average value of the relative expression levels of CRPK1 is up-regulated, but not significant.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

<110> university of southwest

<120> Chimonanthus praecox CpCBL8 gene, protein encoded by same and application thereof

<160> 44

<170> SIPOSequenceListing 1.0

<210> 1

<211> 663

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 1

atgggctgct tctgcacaaa gcatatcaaa catacaccag gatatgagga gcctactgtt 60

cttgcttctg aaacgacatt tacagtgagt gaagttgaag ctttatatga gctatttaag 120

aaactaagca gttccataat tgatgatgga cttattcaca aggaagagtt tcagcttgct 180

ctattcaata acagcaacaa gcgcaatctt tttgctgaca ggatatttga tttgttcgat 240

gtcaaacgta atggagttat cgagtttggg gaatttgttc gatcattgag cgtttttcat 300

ccaaatgcct cagaatcaga gaaaattgca tttgcattta gattgtatga cttaaggcac 360

actggcttca ttgaacgtga ggagttgaag gaaatggtgt tggccctctt gaacgagtct 420

gacctgcgcc tttcagatga tgtagttgaa acaattgttg acaagacatt cagtgatgca 480

gatttcaatg gagatgggaa aatagattca gaagagtgga aggaatttgt atcaagaaac 540

ccgtctttga tgaagaatat gacccttcca tatttgaagg acataaccat tgcatttcca 600

agctttgttt tgaattctga agttgaagat tcagatatga attgtcaaac tctctcatct 660

tga 663

<210> 2

<211> 220

<212> PRT

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 2

Met Gly Cys Phe Cys Thr Lys His Ile Lys His Thr Pro Gly Tyr Glu

1 5 10 15

Glu Pro Thr Val Leu Ala Ser Glu Thr Thr Phe Thr Val Ser Glu Val

20 25 30

Glu Ala Leu Tyr Glu Leu Phe Lys Lys Leu Ser Ser Ser Ile Ile Asp

35 40 45

Asp Gly Leu Ile His Lys Glu Glu Phe Gln Leu Ala Leu Phe Asn Asn

50 55 60

Ser Asn Lys Arg Asn Leu Phe Ala Asp Arg Ile Phe Asp Leu Phe Asp

65 70 75 80

Val Lys Arg Asn Gly Val Ile Glu Phe Gly Glu Phe Val Arg Ser Leu

85 90 95

Ser Val Phe His Pro Asn Ala Ser Glu Ser Glu Lys Ile Ala Phe Ala

100 105 110

Phe Arg Leu Tyr Asp Leu Arg His Thr Gly Phe Ile Glu Arg Glu Glu

115 120 125

Leu Lys Glu Met Val Leu Ala Leu Leu Asn Glu Ser Asp Leu Arg Leu

130 135 140

Ser Asp Asp Val Val Glu Thr Ile Val Asp Lys Thr Phe Ser Asp Ala

145 150 155 160

Asp Phe Asn Gly Asp Gly Lys Ile Asp Ser Glu Glu Trp Lys Glu Phe

165 170 175

Val Ser Arg Asn Pro Ser Leu Met Lys Asn Met Thr Leu Pro Tyr Leu

180 185 190

Lys Asp Ile Thr Ile Ala Phe Pro Ser Phe Val Leu Asn Ser Glu Val

195 200 205

Glu Asp Ser Asp Met Asn Cys Gln Thr Leu Ser Ser

210 215 220

<210> 3

<211> 21

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 3

gcttgattgg ttctcatgtt g 21

<210> 4

<211> 20

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 4

ttcacgaacc tcagacattg 20

<210> 5

<211> 26

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 5

cgagctcatg ggctgcttct gcacaa 26

<210> 6

<211> 31

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 6

cgggatccag atgagagagt ttgacaattc a 31

<210> 7

<211> 21

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 7

gcttgattgg ttctcatgtt g 21

<210> 8

<211> 20

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 8

ttcacgaacc tcagacattg 20

<210> 9

<211> 25

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 9

tagtgacaag acagtaggtg gaggt 25

<210> 10

<211> 24

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 10

gtaggttcca gtcctcactt catc 24

<210> 11

<211> 25

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 11

gttatggttg ggatgggaca gaaag 25

<210> 12

<211> 22

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 12

gggcttcagt aaggaaacag ga 22

<210> 13

<211> 29

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 13

cgggatccat gggctgcttc tgcacaaag 29

<210> 14

<211> 30

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 14

cgagctcgaa ctggttgagg catgtgcagt 30

<210> 15

<211> 23

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 15

aggatatgag gagcctactg ttc 23

<210> 16

<211> 20

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 16

gattgcgctt gttgctgtta 20

<210> 17

<211> 20

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 17

ggaaggatct gtacggtaac 20

<210> 18

<211> 20

<212> DNA

<213> Chimonanthus praecox (Chimonanthus praecox)

<400> 18

tgtgaacgat tcctggacct 20

<210> 19

<211> 24

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 19

ttccaagtcc tagcaaccct cacc 24

<210> 20

<211> 22

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 20

ttcttcctcc gcagccactc tc 22

<210> 21

<211> 20

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 21

agctgcagaa aacgagaagc 20

<210> 22

<211> 22

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 22

tgaagaacaa ggtaacccaa tg 22

<210> 23

<211> 20

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 23

attggttcaa gcaccacctc 20

<210> 24

<211> 20

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 24

caagaagctc ccaacgaaag 20

<210> 25

<211> 20

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 25

tagggctcaa caggagcagt 20

<210> 26

<211> 20

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 26

cagccaaggt tgcagttgta 20

<210> 27

<211> 20

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 27

gcatgtctca acttcgctga 20

<210> 28

<211> 20

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 28

atcgtctcct ccatgtccag 20

<210> 29

<211> 21

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 29

tgacgtgtcc ttatggagct a 21

<210> 30

<211> 21

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 30

ctgcactcaa aaacatttgc a 21

<210> 31

<211> 22

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 31

gatgacgacg tatcgttatg ga 22

<210> 32

<211> 26

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 32

tacactcgtt tctcagtttt acaaac 26

<210> 33

<211> 26

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 33

gcttcagatt tcgtgacgga taaaac 26

<210> 34

<211> 26

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 34

gcaaaacatt aaagaatgtg acggtg 26

<210> 35

<211> 24

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 35

aaagcagagt ggtgttggta ccgt 24

<210> 36

<211> 24

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 36

tcatcgagga tgttgccgtc actt 24

<210> 37

<211> 20

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 37

cagtgtcgga gagtgtggtg 20

<210> 38

<211> 20

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 38

acagctggtg aatcctctgc 20

<210> 39

<211> 22

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 39

ggttgctcct ggttatcgtg tc 22

<210> 40

<211> 22

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 40

tcatcagtgg cctgacgtat ct 22

<210> 41

<211> 22

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 41

gagcagaagg aagagagtag ga 22

<210> 42

<211> 22

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 42

ggattccaga gcaaacagaa ga 22

<210> 43

<211> 20

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 43

ggaaggatct gtacggtaac 20

<210> 44

<211> 20

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 44

tgtgaacgat tcctggacct 20

Claims

1. Chimonanthus praecox CpCBL8 protein is a protein consisting of the amino acids shown in SEQ ID No. 2.

2. A gene encoding the wintergreen CpCBL8 protein of claim 1.

3. The gene of claim 2, wherein the sequence is set forth in SEQ ID No. 1.

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

5. An engineered bacterium comprising the gene of claim 2 or 3.

6. Use of a gene according to claim 2 or 3 for delaying flowering, improving drought tolerance, improving salt tolerance and/or improving sensitivity to low temperatures in plants.

7. The use according to claim 6, wherein the gene is transferred into the genome of a plant and overexpressed in a transgenic plant, such that the plant is delayed in flowering, improved drought tolerance, improved salt tolerance and/or improved sensitivity to low temperatures.

8. A method for delaying flowering in plants, characterized in that a vector comprising the gene according to claim 2 or 3 is transferred into the genome of said plant and overexpressed in the transgenic plant.

9. A method for increasing drought tolerance, salt tolerance and/or sensitivity to low temperatures in a plant, characterized in that a vector comprising the gene according to claim 2 or 3 is transferred into the genome of said plant and overexpressed in the transgenic plant.