New tea tree gene CsCRAP and cold-resistant application thereof

Abstract

The invention belongs to the technical field of gene engineering, and discloses a tea tree CsCRAP gene and cold-resistant application thereof. The CsCRAP gene is a novel cold-resistant tea tree gene, the nucleotide sequence is shown as SEQ ID NO.1, the amino acid sequence of the encoded protein is shown as SEQ ID NO.2, and the protein is located on a cell membrane. Low-temperature stress can obviously induce up-regulated expression of the CsCRAP gene, and overexpression of the gene in a tea tree body can obviously relieve low-temperature damage of leaves and improve low-temperature tolerance; and the low-temperature damage is obviously aggravated by inhibiting the expression. The invention also provides a recombinant plasmid and a transgenic engineering bacterium containing the gene. It is verified for the first time that the CsCRAP gene positively regulates the cold resistance of the tea trees, core gene resources are provided for cold-resistant mechanism research and cold-resistant breeding of the tea trees, and the CsCRAP gene can be used for genetic improvement of the tea trees, low-temperature tolerance of the tea trees is improved, and sustainable development of the tea industry is promoted.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the CsCRAP gene in tea trees and its application in regulating the cold resistance of tea trees. Background Technology

[0002] The tea plant (Camellia sinensis (L.) O. Kuntze) is a plant belonging to the genus Camellia in the family Theaceae. Tea, made from its buds and leaves, is one of the world's three major non-alcoholic beverages, containing various beneficial components and possessing health benefits. The development of the tea industry is crucial to people's livelihoods and the national economy. In recent years, tea trees have frequently suffered from frost and late spring cold snaps during the winter and early spring, resulting in a significant decline in tea yield and quality, and in severe cases, the death of tea trees. Tea trees prefer warm temperatures and are sensitive to cold; low temperatures have become one of the most significant and serious natural disasters in tea production, threatening the healthy and sustainable development of the tea industry.

[0003] To cope with cold stress, tea plants have evolved a series of molecular mechanisms to alleviate the cellular metabolic disorders caused by cold stress, mainly including calcium ion signaling pathways, ICE-CBF-COR signaling pathways, carbohydrate metabolism pathways, antioxidant pathways, and transcription factor regulatory pathways. Current research on tea plant cold resistance largely focuses on the functional analysis of single or a few genes, with limited research on systemic network regulatory mechanisms. The discovery and functional study of key new cold-resistance genes urgently need to be strengthened. The tea plant CsCRAP gene is a novel cold-resistance gene discovered through large-scale gene co-expression network analysis using transcriptome big data. Its expression is significantly upregulated by low temperature. Exploring the biological function of the tea plant CsCRAP gene in regulating tea plant cold resistance will help clarify the complex molecular mechanisms of tea plant cold resistance, provide new insights into the study of tea plant cold resistance mechanisms, and provide important theoretical basis for breeding tea plant resistance traits.

[0004] The purpose of this invention is to provide a CsCRAP gene for tea trees and its application in regulating the cold resistance of tea trees, to solve the technical problems in the prior art of lacking key new genes for cold resistance in tea trees, unsystematic research on the molecular mechanism of cold resistance, and lack of core gene resources for cold resistance genetic improvement, to provide new ideas for the research on the cold resistance mechanism of tea trees, and to provide a theoretical and practical reference basis for the breeding of tea tree resistance traits. Summary of the Invention

[0005] The purpose of this invention is to provide a tea tree CsCRAP gene and its application in regulating the cold resistance of tea trees, so as to solve the above-mentioned problems.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The present invention proposes a CsCRAP gene for tea plants, characterized in that the nucleotide sequence of the CsCRAP gene for tea plants is shown in SEQ ID NO.1.

[0008] A tea plant CsCRAP protein, the amino acid sequence of which is shown in SEQ ID NO.2, is encoded by the tea plant CsCRAP gene.

[0009] A recombinant plasmid, wherein the recombinant plasmid is the tea plant CsCRAP gene as described in claim 1.

[0010] A genetically engineered bacterium, wherein the genetically engineered bacterium contains the recombinant plasmid described above.

[0011] An application of the tea tree CsCRAP gene in regulating plant cold tolerance involves overexpressing the tea tree CsCRAP gene in the plant to improve the plant's low-temperature tolerance.

[0012] The application of a tea plant CsCRAP protein in regulating plant cold tolerance, wherein the tea plant CsCRAP protein is used to enhance the plant's tolerance to low temperature stress.

[0013] A method to improve the cold resistance of tea trees involves overexpressing the tea tree CsCRAP gene as described in claim 1 within the tea tree to alleviate low-temperature damage to tea leaves.

[0014] The beneficial effects of this invention are as follows:

[0015] This invention is the first to clone and verify the tea plant CsCRAP gene and protein that regulate the tea plant's response to low-temperature stress. Both play a regulatory role in the tea plant's response to low-temperature stress, determining the formation of cold resistance. This invention provides a recombinant plasmid containing the tea plant CsCRAP gene and a transgenic engineered bacterium, enriching the research resources on tea plant cold resistance genes, providing new ideas for the study of tea plant cold resistance mechanisms, and offering a theoretical and practical reference basis for breeding tea plant resistance traits. It can solve the industry problem of reduced tea production and tea plant mortality caused by low-temperature disasters, promoting the sustainable development of the tea industry. Attached Figure Description

[0016] Figure 1 This is a diagram showing the expression of the CsCRAP gene and protein in tea plants. Among them, Figure 1 A is a diagram illustrating the expression pattern of the CsCRAP gene in tea plants under a 4℃ low-temperature treatment. Figure 1 B shows the difference in expression of CsCRAP protein in different tissues of the tea plant.

[0017] Figure 2Subcellular localization map of the CsCRAP gene in tea plant leaves.

[0018] Figure 3 The diagram shows the experimental pattern of CsCRAP gene overexpression in tea leaves, the change in expression level, the phenotypic analysis of overexpressed leaves under low temperature treatment, the Fv / Fm value, and the malondialdehyde content analysis.

[0019] Figure 4 Experimental diagrams for inhibiting the CsCRAP gene in tea plants using antisense oligonucleotides, including expression level changes, chlorophyll fluorescence in damaged plants, Fv / Fm ratio, and malondialdehyde (MDA) content analysis. Detailed Implementation

[0020] The present invention will be described in further detail below with reference to specific preparation and application examples and data. It should be understood that these examples are merely illustrative of the invention and are not intended to limit the scope of the invention in any way.

[0021] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. All primers used are indicated upon their first appearance, and subsequent use of the same primer will use the same indication as the initial indication.

[0022] Example 1:

[0023] The cloning and sequence structure analysis of the CsCRAP gene are carried out in the following steps:

[0024] Using the national-level superior tea variety Shuchazao as material, planted in the agricultural industrial park of Anhui Agricultural University, total RNA was extracted from young leaves. The RNA content and quality were detected by spectrophotometer using the RNAprep Pure Plant Kit according to the instructions.

[0025] First-strand cDNA generation via reverse transcription: Using 1 μg RNA as a template, the reaction system was prepared according to the PrimeScript II 1st StrandcDNA Synthesis Kit. Oligo dT Primer, Random 6 mers, dNTPMixture, and RNase-free dH2O were added to bring the total volume to 10 μL. The mixture was denatured at 65°C for 5 min and placed on ice. Then, 5×PrimerScript buffer, RNase Inhibitor, PrimeScript RTase, and dH2O were added to bring the total volume to 20 μL. The mixture was incubated at 42°C for 45 min and then at 95°C for 5 min to inactivate the reverse transcriptase.

[0026] PCR amplification was performed using cDNA first strand as the RT-PCR template. The upstream primer sequence was 5'-ATGTCTTTCCCGATACACTTGACCTTC-3', and the downstream primer sequence was 5'-TCATGATCGTCTGCTTACGAGAGG-3'. The 50 μL PCR reaction mixture consisted of: 25 μL 2×PhantaMax buffer, 1.0 μL dNTP Mix, 2 μL each of the upstream and downstream primers, 1 μL PhantaMax Supper-Fidelity DNA Polymerase, 2 μL template, and 17 μL ddH2O. The reaction program was: 95℃ for 3 min, 95℃ for 15 sec, 60℃ for 15 sec, 72℃ for 30 sec, 35 cycles, 72℃ for 5 min, and a final temperature of 4℃.

[0027] After purification and recovery, the PCR product was ligated into the PGEX4T-1 vector to obtain the PGEX4T-1::CsCRAP recombinant plasmid. This plasmid was transformed into *E. coli* competent cells DH5α. Positive clones were verified by colony PCR and then sequenced. The nucleotide sequence of the *CsCRAP* gene from the tea plant is shown in SEQ ID NO.1 of the sequence listing, as follows:

[0028] ATGTCTTTCCCGATACACTTGACCTTCTTCCTGTCATATTGCTCTTCCTCAAAAGCTTTTCATGGTACATCAACTATGAGTACATGTTTGAAGGTTTCTTTGATCATGTCAAACTTGTGTTCATATTGTCACCATTGATACTTCTGCTAGTTATATCTGTCCTCTCCAAC TTTGGAACCGGGCCACGGCGGAGGTCTCCGATTTTCCTACAAGATTCTCCTGATAGGGCTGGTGGAATTCCGTGGCGTGTTGGTTTTCATCCTTATGCTTCTCTTCTTTATGATCTCCTATCAATCTTCCTTTCAAGAGCGTTGGTTCCCTCTCGTAAGCAGACGATCATGA.

[0029] The amino acid sequence of the tea plant CsCRAP protein, encoded by the tea plant CsCRAP gene, is shown in SEQ ID NO.2, as detailed below:

[0030] MSFPIHLTFFLVILLFLKSFSWYINYEYMFEGFFDHVKLVFILSPLILLLVISVLSNFGTGPRRRSPIFLQDSPDRAGGIPWRVGFILMLLLFFMISYQSSFQERWFPLVSRRS.

[0031] Example 2:

[0032] The expression analysis of the CsCRAP gene was performed using the following steps:

[0033] (1) Expression of CsCRAP gene in different tissues of tea plant:

[0034] Gene expression was analyzed using six representative tissues from the national-level superior tea variety, Shuchazaozao. These six tissues included buds, flowers, young leaves, mature leaves, stems, and roots. These samples were also used for total RNA extraction and first-strand cDNA synthesis. The reverse transcription product (cDNA first strand) was diluted 30-fold as a template. A 20 μl reaction mixture was prepared using Hieff™ qPCR SYBR® GreenMaster Mix (No Rox) (Yeasen, Shanghai, China): 2.0 μl of 30-fold diluted reverse transcription product, 0.4 μl each of forward and reverse primers (10 pmol / μl), 10 μl of Hieff™ qPCR SYBR® Green Master Mix, and 7.2 μl of ddH2O. Each reaction was performed in triplicate. Then, on a Bio-rad CFX-96 instrument, the following program was used: ① 95℃ for 5 min ② 95℃ for 10 sec, 60℃ for 30 sec, 72℃ for 30 sec for 39 cycles ③ Melting curves were plotted from 65℃ to 95℃ at a rate of 0.1°C / sec. The upstream primer was (5'- TGTCTTTCCCGATACACTTGACC -3'), and the downstream primer was (5'- CCACCAGCCCTATCAGGAGA -3'). Using the tea plant ACTIN gene as an internal control, the upstream primer was (5'- GCCATATTTGATTGGAATGG-3'), and the downstream primer was (5'- GGTGCCACAACCTTGATCTT-3'). The relative expression levels of CsCRAP in different tissues were calculated using the instrument's built-in analysis software.

[0035] (2) Analysis of the expression pattern of CsCRAP under low temperature stress:

[0036] Tea tree branches of uniform growth were selected for the experiment and placed in a 4℃ incubator. A control group was placed at room temperature. The first or second tender leaves of both the control and treated plants were collected at different time points (0h, 4h, and 12h) in triplicate. The leaves were then frozen in liquid nitrogen at -80℃ for analysis of CsCRAP gene expression. RNA extraction and quantitative PCR were performed using the same methods.

[0037] Figure 1 This study examines the expression changes of CsCRAP under 4℃ low-temperature treatment and the expression differences in different tissues of tea plants; among which... Figure 1 Figure A shows the expression pattern of the CsCRAP gene in tea plants under 4℃ low-temperature treatment. Figure 1 Figure B shows the differential expression of CsCRAP protein in different tissues of the tea plant. Figure 1 As shown, using the tea cultivar Shuchazao as the research material, CsCRAP expression was upregulated under low-temperature treatment, and the expression pattern in different tissues showed tissue specificity. Figure 1 The results of qPCR showed that CsCRAP expression was significantly induced and upregulated by low temperature. CsCRAP was highly expressed in older leaves and lowly expressed in other tissues, suggesting that CsCRAP may be closely related to the tea plant's response to low temperature stress.

[0038] Example 3:

[0039] The subcellular localization of the CsCRAP gene in tobacco leaves was determined using the following steps:

[0040] (1) Construction of CsCRAP-PCAMBIA1305_GFP vector:

[0041] PCR amplification was performed using the plasmid PGEX4T-1::CsCRAP as a template. The upstream primer was: (5'-GGACAGCCCAGATCAACTAGTATGTCTTTCCCGATAC

[0042] ACTTGACC -3'), downstream primer: (5'-GCCCTTGCTCACCATGGATCCT

[0043] GATCGTCTGCTTACGAGAGGG -3'). PCR products were used to confirm specificity by 1% agarose gel electrophoresis, followed by purification and elution using a FastPure Gel DNA Extraction Mini Kit. The PCAMBIA1305_GFP vector was linearized by double enzyme digestion, using Spe1 and BamH1 as digestion sites. The digestion system was: 1.4 μl ddH2O4, 5 μl 10ⅹK Buffer, 1.6 μl PCAMBIA1305_GFP Vector, 1 μl Spe1, and 1 μl BamH1, incubated at 37°C for 2 h. The digestion products were confirmed to have been cleaved by 1% agarose gel electrophoresis, followed by purification and elution. The linearized PCAMBIA1305_GFP vector and purified PCR product were recombinantly digested using a recombinase (GenRec Assembly Master Mix Kit, GENERAL BIOL). The reaction mixture consisted of 5 μl of 2×GenRec Assembly Master Mix, 1.5 μl of PCR product, and 3.5 μl of vector, incubated at 50 °C for 40 min. The resulting product was then transformed into competent E. coli DH5α cells (WEIDI, Shanghai, China). After positive clones were observed and verified by colony PCR, single clones were selected for sequencing to obtain the CsCRAP-PCAMBIA1305_GFP vector.

[0044] (2) Injection of tobacco using Agrobacterium-mediated permeation method:

[0045] The CsCRAP-PCAMBIA1305_GFP vector was transformed into Agrobacterium GV3101 using the freeze-thaw method, and positive clones were identified by colony PCR. Single clones verified by colony PCR were picked and inoculated into 3 ml of liquid LB medium (containing 50 μg / ml rifampicin). ＋ and 50mg / ml Kan ＋ Incubate at 28℃ and 200 rpm for 16-18 hours until OD reaches its maximum. 600 =0.8-1.2. Inoculate 3 ml of overnight cultured Agrobacterium into 50 ml of liquid LB medium containing (50 μg / ml rifampicin). ＋ and 50mg / ml Kan ＋ Incubate at 28℃ and 200 rpm for 6-8 hours, collect bacterial cells by centrifugation, and treat with 10 mM MgCl2 and 10 mM... 2- The bacterial cells were resuspended in a resuspending solution with (N-morpholino)ethanesulfonic acid adjusted to pH 5.6 until OD was reached. 600=0.75-0.78. Add 100 μM acetylsyringone (As) and DAPI to the bacterial solution, incubate in the dark at 28°C for 2 h, then use a disposable 1 ml syringe (without needle) to draw up the bacterial solution and inject it into the lower epidermis of tobacco leaves, allowing the bacterial solution to penetrate the entire leaf tissue. After treatment in the dark at room temperature for 48 h following injection, observe and photograph GFP fluorescence using a laser confocal microscope.

[0046] Figure 2 This is a subcellular localization map of the gene CsCRAP in tobacco leaves in Example 3 of the present invention, where DAPI is a nuclear dye, GFP is green fluorescent protein, BF (Bright Field) is a bright field image of CsCRAP-PCAMBIA1305_GFP, and Merge (Merged) is an image of the CsCRAP-PCAMBIA1305_GFP fusion. Figure 2 As shown, GFP: Green fluorescent protein; Bright Field: Bright field image of CsCRAP-PCAMBIA1305_GFP; Merged: Image of CsCRAP-PCAMBIA1305_GFP fusion. (Source: [Insert Source Here]) Figure 2 It can be seen that the green fluorescence of CsCRAP can be detected in the cell membrane of tobacco leaves through fusion expression with EGFP, indicating that the CsCRAP protein is localized in the cell membrane.

[0047] Example 4:

[0048] The specific steps for verifying the function of the CsCRAP gene in tea plants are as follows:

[0049] 1. Transient overexpression experiment in vivo:

[0050] (1) Construction of CsCRAP-PCAMBIA1305 vector:

[0051] Using cDNA plasmid as template, upstream primer: (5'-CAGATCACTAGTATG)

[0052] TCTTTCCCGATACACTTGACCTTC -3'), downstream primer: (5'- CACCA

[0053] PCR amplification was performed using the vector TGGATCCTCATGATCGTCTGCTTACGAGAGG -3'). The PCR product was confirmed for specificity by 1% agarose gel electrophoresis, followed by purification and elution. The PCAMBIA1305 vector was linearized by double enzyme digestion, using Spe1 and BamH1 as digestion sites. The digestion system was: 41.4 μl ddH2O, 5 μl 10ⅹK Buffer, 1.6 μl PCAMBIA1305_GFPVector, 1 μl Spe1, and 1 μl BamH1, incubated at 37℃ for 2 h. The digestion product was confirmed to have been cleaved by 1% agarose gel electrophoresis, followed by purification and elution. Recombinant reactions were performed using a recombinase (GenRec Assembly Master Mix Kit, GENERALBIOL) to cut the linearized PCAMBIA1305 vector and purified PCR product. The reaction mixture consisted of 5 μl of 2×GenRec Assembly Master Mix, 1.5 μl of PCR product, and 3.5 μl of vector, incubated at 50 °C for 40 min. The resulting product was then transformed into competent E. coli DH5α cells (WEIDI, Shanghai, China). After positive clones emerged and were verified by colony PCR, single clones that were verified were selected for sequencing to obtain the CsCRAP-PCAMBIA1305 vector.

[0054] (2) Agrobacterium injection permeation method for tea leaves:

[0055] The CsCRAP-PCAMBIA1305 vector was transformed into Agrobacterium GV3101 using the freeze-thaw method, and positive clones were identified by colony PCR. Single clones verified by colony PCR were picked and inoculated into 3 ml of liquid LB medium (containing 50 μg / ml Rifampicin). ＋ and 50mg / ml Kan ＋ Incubate at 28℃ and 200 rpm for 16-18 hours until OD reaches its maximum. 600 =0.8-1.2. Inoculate 3 ml of overnight cultured Agrobacterium into 50 ml of liquid LB medium containing (50 μg / ml rifampicin). ＋ and 50mg / ml Kan ＋ Incubate at 28℃ and 200 rpm for 6-8 hours, collect bacterial cells by centrifugation, and treat with 10 mM MgCl2 and 10 mM... 2- The bacterial cells were resuspended in a resuspending solution with (N-morpholino)ethanesulfonic acid adjusted to pH 5.6 until OD was reached. 600=0.9-1. Add 1 / 1000 100μM acetylsylgenone (As) to the bacterial solution and incubate in the dark at 28℃ for 2 hours. Select healthy early-maturing tea seedlings of the same growth period, make a small incision on the lower epidermis of the leaves 1-2 cm from the leaf tip or petiole, and inject the bacterial solution using a disposable 1ml syringe (without the needle) to allow the solution to penetrate the entire leaf tissue. After injection, treat the seedlings in the dark at room temperature for 24 hours, then resume normal culture for 24 hours. Quickly fix the injected leaves with liquid nitrogen and store them at -80℃.

[0056] (3) Low-temperature treatment and biochemical index determination of overexpression samples:

[0057] To investigate the role of CsCRAP in the response of tea plants to low-temperature stress, CsCRAP-overexpressing samples were treated at -2℃ for 2 h, then allowed to recover to room temperature for 30 min, and changes in chlorophyll fluorescence intensity (FV / FM) were observed. Subsequently, the malondialdehyde (MDA) content of the low-temperature treated samples was determined according to the method described in the kit (catalog number: BC0020) (Solarbio, Beijing, China).

[0058] 2. In vivo antisense oligonucleotide inhibition experiment:

[0059] (1) Antisense oligonucleotide primer design:

[0060] Based on the coding sequence of the CsCRAP gene, the sense strand (sODN) and antisense strand (AsODN) of its oligonucleotides were designed using the nucleic acid statistical folding and regulatory RNA research software developed by the Bioinformatics Center of the New York State Department of Health (http: / / sfold.wadsworth.org / cgi-bin / soligo.pl). The primer sequences are shown below:

[0061] AsODN-1, (5' - GGAAGAGCAATATGACAAGGA -3');

[0062] AsODN-2, (5' - AAAATCGGAGACCTCCGCCGT -3');

[0063] AsODN-3, (5' - AGGTCAAGTGTATCGGGAAAGACAT -3');

[0064] AsODN-4, (5' - GAATCTTGTAGGAAAATCGGAGAC -3');

[0065] Primers were dissolved in sterile water to prepare oligonucleotide primer solutions, with sterile water as a control. New tea shoots of the same growth period and in good condition were selected, and their bottom ends were immersed in 1.5 ml centrifuge tubes containing 1 ml of 20 μM primer solution. After culturing the centrifuge tubes in a room temperature light incubator for 6 hours, the shoots were quickly fixed with liquid nitrogen and stored at -80°C.

[0066] (2) Analysis of antisense oligonucleotide inhibition of CsCRAP expression:

[0067] Total RNA was extracted from both the treated and control samples, and first-strand cDNA was synthesized via reverse transcription. The expression of the CsCRAP gene was then detected using qPCR. The results showed that CsCRAP expression was significantly inhibited.

[0068] (3) Low-temperature treatment and biochemical index determination of antisense oligonucleotide-inhibited samples:

[0069] To investigate the role of CsCRAP in the response of tea plants to low-temperature stress, samples with inhibited CsCRAP expression were treated at -2℃ for 1 h, then allowed to recover to room temperature for 30 min, and changes in chlorophyll fluorescence intensity (FV / FM) were observed. The malondialdehyde (MDA) content of the subsequently low-temperature treated samples was determined according to the kit (catalog number: BC0020) (Solarbio, Beijing, China). Approximately 0.1 g of freshly ground sample powder was added to 1 ml of extraction buffer and homogenized in an ice bath. The mixture was centrifuged at 8000 g at 4℃ for 10 min, and the supernatant was placed on ice for testing. In the experimental group, 600 μl of MDA detection working solution, 200 μl of the test sample, and 200 μl of reagent three were added sequentially. In the control group, 600 μl of MDA detection working solution, 200 μl of distilled water, and 200 μl of reagent three were added sequentially. The mixture was incubated in a 100℃ water bath for 60 min, then cooled in an ice bath and centrifuged at 10000 g at room temperature for 10 min. Transfer the supernatant to a 1 ml glass cuvette and measure the absorbance of each sample at 450 nm, 532 nm, and 600 nm. Calculate ΔA450 = A450 for each sample. 测定 -A450 空白 ΔA532=A532 测定 -A532 空白 ΔA600=A600 测定 -A600 空白 MDA content (nmol / g) = 5 * [6.45 * (ΔA532 - ΔA600) - 1.29 * ΔA450] / W.

[0070] Figure 3In the diagram, A, B, C, D, and E represent the experimental pattern of gene CsCRAP overexpression in tea leaves, the graph of expression level change, the phenotypic analysis of overexpressed leaves under low-temperature treatment, the Fv / Fm value graph, and the malondialdehyde content analysis graph, respectively. Figure 3 As shown, in vivo overexpression of CsCRAP significantly alleviated low-temperature damage to tea leaves, resulting in a significant increase in the Fv / Fm ratio and a significant decrease in malondialdehyde content.

[0071] Figure 4 In the diagram, A, B, C, D, and E represent the experimental model, expression level change, chlorophyll fluorescence of damaged plants, Fv / Fm value, and malondialdehyde content analysis of antisense oligonucleotide inhibition of CsCRAP in this embodiment of the invention, respectively. Figure 4 As shown, the expression level of CsCRAP was significantly inhibited in the antisense oligonucleotide inhibition experiment compared with the control. After low-temperature treatment, compared with the control group, the plants with inhibited CsCRAP expression showed greater damage, a significantly lower Fv / Fm ratio, and a significantly higher malondialdehyde content. These results indicate that the CsCRAP gene can positively regulate the low-temperature tolerance of tea plants.

[0072] Example 5:

[0073] The specific steps for constructing recombinant plasmids and transgenic engineered bacteria are as follows:

[0074] The tea tree CsCRAP gene from Example 1 was inserted into the PGEX4T-1 Vector, PCAMBIA1305, and PCAMBIA1305_GFP vectors to obtain recombinant plasmids. The recombinant plasmids were then transformed into Escherichia coli DH5α and Agrobacterium GV3101 to obtain transgenic engineered bacteria, which can be used for gene cloning, subcellular localization, and transient transformation of tea trees.

[0075] Example 6:

[0076] The specific steps for the cold-resistant breeding application of tea trees are as follows:

[0077] Using the CsCRAP gene of tea tree in Example 1 as a target, the gene was overexpressed in the tea tree through genetic engineering to obtain tea tree plants with enhanced cold resistance. Combined with conventional breeding techniques, stable and heritable cold-resistant tea tree varieties were selected to reduce the impact of low temperature disasters on tea production.

[0078] In summary, the expression pattern of CsCRAP is associated with the tea plant's response to low-temperature stress, and overexpression of this gene can increase the plant's cold resistance. Conversely, inhibiting CsCRAP expression using antisense oligonucleotide technology significantly increases leaf damage in tea plants under low temperatures. These results indicate that the CsCRAP protein and its encoding gene are involved in the tea plant's response to low-temperature stress. Cloning this gene not only facilitates the exploration of the role of new genes in the tea plant's cold resistance process but also helps to promote the genetic improvement process aimed at enhancing the cold resistance of tea plants, thus contributing to the sustainable development of the tea industry. This invention has significant application value.

[0079] This invention marks the first cloning and verification of the CsCRAP gene, which regulates the tea plant's response to low-temperature stress and plays a crucial regulatory role. The invention also provides a recombinant plasmid containing the CsCRAP gene and a transgenic engineered bacterium. This invention enriches the research on cold-resistance genes in tea plants, provides new insights into the mechanisms of cold resistance in tea plants, and offers a theoretical and practical reference basis for breeding tea plants with resistance traits.

[0080] The above is an exemplary description of the invention. Obviously, the specific implementation of the invention is not limited to the above-described manner. Any non-substantial improvement made using the inventive concept and technical solution of the invention, or the direct application of the inventive concept and technical solution to other situations without modification, is within the protection scope of the invention.

Claims

1. A CsCRAP gene for tea plants, characterized in that, The nucleotide sequence of the CsCRAP gene in the tea plant is shown in SEQ ID NO.

1.

2. A tea plant CsCRAP protein, characterized in that, The CsCRAP protein of the tea plant has the amino acid sequence shown in SEQ ID NO.2, and the CsCRAP protein of the tea plant is encoded by the CsCRAP gene of the tea plant as described in claim 1.

3. A recombinant plasmid, characterized in that, The recombinant plasmid contains the tea plant CsCRAP gene as described in claim 1.

4. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria comprises the recombinant plasmid as described in claim 4.

5. An application of the tea tree CsCRAP gene as described in claim 1 in regulating plant cold resistance, characterized in that, The CsCRAP gene of the tea plant was overexpressed in the plant to improve the plant's low-temperature tolerance.

6. The application of the tea plant CsCRAP protein as described in claim 2 in regulating plant cold resistance, characterized in that, The tea plant CsCRAP protein is used to enhance the plant's tolerance to low-temperature stress.

7. A method for improving the cold resistance of tea trees, characterized in that, Overexpression of the tea plant CsCRAP gene as described in claim 1 in tea plants can alleviate low-temperature damage to tea leaves.

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