Tea tree expansin CsEXPA10 gene and application thereof in regulating and controlling cold resistance of tea tree

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a tea tree expansin CsEXPA10 gene and application thereof in regulating and controlling cold resistance of tea trees. The gene is a tea tree EXPANSIS family gene, the nucleotide sequence is shown as SEQ ID NO. 1, and the sequence of an encoded protein is shown as SEQ ID NO. 2. The gene is in a down-regulation expression mode at the low temperature of 4 DEG C and is a negative regulation factor of low-temperature response of the tea tree; overexpression of the gene can aggravate low-temperature damage of tea tree leaves, and low-temperature damage of tea tree shoots can be remarkably relieved by inhibiting expression of the gene through an antisense oligonucleotide technology. The invention further constructs recombinant plasmids and transgenic engineering bacteria containing the gene, the function of the gene in low-temperature stress response of the tea tree is defined for the first time, the research blank of the expansion protein in the field of cold-resistant regulation of the tea tree is filled, and a new thought is provided for research of a cold-resistant mechanism of the tea tree; and a gene target and a technical support are provided for genetic improvement of the cold resistance character of the tea tree.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the tea tree expansion protein CsEXPA10 gene and its application in regulating the cold resistance of tea trees. Background Technology

[0002] The tea plant (Camellia sinensis (L.) O. Kuntze) is an important economic crop primarily harvested for its tender buds, and it thrives in warm climates while being sensitive to cold. Tea leaves can be processed into beverages, containing various beneficial components and possessing health benefits. The development of the tea industry has both livelihood and national economic value. In recent years, tea trees have frequently suffered from frost and late spring cold snaps during the winter and early spring. Damage to the new shoots of tea trees leads to significant losses in tea yield and quality, and in severe cases, can even cause the death of the tea trees. Low temperatures have become one of the most significant and serious natural disasters in tea production, seriously threatening the healthy and sustainable development of the tea industry.

[0003] Expansis are important components of plant cell walls and are widely distributed in various plant cell tissues. Under normal plant growth conditions, their levels are low, but they can be rapidly induced to increase several times or even hundreds of times during specific developmental stages or when stimulated by external environmental factors. Existing research indicates that expansis participate in multiple growth and developmental stages, including seed germination, root hair initiation and elongation, leaf growth and development, petiole abscission, pollen tube elongation, and fruit ripening. They also participate in various stress responses, such as drought resistance, disease resistance, and heat tolerance. However, the role of expansis in the tea plant's response to low-temperature stress remains unclear. The lack of specific genes for genetic improvement of tea plant cold resistance hinders research into the mechanisms of cold resistance at the genetic level and makes targeted breeding of cold-resistant traits in tea plants difficult. This has become a key issue restricting the development of cold-resistant breeding technology for tea plants. Summary of the Invention

[0004] The purpose of this invention is to provide a tea tree expansion protein CsEXPA10 gene and its application in regulating tea tree cold resistance, thereby overcoming the deficiency in the existing technology where the function of expansion proteins in tea tree low-temperature stress response is unclear, clarifying the regulatory relationship between the CsEXPA10 gene and tea tree cold resistance, providing new ideas for the study of tea tree cold resistance mechanisms, and providing specific gene targets and practical application schemes for breeding tea tree cold resistance traits, thus solving the technical problem of reduced yield and quality of tea trees due to low-temperature stress.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] The present invention proposes a tea plant expansin CsEXPA10 gene, which is a gene of the tea plant EXPANSIS family, and its nucleotide sequence is shown in SEQ ID NO.1 of the sequence listing.

[0007] Preferably, the amino acid sequence of the protein encoded by the tea tree expansion protein CsEXPA10 gene is shown in the sequence listing SEQ ID NO.2.

[0008] A recombinant plasmid containing the tea tree expansion protein CsEXPA10 gene, the recombinant plasmid comprising: a PGEX4T-1::CsEXPA10 plasmid obtained by ligating the CsEXPA10 gene to a PGEX4T-1 vector, or a CsEXPA10-PCAMBIA1305 vector obtained by ligating the CsEXPA10 gene to a PCAMBIA1305 vector.

[0009] A transgenic engineered bacterium containing a recombinant plasmid, wherein the transgenic engineered bacterium is an engineered bacterium obtained by transforming the recombinant plasmid into Escherichia coli DH5α, or an engineered bacterium obtained by transforming the recombinant plasmid into Agrobacterium GV3101.

[0010] An application of the tea tree expansion protein CsEXPA10 gene in regulating the cold resistance of tea trees: inhibiting the expression of the CsEXPA10 gene in tea trees and improving the low temperature tolerance of tea trees.

[0011] Preferably, the application is applicable to low-temperature stress scenarios caused by frost or late spring cold snaps in tea trees, wherein the low temperature range is -2℃ to 4℃, and the application is used for genetic improvement breeding of cold resistance traits in tea trees.

[0012] The beneficial effects of this invention are as follows:

[0013] (1) This invention cloned and verified for the first time the CsEXPA10 gene, which regulates the response of tea trees to low temperature stress. It was found that the gene belongs to the EXPANSIS family of tea trees and is a negative regulator of the low temperature response of tea trees. This fills the research gap of the expansion protein in the field of tea tree low temperature stress response in the existing technology and provides a new idea for the study of the cold resistance mechanism of tea trees.

[0014] (2) This invention clarifies the nucleotide sequence of the CsEXPA10 gene and the amino acid sequence of its encoded protein. It also provides cloning primers, detection primers, recombinant plasmids containing the gene, and transgenic engineered bacteria, providing complete technical support for the subsequent research and practical application of the gene and enriching the research system of the function of expansion protein in tea.

[0015] (3) This invention confirms that inhibiting the expression of the CsEXPA10 gene can significantly improve the low temperature tolerance of tea trees, and provides specific inhibition techniques, namely antisense oligonucleotide technology, which provides clear gene targets and operable technical solutions for breeding tea trees with resistance traits. It can promote the genetic improvement process aimed at enhancing the cold resistance of tea trees, effectively alleviate the impact of low temperature stress on tea tree yield and quality, and promote the sustainable development of the tea industry.

[0016] (4) This invention verified the regulatory function of the CsEXPA10 gene on the cold resistance of tea trees from both positive and negative aspects through in vivo transient overexpression experiment and antisense oligonucleotide inhibition experiment. The experimental results are reliable and lay a solid experimental foundation for the practical application of this gene. Attached Figure Description

[0017] Figure 1 This is a diagram illustrating the expression pattern of the CsEXPA10 gene in tea plants. Among them, Figure 1 Figure 1A shows the expression pattern of the CsEXPA10 gene in tea plants under 4℃ low-temperature treatment for 0h, 6h, and 24h. Figure 1B shows the expression of the CsEXPA10 gene in different tissues of tea plants.

[0018] Figure 2 This is a composite image showing the results of functional validation of the CsEXPA10 gene in tea plants. Figure 2 Figure 2A shows the expression analysis of the CsEXPA10 gene overexpression in tea leaves. Figure 2B shows the phenotypic analysis of CsEXPA10 overexpressing leaves under -2℃ low-temperature treatment. Figure 2C shows the Fv / Fm values ​​of CsEXPA10 overexpressing leaves under -2℃ low-temperature treatment. Figure 2D shows the malondialdehyde content analysis of CsEXPA10 overexpressing leaves under -2℃ low-temperature treatment. Figure 2 E represents the fluorescence of damaged chlorophyll in tea plants where the antisense oligonucleotide CsEXPA10 was inhibited. Figure 2 F is a graph showing the change in the expression level of this gene in tea plants where antisense oligonucleotides inhibit CsEXPA10. Figure 2 G represents the Fv / Fm values ​​of tea plants treated with antisense oligonucleotides that inhibited CsEXPA10 at -2℃. Figure 2 H represents the malondialdehyde (MDA) content analysis of tea plants treated with antisense oligonucleotides that inhibited CsEXPA10 at -2℃. Detailed Implementation

[0019] 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.

[0020] 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.

[0021] Example 1:

[0022] The cloning and sequence structure analysis of the CsEXPA10 gene were performed using the following steps:

[0023] 1. Extraction of total RNA:

[0024] Tender leaves of the national-level superior tea variety "Shuchazao" were collected, and total RNA was extracted using the RNAprep Pure Plant Kit. The content and quality of RNA were detected by Nano Drop and gel electrophoresis, and qualified RNA was selected for subsequent experiments.

[0025] 2. Synthesis of the first strand of cDNA:

[0026] Take 1 μg of the above qualified RNA as a template and prepare the reaction system according to the PrimeScript II 1st Strand cDNASynthesis Kit instructions: add 0.6 μl of Oligo dT Primer (50 μM), 0.4 μl of Random 6mers (50 μM), and 1 μl of dNTP Mixture (10 mM each), and add RNase-free dH2O to a final volume of 10 μl. Denature at 65°C for 5 min and immediately cool on ice. Then add 4 μl of 5×PrimerScript buffer, 0.5 μl of RNase Inhibitor (40 U), and 1 μl of PrimeScript RTase (200 U) to the above reaction solution, and add dH2O to a final volume of 20 μl. Incubate at 42°C for 45 min and heat at 95°C for 5 min to inactivate the reverse transcriptase to obtain the first strand of cDNA. Take an appropriate amount of the reverse transcription product for subsequent PCR amplification.

[0027] 3. PCR amplification of the CsEXPA10 gene:

[0028] Using the first strand of cDNA as a template for RT-PCR, PCR amplification was performed using cloning primer pairs. The upstream primer sequence (as shown in SEQ ID NO.3 in the sequence listing) was 5'-ATGTCTATTCAAGCATTTTTCATTGTTTCTCT-3', and the downstream primer sequence (as shown in SEQ ID NO.4 in the sequence listing) was 5'-TTAGAATTGACCGCCTTCA

[0029] AATGTTTGG-3'; The PCR reaction system was 50 μl, including 25 μl of 2×Phanta Max buffer, 1.0 μl of dNTPMix, 2 μl each of forward and reverse primers, 1 μl of Phanta Max Supper-Fidelity DNA Polymerase, 2 μl of template, and 17 μl of ddH2O; The PCR reaction program was: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 sec, 59℃ annealing for 15 sec, 72℃ extension for 30 sec, for a total of 35 cycles, with a final extension at 72℃ for 5 min, and the reaction was terminated at 4℃.

[0030] 4. Gene cloning and sequencing:

[0031] After purification and recovery of the CsEXPA10 gene product obtained by PCR amplification, it was ligated into the PGEX4T-1 Vector to obtain the PGEX4T-1::CsEXPA10 plasmid. This plasmid was transformed into E. coli DH5α competent cells and cultured until positive clones grew. After verification by colony PCR, single clones that were verified were selected for sequencing. The nucleotide sequence of the tea plant expansile protein CsEXPA10 gene was completely consistent with SEQ ID NO.1 in the sequence listing, and the amino acid sequence of the protein encoded by the obtained tea plant expansile protein CsEXPA10 gene was completely consistent with SEQ ID NO.2 in the sequence listing.

[0032] SEQ ID NO.1 is the nucleotide sequence of the tea plant dilatation protein CsEXPA10 gene, specifically:

[0033] ATGTCTATTCAAGCATTTTTCATTGTTTCTCTTCCACTCTTCCTCTTCTTCAATTTGTGCCTCCATGGCGCTTATGGTGACTATGGAGGAGGGTGGCAAGGCGCTCATGCAACTTTTTATGGTGGGGGTGATGCAACCGGCACAATGGGAGGTGCCTGTGGATATGGAAATTTGTATAGCCAAGGTTATGGCACCAACACGGCAGCACTTAGCACTGCTCTGTTCAACAATGGCTTGAGTTGTGGGGCGTGCTATCAGATCAAATGCAATGATGACCCACAATGGTGCCTCCCTGGAACCATCACTGTGACTGCTACAAACTTTTGCCCACCTAACCCTTCTCAGTCTAATGACAATGGAGGCTGGTGCAACCCTCCCCTCCAACATTTTGATCTAGCAGAGCCTGCTTTCTTGCAAATTGCCCAATACAGAGCTGGAATTGTTCCCGTATCTTTCCAAAGGGTGCCTTGTGTAAAGAAAGGAGGAATAAGATTTACAATCAATGGTCACTCTTACTTCAATCTGGTCTTAGTTAGCAACGTTGGAGGCGCAGGGGACGTCCAGTCGGTGTCGATCAAAGGGTCCAACACAGGATGGCAACCCATGTCAAGGAATTGGGGCCAAAACTGGCAAAGTAACTCCTTACTCAATGGTCAGTCTCTCTCATTTCAAGTCACCACTAGTGATGGAAGGACCGTTACCAGCTACAATGCGGTGCCAGCTAATTGGCAGTTTGGCCAAACATTTGAAGGCGGTCAATTCTAA。

[0034] SEQ ID NO.2 is the amino acid sequence of the protein encoded by the tea tree expansin CsEXPA10 gene, specifically:

[0035] MSIQAFFIVSLPLFLFFNLCLHGAYGDYGGGWQGAHATFYGGGDATGTMGGACGYGNLYSQGYGTNTAALSTALFNNGLSCGACYQIKCNDDPQWCLPGTITVTATNFCPPNPSQSNDNGGWCNPPL QHFDLAEPAFLQIAQYRAGIVPVSFQRVPCVKKGGIRFTINGHSYFNLVLVSNVGGAGDVQSVSIKGSNTGWQPMSRNWGQNWQSNSLLNGQSLSFQVTTSDGRTVTSYNAVPANWQFGQTFEGGQF.

[0036] Example 2:

[0037] The expression analysis of the CsEXPA10 gene was performed using the following steps:

[0038] 1. Analysis of the expression pattern of the CsEXPA10 gene in different tissues of tea plants:

[0039] Six representative tissues of Shuchazao were selected: tender buds, flowers, tender leaves, mature leaves, stems, and roots. Total RNA was extracted from each tissue and the first strand of cDNA was synthesized. The reverse transcription product was diluted 30 times as a template for qPCR.

[0040] qPCR primer pairs were used for detection. The upstream primer sequence was 5'-GGGTCCAACACAGGATGGC-3', and the downstream primer sequence was 5'-CCAATTAGCAGGCACCGCAG-3'. The tea plant ACTIN gene was used as a quantitative internal control. Its upstream primer sequence was 5'-GCCATATTTGATTGGAATGG-3', and the downstream primer sequence was 5'-GGTGCCACAACCTTGATCTT-3'.

[0041] The qPCR reaction volume was 20 μl, including 2.0 μl of reverse transcription product diluted 30-fold, 0.4 μl each of forward and reverse primers (10 pmol / μl), and 10 μl of Hieff. TM qPCR SYBR ®Green Master Mix and 7.2 μl ddH2O were used, with three replicates for each reaction. qPCR reactions were performed on a Bio-rad CFX-96 instrument. The reaction program was: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 10 sec, 60℃ annealing for 30 sec, and 72℃ extension for 30 sec, for a total of 39 cycles. Finally, melting curves were plotted from 65℃ to 95℃ at a rate of 0.1°C / sec. The relative expression levels of the CsEXPA10 gene in different tissues were calculated using the instrument's built-in analysis software. The results are shown in Figure 1(B). Figure 1 (B) is a graph showing the expression of the CsEXPA10 gene in different tissues of tea plants. The horizontal axis represents the tissue type of tea plants, including flowers, terminal buds, young leaves, mature leaves, stems, and roots. The vertical axis represents the relative expression level of the CsEXPA10 gene. The results show that the gene is highly expressed in flowers and lowly expressed in mature leaves, exhibiting tissue specificity.

[0042] 2. Analysis of the expression pattern of the CsEXPA10 gene under low temperature stress:

[0043] Tree branches of early-maturing tea plants with uniform growth were selected and divided into treatment and control groups. The treatment group was subjected to a 4℃ low-temperature treatment, while the control group was kept at room temperature. The first or second tender leaves of both groups were collected at 0h, 6h, and 24h, with three replicates at each time point. After collection, the leaves were frozen in liquid nitrogen and stored at -80℃. RNA was extracted and cDNA was synthesized according to the method described in "Analysis of Expression Pattern of CsEXPA10 Gene in Different Tissues of Tea Trees" and qPCR was performed to analyze the changes in the expression level of the CsEXPA10 gene. The results are shown in Figure 1(A). Figure 1(A) shows the expression pattern of the CsEXPA10 gene in tea trees under 4℃ low-temperature treatment at 0h, 6h, and 24h. The horizontal axis represents the low-temperature treatment time, and the vertical axis represents the relative expression level of the CsEXPA10 gene. The results show that the gene exhibits a downregulated expression pattern under low-temperature treatment, indicating that the CsEXPA10 gene is directly related to the tea tree's response to low-temperature stress, suggesting that it may negatively regulate the cold resistance of tea trees.

[0044] Example 3:

[0045] The functional verification of the CsEXPA10 gene in tea plants was carried out through the following steps:

[0046] 1. Transient overexpression experiment in vivo:

[0047] (1) Construction of the CsEXPA10-PCAMBIA1305 vector:

[0048] Using cDNA plasmid as a template, PCR amplification was performed using primers with restriction enzyme sites. The upstream primer sequence was 5'-CAGATCACTAGTATGTCTATTCAAGCATTTT

[0049] TCATTGTTTCTCT-3', the downstream primer sequence is 5'-CACCATGGATCCTTAG

[0050] AATTGACCGCCTTCAAATGTTTTGG-3';

[0051] The PCR products were purified and eluted after confirming their specificity by 1% agarose gel electrophoresis. The PCAMBIA1305 vector was linearized by double enzyme digestion at both Spe1 and BamH1 sites. The digestion system consisted of 41.4 μl ddH2O, 5 μl 10ⅰK Buffer, 1.6 μl PCAMBIA1305_GFP Vector, 1 μl Spe1, and 1 μl BamH1, incubated at 37℃ for 2 h. The digested products were purified and eluted after confirming their specificity by 1% agarose gel electrophoresis. Recombinant reactions were then performed on the linearized PCAMBIA1305 vector and the purified PCR products using recombinase (GenRec Assembly Master Mix Kit, GENERALBIOL). The recombination system consisted of 5 μl 2×GenRec Assembly Master Mix, 1.5 μl PCR product, and 3.5 μl Vector, incubated at 50℃ for 40 min. The recombinant products were then transformed into *E. coli* DH5α. Competent cells were cultured until positive clones appeared. After colony PCR verification, correct single clones were picked and sequenced to obtain the CsEXPA10-PCAMBIA1305 vector.

[0052] (2) Agrobacterium injection permeation method to achieve CsEXPA10 gene overexpression:

[0053] The CsEXPA10-PCAMBIA1305 vector was transformed into Agrobacterium GV3101 using the freeze-thaw method. Positive clones were identified by colony PCR. Verified single clones were picked and inoculated into 3 ml of a solution containing 50 μg / ml rifampicin. ＋ and 50mg / ml Kan ＋ In liquid LB medium, incubate at 28℃ and 200 r / min for 16-18 h until OD600 = 0.8-1.2;

[0054] Take 3 ml of the above-mentioned overnight Agrobacterium culture and inoculate it with 50 μg / ml rifampicin. ＋ and 50mg / ml Kan ＋In liquid LB medium, incubate at 28℃ and 200 rpm for 6-8 hours, collect bacterial cells by centrifugation, and then treat with 10 mM MgCl2 and 10 mM... 2- The bacterial cells were resuspended in a resuspension solution with pH adjusted to 5.6 using (N-morpholino)ethanesulfonic acid until OD600 = 0.9-1.

[0055] Add 1 / 1000 volume of 100μM acetylsyringone As to the bacterial solution and incubate in the dark at 28℃ for 2 hours. Select healthy early-maturing tea seedlings of the same period and make tiny wounds on the lower epidermis of the leaves, 1-2 cm away from the leaf tip or petiole. Use a 1ml disposable syringe with the needle removed to draw up the bacterial solution and inject it, allowing the bacterial solution to penetrate the entire leaf tissue. After injection, treat the tea seedlings in the dark at room temperature for 24 hours, and then resume normal culture for 24 hours. Take the injected leaves, fix them quickly with liquid nitrogen, and store them in a -80℃ freezer.

[0056] (3) Determination of physiological and biochemical indicators of overexpression samples at low temperature:

[0057] Leaf samples overexpressing the CsEXPA10 gene were subjected to low-temperature treatment at -2℃ for 2 h, and then restored to room temperature for 30 min before the chlorophyll fluorescence intensity Fv / Fm value was measured. The malondialdehyde content of the samples was determined according to the method of the Solarbio kit No. BC0020. The results are shown in Figures 2(A)-(D). Figure 2 (A) is an expression analysis diagram of the CsEXPA10 gene overexpression in tea leaves. The horizontal axis represents the experimental group, including the empty vector control group EV and the CsEXPA10 overexpression group OE-CsEXPA10. The vertical axis represents the relative gene expression level. The results show that the CsEXPA10 gene expression level in the overexpression group is significantly increased. Figure 2 (B) is a phenotypic analysis of CsEXPA10 overexpressing leaves under -2℃ low temperature treatment. The horizontal axis represents the experimental groups EV and OE-CsEXPA10, and the vertical axis represents the phenotypic indicators related to the degree of leaf damage. The results show that the degree of low temperature damage in the overexpressing group is significantly higher than that in the control group. Figure 2 (C) is a graph showing the Fv / Fm values ​​of CsEXPA10 overexpressing leaves under -2℃ low temperature treatment. The horizontal axis represents the experimental groups EV and OE-CsEXPA10, and the vertical axis represents the Fv / Fm value. The results show that the Fv / Fm value of the overexpression group is significantly lower than that of the control group. Figure 2(D) is a graph showing the malondialdehyde (MDA) content analysis of CsEXPA10 overexpressing leaves under -2℃ low-temperature treatment. The horizontal axis represents the experimental groups EV and OE-CsEXPA10, and the vertical axis represents the MDA content. The results show that the MDA content in the overexpression group is significantly higher than that in the control group. Therefore, it can be seen that after CsEXPA10 gene overexpression, the degree of low-temperature damage to the leaves is significantly aggravated, the Fv / Fm value is significantly reduced, and the MDA content is significantly increased, indicating that overexpression of the CsEXPA10 gene reduces the low-temperature tolerance of tea plants.

[0058] 2. Antisense oligonucleotide inhibition experiment in tea plant:

[0059] (1) Design and processing of antisense oligonucleotide primers:

[0060] Based on the coding sequence of the CsEXPA10 gene, positive and negative oligonucleotide primers 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 positive and negative primers were sODN-1 and sODN-2, with sODN-1 sequence being 5'-UGUCGAUCAAAGGGUCCAACACAG-3' and sODN-2 sequence being 5'-GUCGAUCAAAGGGUCCAACACAGG-3'. The negative primers were AsODN-1 and AsODN-2, with AsODN-1 sequence being 5'-CTGTGTTGGACCC.

[0061] TTTGATCGACA-3', AsODN-2 sequence is 5'-CCTGTGTTGGACCCT

[0062] TTGATCGAC-3'; Dissolve the primers in sterile water to prepare a 20 μM primer solution, using the positive strand primer solution as a control.

[0063] (2) Treatment of tea shoots with antisense oligonucleotides:

[0064] Select healthy new shoots of tea trees from the same period and immerse their bases in a 1.5 ml centrifuge tube containing 1 ml of the above-mentioned 20 μM primer solution. Incubate the tubes in a room temperature light incubator for 6 hours. After incubation, fix the new shoots with liquid nitrogen and store them in a -80℃ refrigerator.

[0065] (3) Detection of suppressed expression and determination of physiological and biochemical indicators at low temperature:

[0066] Total RNA was extracted from the new shoots of tea plants in both the treatment and control groups. The first strand of cDNA was synthesized, and the expression level of the CsEXPA10 gene was detected by qPCR. The results are as follows: Figure 2 As shown in (F). Figure 2 (F) is a graph showing the change in the expression level of CsEXPA10 in tea plants where antisense oligonucleotides inhibited it. The horizontal axis represents the experimental groups sODN and AsODN-CsEXPA10, and the vertical axis represents the relative gene expression level. The results show that the expression level of CsEXPA10 gene in the treatment group was significantly inhibited.

[0067] Tea shoot samples with suppressed gene expression were treated at -2℃ for 1 hour, and after recovering to room temperature for 30 minutes, the chlorophyll fluorescence intensity (Fv / Fm) was measured. Simultaneously, the malondialdehyde (MDA) content of the subsequently treated samples was determined according to the method of the Solarbio (Beijing, China) kit (catalog number BC0020). The specific procedure was as follows: 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 8000g at 4℃ for 10 minutes, and the supernatant was placed on ice for testing. In the experimental group, 600 μl of MDA detection working solution, 200 μl of the sample to be tested, 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 minutes, then cooled in an ice bath and centrifuged at 10000g at room temperature for 10 minutes. 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 The MDA content was calculated using the formula: MDA content (nmol / g) = 5 * [6.45 * (ΔA532 - ΔA600) - 1.29 * ΔA450] / W; the results are shown in Figures 2 (E), 2 (G), and 2 (H). Figure 2 (E) is a chlorophyll fluorescence image of tea plants damaged by antisense oligonucleotide inhibition of CsEXPA10. The horizontal axis represents the experimental group, including the positive control group sODN and the antisense oligonucleotide treatment group AsODN-CsEXPA10. The vertical axis represents the chlorophyll fluorescence intensity. The results show that the degree of chlorophyll fluorescence damage in the treatment group is significantly lower than that in the control group. Figure 2(G) is a graph showing the Fv / Fm values ​​of tea plants inhibited by antisense oligonucleotides CsEXPA10 under -2℃ low temperature treatment. The horizontal axis represents the experimental groups sODN and AsODN-CsEXPA10, and the vertical axis represents the Fv / Fm value. The results show that the Fv / Fm value of the treatment group is significantly higher than that of the control group. Figure 2 (H) is a graph showing the malondialdehyde (MDA) content analysis of tea plants with antisense oligonucleotide inhibition of CsEXPA10 under -2℃ low-temperature treatment. The horizontal axis represents the experimental groups sODN and AsODN-CsEXPA10, and the vertical axis represents the MDA content. The results show that the MDA content in the treatment group was significantly lower than that in the control group. Therefore, it can be seen that overexpression of CsEXPA10 significantly exacerbates low-temperature damage to tea leaves, leading to a significant decrease in the Fv / Fm ratio and a significant increase in MDA content. In the antisense oligonucleotide inhibition experiment, the expression level of CsEXPA10 was significantly inhibited compared to the control. After low-temperature treatment, compared to the control group, the plants with inhibited CsEXPA10 expression showed less damage, a significantly increased Fv / Fm ratio, and a significantly decreased MDA content. These results indicate that the CsEXPA10 gene can negatively regulate the low-temperature tolerance of tea plants.

[0068] In summary, CsEXPA10 expression responds to low-temperature stress in tea plants, and overexpression of this gene significantly reduces the cold tolerance of tea trees. Conversely, inhibiting CsEXPA10 expression using antisense oligonucleotide technology significantly reduces leaf damage under low temperatures. These results indicate that the CsEXPA10 protein and its encoding gene are negative regulators of the tea tree's low-temperature response and play a crucial role in tea tree cold tolerance. Cloning this gene not only facilitates the investigation of the role of dilatation proteins in tea tree cold tolerance but also contributes to promoting genetic improvement aimed at enhancing tea tree cold tolerance, thus promoting the sustainable development of the tea industry and possessing significant application value.

[0069] This invention marks the first cloning and verification of the dilatation protein CsEXPA10, which regulates the tea plant's response to low-temperature stress, and demonstrates its crucial regulatory role in this process. The invention also provides a recombinant plasmid containing the CsEXPA10 gene and a transgenic engineered bacterium. This invention enriches the research on dilatation proteins in tea plants, provides new insights into the cold resistance mechanism of tea, and offers a theoretical and practical reference basis for breeding tea plants with resistance traits.

[0070] 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 tea plant dilatation protein CsEXPA10 gene, characterized in that, The tea plant expansion protein CsEXPA10 gene is a gene of the tea plant EXPANSIS family, and its nucleotide sequence is shown in SEQ ID NO.1 of the sequence listing.

2. The tea plant dilatation protein CsEXPA10 gene according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the tea plant expansion protein CsEXPA10 gene is shown in the sequence listing SEQ ID NO.

2.

3. A recombinant plasmid containing the tea plant expansion protein CsEXPA10 gene as described in claim 1, characterized in that, The recombinant plasmids include: the PGEX4T-1::CsEXPA10 plasmid obtained by ligating the CsEXPA10 gene to the PGEX4T-1 vector, or the CsEXPA10-PCAMBIA1305 vector obtained by ligating the CsEXPA10 gene to the PCAMBIA1305 vector.

4. A transgenic engineered bacterium containing the recombinant plasmid of claim 3, characterized in that, The genetically engineered bacteria are either engineered bacteria obtained by transforming recombinant plasmids into Escherichia coli DH5α, or engineered bacteria obtained by transforming recombinant plasmids into Agrobacterium GV3101.

5. The application of the tea tree dilatation protein CsEXPA10 gene as described in claim 1 in regulating the cold resistance of tea trees, characterized in that, Inhibiting the expression of the CsEXPA10 gene in tea plants can improve their low-temperature tolerance.

6. The application of the tea tree dilatation protein CsEXPA10 gene according to claim 5 in regulating the cold resistance of tea trees, characterized in that, This application is applicable to low-temperature stress scenarios caused by frost and late spring cold snaps in tea trees, with the temperature range being -2℃ to 4℃. Furthermore, this application is used for genetic improvement breeding of cold-resistant traits in tea trees.

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