Application and method of tea tree CsGT-3b gene in improvement of plant disease resistance

Abstract

The invention discloses application of a tea tree CsGT-3b gene in improving the disease resistance of plants and a method of the tea tree CsGT-3b gene. The CsGT-3b gene is a Trihelx transcription factor family gene, the CsGT-3b gene is constructed to a plant expression vector and is transferred into tobacco, a transgenic tobacco plant is obtained through stable genetic transformation, and the CsGT-3b gene shows relatively strong fungal inhibitory activity on botrytis cinerea inoculation; according to the present invention, the CsGT-3b gene on the tea tree leaf is subjected to instantaneous silencing by using the antisense oligonucleotides (AsODNs) technology, such that the disease resistance of the plant on the lasiodiplodia theobroma is significantly weakened, and the CsGT-3b gene on the tea tree leaf is subjected to the enzyme-linked immunosorbent assay so as to significantly reduce the disease resistance of the plant on the lasiodiplodia theobroma; therefore, the CsGT-3b gene has the effect of improving the disease resistance of the plants, can be used as a disease-resistant gene, can improve the disease resistance of the plants by being introduced into the plants such as tobacco, tea trees or vegetables, and has a wide market application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and genetic engineering, specifically relating to the application and method of the CsGT-3b gene in tea trees in improving plant disease resistance. Background Technology

[0002] Tea (Camellia sinensis (L.) O. Kuntze) is a perennial evergreen shrub or small tree, used as an important economic crop. Our research group has isolated and identified several pathogenic fungi from tea leaves in various regions, including *Botrytis cinerea*, *Lasiodiplodia theobromae*, *Didymellasegeticola*, and *Epicoccum sorghinum*, which can cause diseases in tea leaves. These pathogenic fungi have a serious impact on the yield and quality of tea leaves. Previously, fungicides and other chemical pesticides were mainly used to control fungal diseases in crops. However, safety issues such as pesticide residues, damage to non-target organisms, and increased fungal resistance have led the scientific community to pay increasing attention to the use of pesticides. Developing plants with high resistance to fungal diseases through genetic engineering technology can serve as an effective way to control fungal diseases in crops.

[0003] Plant GT-3b is a member of the Trihelix transcription factor family and participates in various physiological and biochemical processes in plants. Utilizing genetic engineering to cultivate resistant plant varieties and materials has significant advantages and irreplaceable importance. It not only facilitates the large-scale production of plant varieties such as tea, vegetables, and tobacco with high resistance to fungal diseases but also reduces the use of chemical pesticides and environmental pollution. However, to date, there have been no reports on the disease resistance function of the GT-3b gene in tea. Summary of the Invention

[0004] In view of this, one of the objectives of the present invention is to provide an application of the CsGT-3b gene in tea trees, which plays a role in improving the disease resistance of plants and can be applied to improve the disease resistance of plants. The nucleotide sequence of the CsGT-3b gene is shown in SEQ ID NO.1, or the amino acid sequence of the protein encoded by the CsGT-3b gene is shown in SEQ ID NO.2.

[0005] Preferably, the disease resistance is resistance to diseases caused by pathogens including Botrytis cinerea or Diplostomum cocovenenans.

[0006] Preferably, the plant includes tobacco or tea tree.

[0007] A second objective of this invention is to provide a method for improving plant disease resistance, comprising introducing the CsGT-3b gene into a target plant to obtain plants with improved disease resistance; the nucleotide sequence of the CsGT-3b gene is shown in SEQ ID NO.1.

[0008] Preferably, the disease resistance is resistance to diseases caused by pathogens including Botrytis cinerea or Diplostomum cocovenenans.

[0009] Preferably, the CsGT-3b gene is introduced into the target plant via a plant expression vector.

[0010] Preferably, the plant expression vector includes the pBI121 vector. Other vectors, such as Ti-type plasmid vectors or viral vectors, can also be used.

[0011] Preferably, the import is performed via the leaf disc method.

[0012] Preferably, the plant includes tobacco or tea tree.

[0013] A third objective of this invention is to provide a plant with high disease resistance, wherein the expression of the CsGT-3b gene is increased in the plant.

[0014] This invention relates to the CsGT-3b gene, a Trihelix transcription factor family gene. CsGT-3b was constructed into a plant expression vector and transferred into tobacco. Transgenic tobacco plants were obtained through stable genetic transformation, exhibiting strong fungal inhibitory activity against *Botrytis cinerea* inoculation. Transient silencing of the CsGT-3b gene on tea leaves using AsODNs technology significantly reduced the plant's resistance to *Diplostomum cocovenenans*. Therefore, the CsGT-3b gene has the function of enhancing plant disease resistance and can be used as a disease resistance gene. Its introduction into plants such as tobacco, tea, or vegetables can improve plant disease resistance and has broad market application prospects. This invention provides a new method for improving plant resistance to fungal diseases. Cultivating disease-resistant plants through genetic engineering overcomes the shortcomings of traditional breeding methods, shortening the breeding cycle, simplifying the operation, and easily obtaining highly resistant materials. Attached Figure Description

[0015] Figure 1 This is an electrophoresis image of the CsGT-3b gene fragment from the tea plant, as described in this invention.

[0016] Figure 2 This is the phylogenetic tree of the CsGT-3b gene in tea plants according to the present invention;

[0017] Figure 3 This is an observation diagram of the subcellular localization of the CsGT-3b gene in tea plants in tobacco leaves according to the present invention.

[0018] Figure 4This is a PCR detection diagram of transgenic tobacco after overexpression of the CsGT-3b gene in this invention;

[0019] Figure 5 This is a diagram showing the enhanced resistance of tobacco to Botrytis cinerea after overexpression of the gene CsGT-3b according to the present invention; wherein, A is a phenotypic diagram of lesions after inoculation with Botrytis cinerea, B is a diagram of the expression level of the gene CsGT-3b in the Nicotiana benthamiana strain, and C is a comparison diagram of lesion area after inoculation with Botrytis cinerea.

[0020] Figure 6 This invention provides a phenotypic analysis of *Diplostomum cocovenenans* and a quantitative analysis of the CsGT-3b gene in tea plants inoculated with AsODN-suppressed CsGT-3b. Specifically, A represents the lesion phenotype of *Diplostomum cocovenenans* in tea plants inoculated with AsODN-suppressed CsGT-3b; B represents the quantitative expression analysis of the CsGT-3b gene in tea plants inoculated with AsODN-suppressed CsGT-3b; and C represents the lesion area of ​​tea plants inoculated with AsODN-suppressed CsGT-3b. Detailed Implementation

[0021] The present invention will be described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. The present invention is not limited to the following embodiments or examples. Any modifications and variations made without departing from the spirit of the present invention should be included within the scope of the present invention. Unless otherwise specified, the experimental materials or reagents used in the following embodiments are all conventional commercially available products.

[0022] 1. Data: The CsGT-3b gene sequence was downloaded from the tea tree genome database of Anhui Agricultural University. The CDS sequence of the CsGT-3b gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0023] 2. The strains used in this embodiment are: Botrytis cinerea (strain number: GZFQ-1 (accession number: CGMCC3.20932) and L. theobromae (strain number: GZHS-2017-010 (accession number: CGMCC3.20151)); all of the above strains were isolated and obtained by the inventors' team and can be obtained from the National Key Laboratory of Green Pesticides of Guizhou University.

[0024] 3. Preparation of some reagents

[0025] 1) Preparation of LB medium:

[0026] Yeast extract 5 g;

[0027] Tryptone 10 g;

[0028] 10 g of sodium chloride (NaCl);

[0029] For liquid culture medium, place the reagent in a 1 L glass beaker, add 1 L of ddH2O, stir with a glass rod until completely dissolved, and dispense 100 mL of liquid culture medium into 250 mL Erlenmeyer flasks. For solid LB medium, add 1.5 g of agar powder to every 100 mL of liquid LB medium.

[0030] 2) Preparation of 1 M morpholinoethanesulfonic acid (MES):

[0031] 2.132 g of morpholine ethanesulfonic acid was dissolved in 10 mL of ddH2O, filtered through an aqueous filter membrane (diameter = 0.22 μm), and stored at room temperature.

[0032] 3) Preparation of 200 mM acetylsuccinone (AS):

[0033] Dissolve 0.039 g of acetylsuccinone in 1 mL of dimethyl sulfoxide (DMSO), and store the solution at -20°C.

[0034] 4) Preparation of 1 M magnesium chloride (MgCl2):

[0035] 2.033 g of magnesium chloride was dissolved in 10 mL of ddH2O, autoclaved at 121°C for 20 min, and then stored in a refrigerator at 4°C.

[0036] 5) Preparation of 50 mg / mL kanamycin (Kana):

[0037] Dissolve 0.5 g of kanamycin in 10 mL of ddH2O, then filter using an aqueous filter membrane (diameter = 0.22 μm). Store the prepared solution at -20°C.

[0038] Experimental Example 1: Synthesis Validation and Sequence Analysis of the Full-Length CDS Fragment of the CsGT-3b Gene

[0039] The gene fragment was directly synthesized into a plasmid by Sangon Biotech (Shanghai) Co., Ltd. based on the CDS sequence of the CsGT-3b gene (as shown in SEQ ID NO. 1). The synthesized sequence was then verified to be correct by PCR and sequencing. The verification method was as follows: the CDS plasmid was used as a template for PCR amplification using primers shown in Table 2. After PCR, 50 μL was excised and purified using a gel, and then 5 μL was subjected to agarose gel electrophoresis. The bands met expectations. Figure 1The synthesized fragment was sent to a biotechnology company (Beijing Qingke Biotechnology) for sequencing. The sequencing results confirmed that the sequence of the synthesized fragment was consistent with the sequence shown in SEQ ID NO.1. Therefore, the CDS plasmid can be used as a template for the amplification of the target gene fragment in subsequent experiments.

[0040] The CsGT-3b protein sequence (SEQ ID NO.2) was subjected to multiple sequence alignment with protein sequences from the Arabidopsis thaliana and rice Trihelix family (http: / / planttfdb.gao-lab.org / ). Amino acid sequence alignment was performed using the Muscle function in MEGA 11 software (version 11.0.9), and a Neighbor-Joining (NJ) tree was constructed with 1000 guide repeats. The phylogenetic tree is shown below. Figure 2 As shown, CsGT-3b is most closely related to the Arabidopsis thaliana AtGT-3b gene and the rice OsGT-3b gene.

[0041] Experimental Example 2: Subcellular localization of the CsGT-3b gene

[0042] Based on the cDNA sequence of the tea plant CsGT-3b gene (as shown in SEQ ID NO.1) and the BamHI and SalI restriction sites of the pCAMBIA2300 vector, homologous recombination primers were designed to contain the homologous arms of the pCAMBIA2300 vector. The primer sequences are shown in Table 1. The subcellular localization vector plasmid of the CsGT-3b gene was constructed using conventional methods.

[0043] The constructed vector plasmid was transformed into Agrobacterium tumefaciens strain GV3101 using the freeze-thaw method and cultured at 28°C for 48 h until colonies appeared. Single colonies were picked and cultured in LB liquid containing Kana resistance for 18–24 h at 28°C until OD (Organic Discharge) was achieved. 600 The bacterial cells were collected at approximately 0.8 μM. They were centrifuged at 4000 rpm for 10 min and resuspended in a suspension of 10 mM MgCl2, 10 mM MES, and 200 μM AS. The OD value was then adjusted. 600 To a concentration of approximately 0.8. Select healthy tobacco plants and inject 1 mL of the infection solution into the lower epidermis of the leaves using a syringe, clearly labeling each injection site. After incubating the injected tobacco plants in the dark for 48 hours, cut tissue sections from the area near the injection site, observe the GFP (Green fluorescent protein) signal using a laser confocal microscope, and photograph and save the images. Figure 3 As shown, the CsGT-3b gene was observed to be located in the cell nucleus.

[0044] Table 1 Primers for constructing the CsGT-3b subcellular localization vector

[0045]

[0046] Example 3: Agrobacterium-mediated plant genetic transformation

[0047] 1. Construction of CsGT-3b gene overexpression vector

[0048] The plant expression vector pBI121 plasmid was extracted using the SanPrep column-based plasmid DNA mini-extraction kit (Shanghai Sangon Biotech) (extraction steps are detailed in the Shanghai Sangon Biotech kit instructions). The vector was linearized by double digestion of two cloning sites, XbaI and SacI. 1 μL of the extracted plasmid was used for 1.5% agarose gel electrophoresis to check its integrity and concentration. The DNA was purified using the Trelief DNA Gel Extraction Kit (Beijing Qingke Biotechnology) (specific steps are detailed in the instructions). The size and concentration of the recovered fragments were determined by 1.5% agarose gel electrophoresis. Homologous recombination primers containing the homologous arms of the pBI121 vector were designed; the primer sequences are shown in Table 2.

[0049] Using the ClonExpress II One Step Cloning Kit (Nanjing Novizan) and its instructions, after determining the concentrations of the vector fragment and the target gene, homologous recombination was performed using the following reagents: 5×CE Ⅱ Buffer (2 μL), Exnase Ⅱ (1 μL), linearized vector (6 μL), and target gene fragment (1 μL). The reaction program was 37℃ for 30 min. After the reaction, the mixture was immediately placed on ice and then transferred to E. coli DH5α. After ligation and transformation, the mixture was plated on LB agar containing Kana antibiotic. The bacterial culture was shaken for 12 h and then analyzed. Positive clones were sent to Qingke Biotechnology Co., Ltd. for sequencing to ensure successful ligation of the target sequence into the pBI121 vector, thus obtaining the overexpression vector.

[0050] Table 2 Primers for overexpression vector construction

[0051]

[0052] 2. CsGT-3b gene genetic transformation of tobacco

[0053] The constructed recombinant overexpression vector was transformed into Agrobacterium using the freeze-thaw method, and then transformed into tobacco for transgenic overexpression. The transgenic recipient tobacco in this experiment was Nicotiana benthamiana. The specific steps of transgenicization are as follows:

[0054] 1) Culture of aseptic tobacco seedlings

[0055] Tobacco seeds were soaked in 75% alcohol for 1 min, then sterilized with 15% H2O2 for 15 min, and then washed three times with water for 3 min each time. They were then spread on MS medium in a clean bench and cultured in a light incubator at 28°C for about 15 days.

[0056] 2) Transformation

[0057] In a clean bench, tobacco leaves were cut into 0.5×0.5 cm pieces. The tobacco leaves were then transferred into the prepared Agrobacterium tumefaciens solution (OD value around 0.8). After 5 min of inoculation, the leaves were blotted dry on sterile filter paper and then inoculated onto MS solid co-medium (1 / 2 MS + 30 g / L sucrose + 8 g / L agar, pH 5.8) and incubated in a constant temperature incubator at 22℃ in the dark for 2 days.

[0058] 3) Embryo induction

[0059] Transfer the leaves from the plate to the induction medium (MS + 0.5 mg / L BA + 30 g / L sucrose + 8 g / L agar + 500 mg / L CEF + 100 mg / L Kana, pH 5.8), with the wound facing up on the surface of the medium. After transfer, seal the plate with sealing film and place it in a light incubator for cultivation. Transfer the leaves to the same medium approximately every 15 days.

[0060] 4) Rooting

[0061] When small shoots emerged on the induction medium, they were transferred to rooting medium (MS + 0.1 mg / L NAA + 30 g / L sucrose + 8 g / L agar + 100 mg / L Kana, pH 5.8) and cultured under light conditions to induce shoot growth and root formation. The control plant was wild-type Nicotiana benthamiana.

[0062] 3. Detection of CsGT-3b gene in transgenic tobacco

[0063] DNA was extracted from transgenic tobacco using the cetyltrimethylammonium bromide (CTAB) method. PCR was performed using universal primers from the pBI121 vector (see Table 3). The PCR program consisted of Taq HS (0.25 μL), dNTP Mixture (4 μL), 10 × PCR Buffer (5 μL), DNA template (2 μL), forward and reverse primers (1 μL), and ddH2O (36.75 μL). The PCR program was 94℃ pre-denaturation for 2 min, 98℃ denaturation for 10 s, 55℃ annealing for 30 s, and 72℃ extension for 50 s, for 35 cycles. A final extension at 72℃ for 5 min was performed. After PCR, 5 μL was collected for agarose gel electrophoresis. Figure 4 As shown, the size of the target band is as expected, indicating successful transgenic modification.

[0064] Table 3 Primers for detecting transgenic plants

[0065]

[0066] Experiment Example 4: Disease Resistance Experiment with Transgenic Tobacco

[0067] Transgenic tobacco plants overexpressing the CsGT-3b gene and wild-type tobacco were inoculated with *Botrytis cinerea*. The lesion area was recorded 1, 2, and 3 days after inoculation, and the data were analyzed for significance. Specifically, *Botrytis cinerea* was inoculated onto PDA solid medium and incubated upside down at 25°C. When the *Botrytis cinerea* reached 3-4 days of growth, leaves were inoculated. Mycelia were broken into 6 mm diameter mycelial discs. Healthy tobacco leaves of similar shape and size without lesions were selected. Four small holes were made in each tobacco leaf, and the mycelial discs were inoculated onto the transgenic tobacco leaves using an inoculation needle, with the mycelial surface in contact with the upper surface of the leaf. The lesion size was recorded 1, 2, and 3 days after inoculation. Using lesion area as the metric, 28 lesion replicates were randomly selected. A ruler was used to take photos, and the images were imported into ImageJ software to calculate the lesion area. GraphPad Prism 9.0 software was used to analyze, organize, and plot the experimental data. Student's t-test was used for significance analysis. Experimental results showed that CsGT-3b transgenic tobacco exhibited significant resistance to the growth of Botrytis cinerea, such as... Figure 5 As shown, the relative expression level of the CsGT-3b gene in transgenic tobacco was significantly higher than that in wild-type tobacco. Figure 5 (B) When Botrytis cinerea was used to induce disease for 1, 2, and 3 days, the average lesion area of ​​the control wild-type tobacco was 1.32, 3.12, and 4.32 cm², respectively. 2 The average lesion area on CsGT-3b transgenic tobacco was 1.00, 2.34, and 3.40 cm², respectively. 2The average lesion area of ​​transgenic tobacco and wild-type tobacco within the same number of days was significantly different, with the lesions being significantly smaller in transgenic tobacco than in wild-type tobacco (*: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001). Figure 5 C). Therefore, CsGT-3b transgenic tobacco has a significant disease resistance effect, indicating that the CsGT-3b gene enhances the disease resistance of tobacco.

[0068] Experimental Example 5: Gene Expression Inhibition and Disease Resistance Experiment of CsGT-3b in AsODNs

[0069] The CDS sequence of the CsGT-3b gene was submitted, and antisense oligonucleotide fragments (OSFs) were designed using the Solido online tool (https: / / sfold.wadsworth.org / cgi-bin / soligo.pl / ). OSFs typically 20 bp in length were designed. Priority was given to selecting OSFs with low binding site disruption energy, and these sequences were reverse-complemented to obtain sense-chain specific OSFs. The artificially synthesized CsGT-3b OSF sequence (as shown in Table 4) was transferred into the young shoots of Fuding Dabaicha (Camellia sinensis cv. Fuding-dabaicha). The treatment group consisted of young shoots containing the antisense OSF of the target gene, while the control group consisted of young shoots containing the sense OSF. The treated young leaves were placed in a light incubator at 28℃ and 75% humidity, with a 16-hour light / 8-hour dark cycle. Leaf samples were collected every 12 hours, and RNA was extracted using a total RNA extraction kit. A total of four samples were collected, and the RNA was reverse transcribed into single-stranded cDNA using a reverse transcription kit. Quantitative primers were designed (sequences shown in Table 5), and the expression level of the target gene CsGT-3b in the treated and control groups was detected using qRT-PCR. Figure 6 As shown in Figure B, quantitative fluorescence detection was performed at 12, 24, 36, and 48 h after silencing the antisense oligonucleotide fragment. The expression level of CsGT-3b in the treatment group decreased significantly at all four time points, with the highest silencing efficiency at 24 h. Therefore, the pathogenicity experiment of *Dioscorea opposita* was conducted at 24 h. Using lesion area as the indicator, the disease incidence was observed after 24 h of *Dioscorea opposita* infection. Twenty-four lesion replicates were randomly selected, and images were taken using a ruler. The images were imported into ImageJ software to calculate the lesion area. The experimental data were analyzed, organized, and plotted using GraphPadPrism 9.0 software, and the significance of differences was analyzed using Student's t-test.

[0070] like Figure 6 As shown in A and 6C, the average lesion area of ​​the control plants was 0.227 cm².2 The average lesion area of ​​silent plants was 0.600 cm². 2 The lesions on the silenced plants were significantly larger than those on the control plants (*: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001). Therefore, silencing the CsGT-3b gene significantly weakened the disease resistance of the plants, further demonstrating that the CsGT-3b gene has a disease resistance function.

[0071] Table 4. CsGT-3b oligonucleotide fragments

[0072]

[0073] Table 5 Quantitative Primers

[0074]

[0075] In summary, this invention constructs CsGT-3b into a plant expression vector and transforms it into tobacco. Transgenic tobacco plants are obtained through stable genetic transformation, and these plants exhibit strong fungal inhibitory activity after inoculation with *Botrytis cinerea*. Furthermore, transient silencing of the CsGT-3b gene on tea leaves using antisense oligonucleotide technology significantly weakens the plant's resistance to *Diplostomum cocovenenans*. Therefore, the CsGT-3b gene plays a role in regulating plant disease resistance and has broad market application prospects in cultivating plants with high disease resistance.

[0076] The conventional techniques and solutions not described in detail in the above embodiments are all well known in the art, and therefore will not be elaborated upon here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. Application of the CsGT-3b gene in regulating plant disease resistance, wherein the nucleotide sequence of the CsGT-3b gene is shown in SEQ ID NO.1, or the amino acid sequence of the protein encoded by the CsGT-3b gene is shown in SEQ ID NO.

2.

2. The application as described in claim 1, characterized in that, The disease resistance refers to resistance to diseases caused by pathogens including Botrytiscinerea or Lasiodiplodia theobromae.

3. The application as described in claim 1, characterized in that, The plants mentioned include tobacco or tea trees.

4. A method for improving plant disease resistance, characterized in that, This includes introducing the CsGT-3b gene into a target plant to obtain plants with improved disease resistance; the nucleotide sequence of the CsGT-3b gene is shown in SEQ ID NO.

1.

5. The method as described in claim 4, characterized in that, The disease resistance refers to resistance to diseases caused by pathogens including Botrytis cinerea or Diplostomum cocovenenans.

6. The method as described in claim 4, characterized in that, The CsGT-3b gene was introduced into the target plant via a plant expression vector.

7. The method as described in claim 6, characterized in that, The plant expression vector includes the pBI121 vector.

8. The method as described in claim 4, characterized in that, The import is performed using the leaf disc method.

9. The method as described in claim 4, characterized in that, The plants mentioned include tobacco or tea trees.

10. A plant with high disease resistance, characterized in that, The expression of the CsGT-3b gene was increased in the plant.

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