Method for transient expression of genes in tea-oil tree leaves

Abstract

The invention belongs to the field of plant biotechnology and genetic engineering, and particularly relates to a method for transient expression of genes in tea-oil tree leaves. The method comprises the following steps: 1) preparing an agrobacterium infection solution of a plant expression vector containing a target gene, wherein the optimum OD600 value of the agrobacterium infection solution is 0.6-0.9; 2) using a disposable sterile syringe needle to perform scribing and injury making on the back of a non-in-vitro mature camellia oleifera leaf with the leaf age of 6-12 months, sucking the infection liquid in the step 1) into a syringe and abutting against the wound for injection until the infection liquid diffuses from the wound to the whole leaf; (3) immediately wrapping the leaves with tin foil paper, and carrying out shading treatment for 24-36 hours; and (4) detecting the expression condition of the target gene of the transgenic leaves on the second to sixth days after treatment. According to the method disclosed by the invention, instantaneous overexpression of the target gene of the camellia oleifera is realized by injecting mature leaves of the camellia oleifera, and functional verification of the target gene in a camellia oleifera body can be quickly and efficiently realized.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology and genetic engineering, and in particular relates to a method for realizing transient expression of a target gene in oil-tea camellia leaves. Background Art

[0002] Camellia oleifera Abel. is a general term for oil-producing species of the genus Camellia in the family Theaceae. It is also known as tea tree, white flower tea, and tea oil tree. It is a small tree or shrub. Camellia oleifera grows in the high hills of the subtropical region in southern China. It is a woody edible oil tree species unique to southern my country. It is known as the world's four major woody oil plants along with oil palm (Elaeisguineensis Jacq.), olive (Canarium album (Lour.) Raeusch.) and coconut (Cocos nucifera L.). Camellia oleifera is an excellent tree species with nutritional value, economic value and ecological value. Camellia oil has the reputation of "Oriental olive oil" and "soft gold in oil". The content of unsaturated fatty acids in camellia oleifera is more than 90%, of which oleic acid accounts for as much as 80%. It is rich in nutrients, easy to absorb, and has many benefits for human health.

[0003] Compared with traditional asexual line selection and hybrid breeding, molecular breeding can improve a specific trait and has great development potential. Existing studies have applied various molecular technologies such as genetics, multi-omics and molecular biology to the breeding of camellia oleifera. Previous researchers have also conducted research on transient transformation of protoplasts of camellia oleifera, but the protoplasts need to be separated, which is a relatively complicated operation. Since the transgenic system of camellia oleifera has not yet been established, when verifying the gene function of woody plants such as camellia oleifera, heterologous expression systems such as Arabidopsis or tobacco are usually used, which are quite different from the native expression and cannot obtain accurate results. Therefore, exploring an efficient transient expression system for camellia oleifera is crucial for the study of gene function.

[0004] Agrobacterium-mediated transient expression provides a method for rapid analysis of genotype function. Agrobacterium carrying a recombinant plasmid is induced and infiltrated into plant leaves and then into plant cells by vacuum infiltration. The efficiency of Agrobacterium-mediated T-DNA transfer in plants is detected by transient expression of the target gene (Kralemann LEM, De Pater S, Shen H, et al. Distinct mechanisms for genomic attachment of the 5'and 3'ends of Agrobacterium T-DNA in plants [J]. Nature Plants, 2022 (5): 8.). Subsequently, people used syringe injection into the leaves of living plants to conduct Agrobacterium-mediated transient gene expression detection, and successfully carried out transient gene transformation on the leaves of Artemisia annua, poplar, tea tree and other plants. The main principle of this method is: insert the target gene into a co-integration vector or a binary vector, transform Agrobacterium tumefaciens, which is induced by phenolic compounds and then injected into plant leaf tissue by vacuum infiltration or needle tube. Agrobacterium is in close contact with plant cells in the leaves, and the induction treatment activates the Vir region gene at the transcriptional level. Vacuum infiltration or injection allows Agrobacterium to contact plant leaf cells, thereby achieving T-DNA transfer into plant cell nuclei. Most T-DNAs are not integrated into the plant genome but temporarily exist in the nucleus and transiently express T-DNA genes with the assistance of plant cell transcription and translation components, usually reaching a peak 2-4 days after plant tissue infection, and then the number of expressing cells and the expression level of each transformed cell decrease (Krenek P, Amajova O, Luptovciak I, et al. Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications [J]. Biotechnology Advances, 2015, 33 (6): 1024-1042.). In recent years, this technology has been continuously improved and developed, and has been widely used in many fields of plant molecular biology, such as analysis of exogenous gene expression, interaction between non-toxic genes and resistance genes, gene silencing, promoter analysis, etc. Therefore, the establishment of an Agrobacterium-mediated transient transformation system for tea leaves is of great significance for the study of gene function in tea leaves. Summary of the invention

[0005] The invention provides a method for transiently expressing genes in tea leaves of oil tea through research and optimization.

[0006] The present invention first provides a method for transiently expressing genes in tea leaves, which is characterized by comprising the following steps:

[0007] 1) Prepare Agrobacterium infection solution containing the plant expression vector of the target gene. The OD of the Agrobacterium infection solution is 600 The values ​​range from 0.6 to 0.9;

[0008] 2) using a disposable sterile syringe to scratch and wound the back of a non-detached mature tea leaf, sucking the infection solution described in step 1) into the sterile syringe and injecting it at the wound until the infection solution spreads from the wound to the entire leaf;

[0009] 3) Then immediately wrap the leaves with tin foil to shade them for 24-36 hours;

[0010] 4) The expression of the target gene in the transgenic leaves was detected 2-6 days after treatment.

[0011] Specifically, the mature tea oil leaves are mature tea oil leaves with a leaf age of 6 to 12 months under natural growth conditions.

[0012] In a specific embodiment, the bacterial liquid in the Agrobacterium infection liquid is collected as follows:

[0013] a) Pour the shaken Agrobacterium liquid containing the recombinant target gene Camellia oleifera gene into a centrifuge tube and centrifuge at room temperature for 10 min, 4000 r / min -1 , discard the supernatant and collect the bacteria;

[0014] b) Resuspend the cells in transformation buffer and adjust the OD 600 The value is 0.6-0.9;

[0015] c) Cover with tin foil to block light and let stand at room temperature for 4 hours.

[0016] Specifically, the expression of transgenic leaves was detected 2-6 days after infection.

[0017] More preferably, the OD of the Agrobacterium infection solution is 600 The values ​​are 0.6 and 0.9.

[0018] The method of the present invention comprises selecting oil tea leaves of suitable age, suitable infection liquid OD 600 The combined effect of multiple factors such as the value, the transient transformation gene expression time, etc. has greatly improved the efficiency of transient transformation of tea leaves. Compared with the existing tea protoplast transient transformation method, the present invention has the advantages of convenient operation, low cost, short cycle, strong practicality, good effect, etc. It can quickly and efficiently realize the transient transformation of the target gene in the tea body, and provides important technical guarantee for the verification of gene function of woody plants and molecular assisted breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the flow chart of transient transformation of target gene in Camellia oleifera leaves.

[0020] Figure 2 The phenotypic changes after transient overexpression of CoERR113 gene in tea leaves of different leaf ages.

[0021] Figure 3 OD of different infection solutions 600 Effects of transient overexpression of CoERR113 gene on leaf phenotype under different treatment values. Note: A: This column is the OD of the infection solution. 600 Phenotypes of transient overexpression and control leaves when the value is 0.6; B: This column is the OD of the infection solution 600 Phenotypes of transient overexpression and control leaves when the value is 0.9; C: This column is the OD of the infection solution 600 The phenotypes of transient overexpression and control leaves when the value was 1.2; the photography time was 8 days after the leaves were infected.

[0022] Figure 4 The expression levels of CoERF113 gene in transient overexpression and control leaves at different times after infection.

[0023] Figure 5 Comparative analysis of SOD, POD and MDA in leaves of transiently overexpressed CoERF113 gene and control group.

[0024] Figure 6 The effects of transient overexpression of different genes on gene expression in oil tea leaves. Note: A: CoACO2 gene transient overexpression and CoACO2 expression in control leaves; B: CoFAD7 gene transient overexpression and CoFAD7 expression in control leaves. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with specific preferred embodiments, but the protection scope of the present invention is not limited thereby. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial sources.

[0026] Example 1: Effect of different leaf ages on the instantaneous conversion efficiency of tea oil leaves

[0027] 1. Preparation of Agrobacterium infection solution

[0028] Agrobacterium carrying the CoERF113 gene was used in our laboratory. The specific operation steps are as follows ( Figure 1 ):

[0029] 1. Plate streaking: First take out the CoERF113 Agrobacterium and the empty Agrobacterium from the low-temperature refrigerator and place them in ice to thaw for 5 minutes. Operate on a clean bench: After the bacterial solution melts, streak the LB plate for inoculation. Cover the lid, seal the culture medium with a sealing film, and indicate the date, strain name, number and other relevant information in a conspicuous place for next use. During the experimental operation, keep in mind the sterile specifications, such as hands and instruments cannot pass over the culture medium. Invert the culture medium and culture it in a 28°C constant temperature box for 48 hours. Take it out when it is observed that the colony is growing well and a monoclonal colony has been cultured.

[0030] 2. Shake: Take out the LB plate with a single clone. On the clean bench, select a 1.5mL centrifuge tube and add 1mL of liquid LB medium. Use a pipette to pick up a round and smooth single colony, and mix it in the centrifuge tube by pipetting to keep the bacteria in the centrifuge tube. After mixing, place at 28℃, 200r·min -1 , and cultured on a shaker for 48 hours.

[0031] 3. Shake vigorously: Select a 250mL conical flask and add 100mL of LB liquid culture medium. Pipette 1mL of the bacterial solution after small shaking and add it to the culture medium. Mix well and place at 28℃, 200r·min -1 , and culture on a shaker for 24 hours.

[0032] 4. OD measurement 600 Value: Use UV spectrophotometer to measure the OD of Agrobacterium solution after shaking 600 Whether the value is between 0.6 and 0.9.

[0033] 5. Centrifugation: centrifuge at 4000r / min -1 , centrifuge for 10 minutes, and discard the supernatant. When using the centrifuge, pay attention to balance.

[0034] 6. Prepare Agrobacterium infection solution: The resuspension solution contains 10mmol·L -1 MES, 10mmol·L -1 Magnesium chloride, 200 μmol·L -1 Acetosyringone, deionized water. Use the resuspension to prepare OD 600 The infection solution of CoERF113 Agrobacterium with a value of 0.9 and the empty vector Agrobacterium was shielded from light with tin foil and allowed to stand at room temperature for 4 hours.

[0035] 2. Agrobacterium infection

[0036] 1. Selection of experimental subjects: 12 healthy and vigorous branches without pests and diseases and with similar growth conditions growing on the same tea tree were selected as experimental subjects. The branches were randomly divided into 2 groups (tender leaf infection group and mature leaf infection group). Three branches were selected from each group for infection with Agrobacterium containing the CoERF113 gene (CoERF113-pCAMBIA1305.1) overexpression vector, and three branches were selected for infection with P1305 (pCAMBIA1305.1) Agrobacterium containing an empty vector as a control. Tender leaves are new leaves with a softer texture (leaf age within 1-3 months) grown from the new shoots (spring shoots) of the year, and mature leaves are fully developed leaves with a harder texture (leaf age within 6-12 months).

[0037] The partial sequence of CoERF113 obtained by sequencing the transcriptome data of the abscission zone of oil-tea camellia fruit was compared with the tea plant genome database by BLAST (http: / / tpia.teaplant.org / index.html) to obtain the sequence of the ERF gene homologous to CoERF113 in tea plants. Based on this, primers were designed to clone the full-length CDS sequence of the CoERF113 gene from the oil-tea camellia variety 'Hua Shuo'. The nucleotide sequence is shown in SEQ ID NO.1, specifically:

[0038] .

[0039] The obtained gene sequence was analyzed for protein domain and predicted for physical and chemical properties. The plasmid containing the target gene and the pCAMBIA1305.1 expression vector plasmid were digested, connected and transformed to construct the CoERF113 overexpression vector CoERF113-pCAMBIA1305.1.

[0040] 2. Infection: In April, when there is sufficient rainfall and the outdoor temperature is between 20℃ and 28℃, use a disposable sterile syringe to scratch the back of the non-detached mature leaves of Camellia oleifera. Use a needle-free syringe to absorb the infection liquid and press it against the wound and inject until the infection liquid spreads from the wound to the whole leaf. After the infection is completed, wrap the leaves with tin foil and shade them for 24 hours. Select 15-20 Camellia oleifera leaves for each branch for treatment. Test and observe the SPAD value (SPAD value is often used in chlorophyll meters to indicate chlorophyll content) and phenotype of the leaves before infection, 4 days after infection, and 8 days after infection.

[0041] 3. Results Analysis

[0042] By comparing different OD 600 The results showed that the effect of Agrobacterium infection on the SPAD value of mature leaves of Camellia oleifera was 600 When the value was 0.9, the SPAD value of the leaves overexpressing the CoERF113 gene on the 8th day after infection was significantly decreased compared with that before infection, while the OD 600 The SPAD values ​​of the control leaves on the 4th and 8th days after infection showed no significant changes compared with those before infection (P < 0.05) (Table 1).

[0043] Table 1 Different OD 600 Effects of Agrobacterium infection on SPAD values ​​of mature leaves of Camellia oleifera

[0044]

[0045] Note: The data in the table represent mean ± standard deviation. Different lowercase letters represent significant differences between different treatments of the same index at P < 0.05.

[0046] Description in OD 600 When the value was 0.9, the chlorophyll content of mature leaves of Camellia oleifera after overexpression of CoERF113 gene decreased. In addition, the color of mature leaves of Camellia oleifera after overexpression of CoERF113 gene tended to turn yellow on the 4th and 8th days after infection compared with the leaves before infection ( Figure 2 ). The above results show that in OD 600 When the value was 0.9, the transient overexpression of CoERF113 gene in mature leaves of Camellia oleifera was successful.

[0047] By comparing different OD 600 The results showed that the influence of Agrobacterium infection on the SPAD value of young leaves of Camellia oleifera was different. 600 There was no significant change in the SPAD values ​​of leaves after infection of young leaves by CoERF113 gene overexpressing Agrobacterium and control compared with those before infection (P < 0.05) (Table 2).

[0048] Table 2 Different OD 600 Effects of Agrobacterium infection on SPAD values ​​of young leaves of Camellia oleifera

[0049]

[0050]

[0051] Note: The data in the table represent mean ± standard deviation. Different lowercase letters represent significant differences between different treatments of the same index at P < 0.05.

[0052] This indicates that the chlorophyll content of young leaves did not decrease after infection by Agrobacterium containing the CoERF113 gene. In addition, the young leaves did not show obvious color changes on the 4th and 8th days after infection ( Figure 2 ). The above results suggest that transient overexpression of CoERF113 gene in young leaves of Camellia oleifera was unsuccessful.

[0053] The above results indicate that the transient transformation method of the present invention can successfully express the recombinant target gene CoERF113 in mature leaves of the woody plant Camellia oleifera.

[0054] Example 2, different infection fluid OD 600 Effect of % on the instantaneous conversion efficiency of Camellia oleifera leaves

[0055] 1. Preparation of Agrobacterium infection solution

[0056] Agrobacterium monoclonal colonies of CoERF113 gene overexpression (ERF) and P1305 empty vector control (CK empty vector group) were obtained by plate streaking method. -1 Kan and 50mg·L -1 The monoclonal bacteria were streaked on the LB solid plate medium of Rif. The medium was inverted and placed in a constant temperature incubator at 28°C for 48 hours. Round and smooth monoclonal colonies were observed, which can be used for subsequent expansion culture. 100 mL of 50 mg·L -1 Kan and 50mg·L -1 Rif LB liquid medium. Pipette 1mL of the shaken bacterial solution and add it to the medium. Pipette and mix repeatedly, then place at 28℃, 200r·min -1 , shake on a shaker for 24 hours. After proliferation, the bacterial solution turns orange-yellow. Use a UV spectrophotometer to measure the OD of the Agrobacterium bacterial solution. 600 Value. Measure its OD 600 The value is in the range of 0.6 to 0.9, which is in line with expectations and can be used for subsequent experiments. The bacterial solution after shaking was divided into centrifuge tubes and centrifuged at 4000 r / min. -1Centrifuge for 10 min and discard the supernatant. Prepare 10 mM MES, 10 mM magnesium chloride, 200 μmol·L -1 A resuspension of acetosyringone and double distilled water. 600 The infection solution with the value of 0.6, 0.9, 1.2 was placed under tin foil to block light at room temperature for 4 hours.

[0057] 2. Infection

[0058] In this experiment, a syringe was used to inject the whole leaf of Camellia oleifera in May 2024. Seven healthy, pest-free and similar growth branches from the same Camellia oleifera tree were selected. They were randomly divided into three groups (treatment group, CK empty group, and CK non-injection group). The three branches in the treatment group were marked as ERF0.6, ERF0.9, and ERF1.2, respectively. The three branches in the CK empty group were marked as CK0.6, CK0.9, and CK1.2, respectively. There were seven groups in the CK non-injection group, i.e. branches that were not injected. Each group selected 15-20 Camellia oleifera leaves for treatment. The specific infection process is as follows: Use a disposable sterile syringe needle to make a line wound on the back of a non-detached mature Camellia oleifera leaf. Use a needle-free syringe to absorb the infection liquid, press it against the wound and inject it until the infection liquid spreads from the wound to the entire leaf. Immediately after the infection is completed, wrap the leaves with tin foil and shade them for 24 hours. On the eighth day after the infection, the different OD 600 The leaf morphology of the ERF overexpression group and the CK empty group treated with the value was observed and photographed, and the SPAD values ​​of the leaves of the treatment group and the control group were detected before infection and 2, 4, 6, and 8 days after infection. For the transgenic leaves with obvious phenotypes such as yellowing of leaves and a significant decrease in chlorophyll, the gene expression level and physiological indicators such as superoxide dismutase (SOD) activity, peroxidase (POD) activity, and malondialdehyde (MDA) content were further detected by fluorescent quantitative PCR to verify the function of the CoERF113 gene and the transformation efficiency of the oil tea leaf transient transformation system.

[0059] 3. Results Analysis

[0060] By comparing different OD 600 The results showed that the leaf color of CoERF113 gene overexpression was significantly different from that of Agrobacterium infection at different OD values. 600 Under the influence of Agrobacterium infection solution with different values, the plants all turned yellow to varying degrees. Figure 3 OD of infection fluid with overexpression of CoERF113 gene 600 When the values ​​were 0.6 and 0.9, the leaf SPAD values ​​on the 8th day after infection decreased significantly by 7.16% and 12.00% compared with those before infection (P < 0.05). 600When the value was 1.2, the SPAD values ​​of the leaves of the CoERF113 gene overexpression group and the control group (CK empty group and CK non-injection group) had no significant changes compared with those before treatment (P < 0.05) (Table 3).

[0061] Table 3 Different OD 600 Effects of different time after Agrobacterium infection on SPAD value of mature leaves of Camellia oleifera

[0062]

[0063] Note: The data in the table represent mean ± standard deviation. Different lowercase letters represent significant differences between different treatments of the same index at P < 0.05.

[0064] By comparing the expression of CoERF113 gene in tea leaves at different times after infection, it was found that the expression of CoERF113 gene in leaves on the 2nd and 6th days after infection was 109.09% and 100.00% higher than that in the CK empty load group on the corresponding days, respectively. On the 8th day, the expression of CoERF113 gene was significantly decreased by 17.86% compared with the control group (P < 0.05) ( Figure 4 The above results show that the transient overexpression of genes generally has a high expression level within 2-6 days, and the expression level will decrease on the 8th day.

[0065] The detection of physiological indicators such as SOD activity, POD activity, and MDA content in leaves after overexpression of CoERF113 gene and infection with P1305 empty vector showed that the SOD activity in leaves after overexpression of CoERF113 gene was significantly increased by 15.43 U·g compared with the control group. -1 FW, POD activity in leaves after overexpression of CoERF113 gene was significantly reduced by 92.66 U·g compared with the control group. -1 There was no significant difference in FW and MDA content between the CoERF113 gene overexpression group and the control group (P < 0.05) ( Figure 5 ).

[0066] Yellowing and decreased SPAD values ​​are characteristics of leaf senescence. In addition, SOD activity, POD activity, and MDA content are also affected by physiological stresses such as leaf senescence. 600When the values ​​were 0.6 and 0.9, the leaves overexpressing the CoERF113 gene turned yellow, the SPAD value decreased, the SOD activity and POD activity changed, and the expression level of the CoERF113 gene increased, indicating that the CoERF113 gene pair would promote the aging of camellia oleifera leaves to a certain extent. This further verified the function of the camellia oleifera transcription factor CoERF113, and also explored the conditions for improving the instantaneous conversion efficiency of camellia oleifera leaves.

[0067] Example 3: Transient transformation of CoACO2 and CoFAD7 genes in mature leaves of Camellia oleifera

[0068] 1. Preparation of Agrobacterium infection solution

[0069] The overexpression vectors of the two genes CoACO2 and CoFAD7 were constructed by double enzyme digestion. The control P1300 was loaded with the pCAMBIA1300.1 vector without the target gene. Mature tea leaves were selected as experimental materials, and these mature leaves were injected and infected with Agrobacterium liquid containing the target gene or the control. The preparation process of Agrobacterium infection liquid is as follows:

[0070] In the presence of 50 mg·L -1 Kan and 50mg·L -1 The monoclonal bacteria were streaked on the LB solid plate medium of Rif. The medium was inverted and placed in a constant temperature incubator at 28°C for 48 hours. Round and smooth monoclonal colonies were observed, which can be used for subsequent expansion culture. 100 mL of 50 mg·L -1 Kan and 50mg·L -1 Rif LB liquid medium. Pipette 1mL of the shaken bacterial solution and add it to the medium. Pipette and mix repeatedly, then place at 28℃, 200r / min -1 , shake on a shaker for 24 hours. After proliferation, the bacterial solution turns orange-yellow. Use a UV spectrophotometer to measure the OD of the Agrobacterium bacterial solution. 600 Value. Measure its OD 600 The value was in the range of 0.6 to 0.9, which was in line with expectations and could be used for subsequent experiments. The bacterial solution after shaking was divided into centrifuge tubes and centrifuged at 4000 r / min. -1 Centrifuge for 10 min and discard the supernatant. Prepare 10 mM MES, 10 mM magnesium chloride, 200 μmol·L -1 A resuspension of acetosyringone and double distilled water was used to prepare OD 600 The value of the infection solution is 0.9. Place it at room temperature for 4 hours under tin foil to block light.

[0071] 2. Infection

[0072] Two healthy, pest-free and similar branches were selected from each of the two tea trees for infection. The branches on the two trees were used for infection with CoACO2 overexpression (CoACO2-pCAMBIA1300.1) and P1300 empty control, and CoFAD7 overexpression (CoFAD7-pCAMBIA1300.1) and P1300 empty control, and 30 mature leaves were selected for infection from each branch. The specific infection process is as follows: a disposable sterile syringe needle is used to scratch and wound the back of a non-detached mature tea leaf. The infection liquid is sucked with a needle-free syringe and then pressed against the wound and injected until the infection liquid spreads from the wound to the entire leaf. After the infection is completed, the leaves are immediately wrapped with tin foil for shading for 24 hours. On the 2nd and 8th days after infection, the gene expression levels of the leaves with different treatments are detected by fluorescent quantitative PCR to verify the transient transformation of different genes in tea leaves.

[0073] 3. Results

[0074] By analyzing the expression of the CoACO2 gene in CoACO2 overexpressed leaves, it was found that on the second day after infection, the expression of the CoACO2 gene was significantly increased by 63.64% compared with the control group, and on the eighth day, the expression of the CoACO2 gene was significantly reduced by 79.34% compared with the control group ( Figure 6 A). By comparing the expression of CoFAD7 gene in CoFAD7 overexpressed leaves, it was found that the expression of CoFAD7 gene increased significantly by 19.35% and 50.82% on the 2nd and 8th day after infection compared with the control group ( Figure 6 B). These results show that the method of transient transformation of tea leaves of the present invention can achieve overexpression of different target genes of tea leaves. In addition, although the expression levels of different genes will be different on the 8th day after transient overexpression, the expression levels of these genes in the overexpressed leaves are all increased compared with the control on the 2nd day after overexpression. Therefore, it is recommended to analyze the expression of the target gene within 2-6 days after infection.

Claims

1. A method for transiently expressing genes in tea leaves, characterized in that: The steps include: 1) Prepare Agrobacterium infection solution containing plant expression vectors containing foreign genes. The OD of the Agrobacterium infection solution is 600 Values ​​range from 0.6 to 0.9; 2) using a sterile syringe needle to scratch and wound the back of a non-detached mature tea oil leaf, sucking the infection solution described in step 1) into the sterile syringe and injecting it at the wound until the infection solution spreads from the wound to the entire leaf; 3) The leaves were then immediately wrapped with tin foil for shading for 24-36 hours, and the expression of the target gene in the transgenic leaves was detected on the 2nd to 6th day after the treatment.

2. The instantaneous conversion method of tea leaves as claimed in claim 1, characterized in that: The mature leaves of Camellia oleifera are mature leaves of Camellia oleifera with a leaf age of 6 to 12 months under a natural growth state.

3. The instantaneous conversion method of tea leaves according to claim 1, characterized in that: The recombinant target gene is a camellia oleifera gene.

4. The instantaneous conversion method of tea leaves according to claim 1, characterized in that: The expression of target genes in transgenic leaves was detected 2-6 days after infection.

5. The instantaneous conversion method of tea leaves according to claim 1, characterized in that: The bacterial liquid in the Agrobacterium infection liquid is cultured as follows: 1) Agrobacterium tumefaciens carrying the exogenous gene plasmid is streaked on a solid LB medium containing the recombinant target gene expression vector and the corresponding antibiotics to activate the strain; 2) Small shaking: Take out the LB plate with monoclonal colonies, pick up round and smooth single colonies, and mix them evenly in a centrifuge tube containing liquid LB medium with corresponding antibiotics so that the bacteria remain in the centrifuge tube. After mixing, place it in a shaker at 26-30°C and 180-240 rpm for 36-60 hours until the bacterial solution becomes turbid and turns orange-yellow; 3) Large shaking: Pipette the bacterial solution after small shaking into the liquid LB medium containing the corresponding antibiotics at a volume ratio of 1:50-100, mix well, and culture at 26-30℃, 180-240r / m on a shaker for 12-36 hours until the bacterial solution becomes turbid and turns orange-yellow.

6. The instantaneous conversion method of tea leaves according to claim 5, characterized in that: Step 2) Incubate at 28°C, 200 rpm, and shake for 48 hours until the bacterial solution becomes turbid and orange-yellow; In step 3), the culture was carried out at 28°C and 200 rpm on a shaker for 24 hours until the bacterial solution became turbid and orange-yellow in color.

7. The instantaneous conversion method of tea leaves according to claim 5, characterized in that: Step 1) The solid LB medium used was 50 mg / L kanamycin + 50 mg / L rifampicin + 10 g / L tryptic peptone + 5 g / L yeast extract + 10 g / L sodium chloride + 15 g / L agar powder, pH = 5.7; In steps 2) and 3), the liquid LB medium used was 50 mg / L kanamycin + 50 mg / L rifampicin + 10 g / L tryptic peptone + 5 g / L yeast extract + 10 g / L sodium chloride, pH = 5.

7.

8. The instantaneous conversion method of tea leaves according to claim 5, characterized in that: The bacterial liquid in the Agrobacterium infection liquid is cultured as follows: a) pouring the bacterial solution into a centrifuge tube and centrifuging at room temperature for 10 min at 4000 r / min, discarding the supernatant and collecting the bacterial cells; b) Resuspend the cells in transformation buffer and adjust the OD 600 The values ​​are 0.6 and 0.9; wherein the conversion buffer contains 10 mmol / L MES, 10 mmol / L magnesium chloride, 200 μmol / L acetosyringone, deionized water, and pH=5.7; c) Cover with tin foil to block light and let stand at room temperature for 4 hours.

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