Method for transiently overexpressing exogenous gene in cyclocarya paliurus leaf and application thereof

Abstract

The application provides a method for transiently overexpressing an exogenous gene in Cyclobalanopsis glauca leaves, which comprises the following steps: double enzyme digestion of a target gene and an overexpression vector, ligation with a ligase, transformation of a recombinant overexpression vector into Agrobacterium, coating on LB solid culture medium, expansion of a single colony in LB liquid culture medium, collection of bacteria, discarding of supernatant, resuspension with an infection liquid, and preparation of an Agrobacterium infection liquid; and the method further comprises the following steps: wounding of the back of a Cyclobalanopsis glauca leaf and a leaf stem, infection of the leaf by the Agrobacterium infection liquid, or wrapping of the wounding part by cotton absorbing the Agrobacterium infection liquid. CpSnRK1α The application further provides application of the method, and the exogenous gene is a Cyclobalanopsis glauca CpSnRK1α The application has the advantages that a simple and efficient method for transiently overexpressing an exogenous gene in Cyclobalanopsis glauca leaves is established for the first time, the effectiveness of the method is verified through application of a Cyclobalanopsis glauca gene, and potential possibilities are provided for subsequent improvement of Cyclobalanopsis glauca varieties through gene regulation.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a method and application of transient overexpression of exogenous genes in Cyclocarya paliurus leaves. Background Technology

[0002] Transient expression technology involves transferring a target gene into target cells within a relatively short period of time, establishing a temporarily efficient expression system within the cell, and obtaining a short-term high level of expression of the target gene. Compared with stable expression, Agrobacterium-mediated transient expression requires less time and can rapidly express exogenous genes within days. It does not require the integration of exogenous genes into the host plant chromosome, and is simple to operate, highly reproducible, and inexpensive, making it an important tool for studying gene function.

[0003] Qingqianliu [ Cyclocarya paliurus (Batal) Iljinsk As a member of the Juglandaceae family ( Juglandaceae This multifunctional tree species has leaves rich in medicinal active ingredients such as flavonoids and triterpenes, which are of great value in anti-oxidation and hypoglycemia. However, a transient overexpression method for *Cyclocarya paliurus* leaves has not yet been established, which limits its gene function analysis and medicinal quality improvement.

[0004] Therefore, there is an urgent need for an efficient and simple method for transient overexpression of exogenous genes in Cyclocarya paliurus leaves to provide technical support for its gene research. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide an efficient and simple method for transient overexpression of exogenous genes in Cyclocarya paliurus leaves and its application.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A method for transient overexpression of exogenous genes in Cyclocarya paliurus leaves includes the following steps: (1) Construction of expression vector: The target gene and the overexpression vector pCAMBIA2300 were double-digested with restriction endonucleases BamHⅠ and XhoⅠ. After the DNA digestion products were purified, they were ligated with ligase to obtain the recombinant overexpression vector. (2) Agrobacterium transformation: The recombinant overexpression vector obtained in step (1) was transformed into Agrobacterium GV3101 by the freeze-thaw method. The transformed Agrobacterium was spread on LB solid medium. After the colonies grew, single colonies were picked for PCR detection. (3) Preparation of Agrobacterium infection solution: The Agrobacterium single clone containing the recombinant overexpression vector that was correctly sequenced in step (2) was amplified in LB liquid medium; after centrifugation to collect the bacterial cells, the supernatant was discarded, and the cells were resuspended in the infection solution and the OD was adjusted. 600 Adjust the concentration to 0.6~1.0; after standing or shaking, obtain the Agrobacterium-infected solution; (4) Agrobacterium infection of Cyclocarya paliurus plants: Scratch the back of Cyclocarya paliurus leaves and infect the Cyclocarya paliurus leaves with the Agrobacterium infection solution prepared in step (3), or wrap the scratched area with cotton soaked in the Agrobacterium infection solution for infection.

[0007] As one of the preferred embodiments of the present invention, in step (1), the double enzyme digestion system is: BamHⅠ1μL, XhoⅠ1μL, plasmid / target gene fragment 1~5μg, CutSmart Buffer 5μL, and ddH2O is added to 50μL; the enzyme digestion product is detected by agarose gel electrophoresis and then purified by gel recovery.

[0008] As one of the preferred embodiments of the present invention, in step (2), the solid culture medium is LB solid culture medium containing 50 μg / mL Kan and 25 μg / mL Rif; in step (3), the liquid culture medium is LB liquid culture medium containing 50 μg / mL Kan and 25 μg / mL Rif.

[0009] As one of the preferred embodiments of the present invention, in step (3), the propagation temperature is 28℃; after propagation, the cells are collected by centrifugation at 5000 rpm / min for 10 min, the supernatant is discarded, and the cells are resuspended in an infection solution containing 10 mM MgCl2, 10 mM MES and 200 μM acetylsylgenone, and the OD is adjusted. 600 Adjust to 0.6~1.0; let stand for 3 hours or shake for 1 hour at 28℃.

[0010] As one of the preferred embodiments of the present invention, in step (4), the infected part of the Cyclocarya paliurus leaf is the back of the leaf or the leaf stem; when the infected part is specifically the back of the leaf, the infected part is soaked in Agrobacterium tumefaciens infection solution for 30-60 seconds; when the infected part is specifically the leaf stem, the infected part is wrapped with cotton soaked in Agrobacterium tumefaciens infection solution.

[0011] As one of the preferred embodiments of the present invention, in step (4), after infection, the cells are cultured in the dark for 2 days, and then cultured in a dark cycle of 24°C, 16h light / 20°C, 8h, with watering regularly during the period.

[0012] As one of the preferred embodiments of the present invention, the method further includes a step (5) of qRT-PCR detection: collecting leaves of infected Cyclocarya paliurus plants, extracting their RNA and reverse transcribing it into cDNA, to... Cp18S The gene was used as an internal control, and the overexpression of the target gene was detected by qRT-PCR.

[0013] In one application of the above method, the exogenous gene is *Cyclocarya paliurus*. CpSnRK1a The gene, whose CDS region sequence is shown in SEQ ID NO.1.

[0014] As one of the preferred embodiments of the present invention, the *Cyclocarya paliurus* CpSnRK1a The CDS region fragment of a gene is amplified in the following ways: Using cDNA reverse transcribed from *Cyclocarya paliurus* leaf RNA as a template, PCR amplification was performed using KOD high-fidelity enzyme. The primers were: CpSnRK1a -F and CpSnRK1a -R, the sequences are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively; the amplification program is: 95℃ for 3 min; 95℃ for 15 sec, 60℃ for 15 sec, 72℃ for 30 sec, 40 cycles; 72℃ for 5 min.

[0015] As one of the preferred embodiments of the present invention, the application is to achieve the effect of *Cyclocarya paliurus* using the above method. CpSnRK1a The transient overexpression of the gene in Cyclocarya paliurus leaves was used to study the role of the gene in the regulation of sugar metabolism and secondary metabolism in Cyclocarya paliurus.

[0016] The advantages of this invention compared to the prior art are: I. Methodology (1) A transient overexpression system for exogenous genes in Cyclocarya paliurus leaves was established for the first time, filling a technical gap in the study of Cyclocarya paliurus gene function; this method optimizes the concentration of the infection solution (OD). 600 =0.6~1.0, with the highest efficiency of 40.12% at 0.8), infection sites (underside of leaves, tender stems) and culture conditions (dark pretreatment + light-dark cycle culture), solved the problem of thin leaves and difficult infection of Cyclocarya paliurus, and achieved efficient expression of exogenous genes; (2) It is easy to operate, efficient and has a short cycle (the whole process from infection to detection can be completed within 32 days). It does not require the establishment of a stable genetic transformation system, which reduces the experimental cost and is suitable for rapid verification of the gene function of Eucommia ulmoides. (3) It is universal and can be applied not only to specific genes, but also to the transient overexpression study of other functional genes of Cyclocarya paliurus, providing a standardized technical tool for Cyclocarya paliurus genetic engineering research.

[0017] II. Application CpSnRK1a As a key gene for sugar metabolism, its transient overexpression in *Cyclocarya paliurus* provides an experimental basis for studying the role of this gene in the regulation of sugar metabolism and secondary metabolism in *Cyclocarya paliurus*. The application of this gene in this invention verifies the effectiveness of the method of this invention, proving that it can be used as a standardized technical means for gene function research in *Cyclocarya paliurus*, and providing potential possibilities for subsequent improvement of *Cyclocarya paliurus* varieties through gene regulation. Attached Figure Description

[0018] Figure 1 This is an image of the appearance of the Cyclocarya paliurus plant during the cultivation process in Example 1; Figure 2 Different OD values ​​in Experiment Example 1 600 Agrobacterium infection solution for different infection sites of Cyclocarya paliurus CpSnRK1a The effect of overexpression efficiency (Figure A shows OD) 600 Results of infection with 0.6% Agrobacterium tumefaciens inoculum; Figure B shows the OD. 600 Results of infection with Agrobacterium tumefaciens inoculum at 0.8; Figure C shows the OD. 600 Results of Agrobacterium tumefaciens infection solution 1.0; where “WT” represents wild-type Cyclocarya paliurus plants, “empty vector” represents empty vector control without target gene, “leaf underside” indicates that Agrobacterium tumefaciens infection solution was applied to the underside of the leaf, and “leaf stem” indicates that Agrobacterium tumefaciens infection solution was applied to the leaf stem. Detailed Implementation

[0019] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents and experimental methods used below are all conventional reagents or methods in the art and will not be described in detail again.

[0020] Example 1 This embodiment of a method for transient overexpression of exogenous genes in Cyclocarya paliurus leaves includes the following steps: (1) Construction of expression carrier For Qingqianliu CpSnRK1a The gene (CDS region sequence shown in SEQ ID NO.1) was cloned. Using cDNA reverse transcribed from *Cyclocarya paliurus* leaf RNA as a template, PCR amplification was performed using KOD high-fidelity enzyme. Primers were as follows: CpSnRK1a -F (SEQ ID NO.2) and CpSnRK1a -R (SEQ ID NO.3); the amplification program was: 95℃ for 3 min; 95℃ for 15 sec, 60℃ for 15 sec, 72℃ for 30 sec, for 40 cycles; 72℃ for 5 min.

[0021] Will CpSnRK1a The gene and overexpression vector pCAMBIA2300 were double-digested with restriction endonucleases BamHI and XhoHI. The double digestion system was as follows: 1 μL BamHI, 1 μL XhoHI, 1 μg plasmid / target gene fragment, 5 μL CutSmart Buffer, and ddH2O was added to a final volume of 50 μL.

[0022] After double enzyme digestion, the DNA was detected by agarose gel electrophoresis, and the corresponding fragments were recovered from the gel for DNA purification. Then, the fragments were ligated using ligase to obtain the recombinant overexpression vector pCAMBIA2300- CpSnRK1a After sequencing verification confirms correctness, it is ready for use.

[0023] (2) Agrobacterium transformation The recombinant overexpression vector pCAMBIA2300- obtained in step (1) was subjected to a freeze-thaw method. CpSnRK1a The bacteria were transformed into Agrobacterium GV3101, and the transformed Agrobacterium was spread on LB solid medium containing 50 μg / mL Kan and 25 μg / mL Rif. After the bacteria grew, single colonies were picked for PCR detection.

[0024] (3) Preparation of Agrobacterium infection solution The correctly sequenced pCAMBIA2300- from step (2) contains the recombinant overexpression vector. CpSnRK1a Agrobacterium single clones were propagated in LB liquid medium containing 50 μg / mL Kan and 25 μg / mL Rif at 28°C; the cells were collected by centrifugation at 5000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in an infection solution containing 10 mM MgCl2, 10 mM MES, and 200 μM acetylsyringone, and the OD was adjusted. 600 The concentration was increased to 0.6; after standing at 28°C for 3 hours, Agrobacterium infection solution was obtained.

[0025] (4) Agrobacterium infection of Cyclocarya paliurus plants The underside of the leaves of *Cyclocarya paliurus* were scratched, and the scratched leaves were immersed in the *Agrobacterium* infection solution prepared in step (3) for 60 seconds for infection (using an infection solution containing an empty vector as a control). After infection, the *Cyclocarya paliurus* plants were cultured in the dark for 2 days, and then cultured in a cycle of 24℃, 16h light / 20℃, 8h dark, with regular watering during the period. Figure 1 This is an image of the appearance of a Cyclocarya paliurus plant during its cultivation process.

[0026] (5) qRT-PCR detection of whether the target gene is overexpressed Leaves of *Cyclocarya paliurus* plants were collected on days 2, 4, 8, 16, 24, and 32 after infection. RNA was extracted and reverse transcribed into cDNA. Cp18S The gene was used as an internal control, and the overexpression of the target gene was detected by qRT-PCR. The qRT-PCR reaction program was as follows: 95℃, 2 min; 95℃, 10 sec, 40 cycles; 60℃, 30 sec.

[0027] Example 2 This embodiment of a method for transient overexpression of exogenous genes in Cyclocarya paliurus leaves includes the following steps: (1) Construction of expression carrier For Qingqianliu CpSnRK1aThe gene (CDS region sequence shown in SEQ ID NO.1) was cloned. Using cDNA reverse transcribed from *Cyclocarya paliurus* leaf RNA as a template, PCR amplification was performed using KOD high-fidelity enzyme. Primers were as follows: CpSnRK1a -F (SEQ ID NO.2) and CpSnRK1a -R (SEQ ID NO.3); the amplification program was: 95℃ for 3 min; 95℃ for 15 sec, 60℃ for 15 sec, 72℃ for 30 sec, for 40 cycles; 72℃ for 5 min.

[0028] Will CpSnRK1a The gene and overexpression vector pCAMBIA2300 were double-digested with restriction endonucleases BamHI and XhoHI. The double digestion system was as follows: 1 μL BamHI, 1 μL XhoHI, 2 μg plasmid / target gene fragment, 5 μL CutSmart Buffer, and ddH2O was added to a final volume of 50 μL.

[0029] After double enzyme digestion, the DNA was detected by agarose gel electrophoresis, and the corresponding fragments were recovered from the gel for DNA purification. Then, the fragments were ligated using ligase to obtain the recombinant overexpression vector pCAMBIA2300- CpSnRK1a After sequencing verification confirms correctness, it is ready for use.

[0030] (2) Agrobacterium transformation Same as Example 1.

[0031] (3) Preparation of Agrobacterium infection solution The correctly sequenced pCAMBIA2300- from step (2) contains the recombinant overexpression vector. CpSnRK1a Agrobacterium single clones were propagated in LB liquid medium containing 50 μg / mL Kan and 25 μg / mL Rif at 28°C; the cells were collected by centrifugation at 5000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in an infection solution containing 10 mM MgCl2, 10 mM MES, and 200 μM acetylsyringone, and the OD was adjusted. 600 After shaking at 28°C for 1 hour, the Agrobacterium-infected solution was obtained.

[0032] (4) Agrobacterium infection of Cyclocarya paliurus plants The leaf stalks of *Cyclocarya paliurus* leaves were scratched, and the scratched areas were wrapped with cotton soaked in the *Agrobacterium* infection solution (with an empty infection solution as a control). The *Cyclocarya paliurus* plants were then cultured in the dark for 2 days, during which the culture process was accompanied by infection. After the above culture was completed, the cotton was removed, and the plants were cultured again under a cycle of 24°C, 16h light / 20°C, 8h darkness, with regular watering during the period.

[0033] (5) qRT-PCR detection of whether the target gene is overexpressed Same as Example 1.

[0034] Example 3 This embodiment of a method for transient overexpression of exogenous genes in Cyclocarya paliurus leaves includes the following steps: (1) Construction of expression carrier For Qingqianliu CpSnRK1a The gene (CDS region sequence shown in SEQ ID NO.1) was cloned. Using cDNA reverse transcribed from *Cyclocarya paliurus* leaf RNA as a template, PCR amplification was performed using KOD high-fidelity enzyme. Primers were as follows: CpSnRK1a -F (SEQ ID NO.2) and CpSnRK1a -R (SEQ ID NO.3); the amplification program was: 95℃ for 3 min; 95℃ for 15 sec, 60℃ for 15 sec, 72℃ for 30 sec, for 40 cycles; 72℃ for 5 min.

[0035] Will CpSnRK1a The gene and overexpression vector pCAMBIA2300 were double-digested with restriction endonucleases BamHI and XhoHI. The double digestion system was as follows: 1 μL BamHI, 1 μL XhoHI, 5 μg plasmid / target gene fragment, 5 μL CutSmart Buffer, and ddH2O was added to a final volume of 50 μL.

[0036] After double enzyme digestion, the DNA was detected by agarose gel electrophoresis, and the corresponding fragments were recovered from the gel for DNA purification. Then, the fragments were ligated using ligase to obtain the recombinant overexpression vector pCAMBIA2300- CpSnRK1a After sequencing verification confirms correctness, it is ready for use.

[0037] (2) Agrobacterium transformation Same as Example 1.

[0038] (3) Preparation of Agrobacterium infection solution The correctly sequenced pCAMBIA2300- from step (2) contains the recombinant overexpression vector. CpSnRK1a Agrobacterium single clones were propagated in LB liquid medium containing 50 μg / mL Kan and 25 μg / mL Rif at 28°C; the cells were collected by centrifugation at 5000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in an infection solution containing 10 mM MgCl2, 10 mM MES, and 200 μM acetylsyringone, and the OD was adjusted. 600 The concentration was increased to 1.0; after standing at 28°C for 3 hours, Agrobacterium-infected solution was obtained.

[0039] (4) Agrobacterium infection of Cyclocarya paliurus plants The underside of the leaves of *Cyclocarya paliurus* were scratched, and the scratched leaves were immersed in the *Agrobacterium* infection solution prepared in step (3) for 60 seconds for infection (using an infection solution containing an empty vector as a control). After infection, the *Cyclocarya paliurus* plants were cultured in the dark for 2 days, and then cultured in a cycle of 24℃, 16h light / 20℃, 8h dark, with regular watering during the period.

[0040] (5) qRT-PCR detection of whether the target gene is overexpressed Same as Example 1.

[0041] Example 4 This embodiment of a method for transient overexpression of exogenous genes in Cyclocarya paliurus leaves includes the following steps: (1) Construction of expression carrier Same as Example 1.

[0042] (2) Agrobacterium transformation Same as Example 1.

[0043] (3) Preparation of Agrobacterium infection solution Same as Example 1.

[0044] (4) Agrobacterium infection of Cyclocarya paliurus plants The underside of the leaves of *Cyclocarya paliurus* were scratched, and the scratched leaves were immersed in the *Agrobacterium* infection solution prepared in step (3) for 60 seconds for infection (using an infection solution containing an empty vector as a control). After infection, the *Cyclocarya paliurus* plants were cultured in the dark for 2 days, and then cultured in a cycle of 24℃, 16h light / 20℃, 8h dark, with regular watering during the period.

[0045] (5) qRT-PCR detection of whether the target gene is overexpressed Same as Example 1.

[0046] Experimental Example 1 This experimental example is used to verify different ODs 600 Agrobacterium infection solution for different infection sites of Cyclocarya paliurus CpSnRK1 α Overexpression efficiency is affected.

[0047] I. Experimental Methods The method is the same as in Example 1, but the OD of the bacterial solution is adjusted during the preparation of the Agrobacterium infection solution. 600 The concentrations of the solution were adjusted to 0.6, 0.8, and 1.0, respectively, and applied to the underside of leaves and the leaf stalk, respectively, to investigate the effects of different concentrations on the infection sites. CpSnRK1aThe effect of overexpression efficiency. Specifically, when the infection site is the underside of the leaf, the infection is carried out by soaking in Agrobacterium infection solution (refer to the underside of leaf infection method in Example 1); when the infection site is the leaf stem, the infection is carried out by wrapping the leaf stem with cotton soaked in Agrobacterium infection solution (refer to the leaf stem infection method in Example 2).

[0048] CpSnRK1a Overexpression efficiency = empty vector injection CpSnRK1a Gene expression level / content CpSnRK1a Gene expression level *100% when gene overexpression vector is injected into the infection solution.

[0049] II. Experimental Results The results are shown in Tables 1-3 and Figure 2 As shown.

[0050] Table 1 OD 600 Effect of Agrobacterium tumefaciens infection solution on the underside of leaves and leaf stems on the overexpression efficiency of Cyclocarya paliurus.

[0051] Table 2 OD 600 Effect of Agrobacterium tumefaciens infection solution on the underside of leaves and leaf stems on the overexpression efficiency of Cyclocarya paliurus.

[0052] Table 3 OD 600 1.0 Effect of Agrobacterium tumefaciens infection solution on leaf underside and leaf stem on the overexpression efficiency of Cyclocarya paliurus

[0053] The results above show that the time to reach the maximum gene overexpression efficiency for all concentrations of infection solutions was 16 days, and the efficiency was highest when the infection site was the stem; the OD of the infection solution... 600 The maximum overexpression efficiency at 0.6 was 22.67%, and the OD of the infection solution was [missing information]. 600 The maximum overexpression efficiency at 0.8 was 40.12%, and the OD of the infection solution was [missing value]. 600 The maximum overexpression efficiency at 1.0 was 24.46%; overall, the OD of the infection solution was... 600 0.8 CpSnRK1a Gene overexpression efficiency is the highest and the duration is the longest.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for transient overexpression of a foreign gene in Cyclocarya paliurus leaves, characterized in that, Includes the following steps: (1) Construction of expression vector: The target gene and the overexpression vector pCAMBIA2300 were double-digested with restriction endonucleases BamHⅠ and XhoⅠ. After the DNA digestion products were purified, they were ligated with ligase to obtain the recombinant overexpression vector. (2) Agrobacterium transformation: The recombinant overexpression vector obtained in step (1) was transformed into Agrobacterium GV3101 by the freeze-thaw method. The transformed Agrobacterium was spread on LB solid medium. After the colonies grew, single colonies were picked for PCR detection. (3) Preparation of Agrobacterium infection solution: The Agrobacterium single clone containing the recombinant overexpression vector that was correctly sequenced in step (2) was amplified in LB liquid medium; after centrifugation to collect the bacterial cells, the supernatant was discarded, and the cells were resuspended in an infection solution containing 10 mM MgCl2, 10 mM MES and 200 M acetylsyl syringone, and the OD was adjusted. 600 To a concentration of 0.8; after standing or shaking, obtain Agrobacterium-infected solution; (4) Agrobacterium infection of Cyclocarya paliurus plants: Scratch the Cyclocarya paliurus leaf stems and wrap the scratched Cyclocarya paliurus leaf stems with cotton soaked in the Agrobacterium infection solution for infection; after infection, culture in the dark for 2 days, and then culture in a cycle of 24℃, 16h light / 20℃, 8h dark, with regular watering during the period.

2. The method for transient overexpression of exogenous genes in Cyclocarya paliurus leaves according to claim 1, characterized in that, In step (1), the double enzyme digestion system is as follows: BamHⅠ 1 μL, XhoⅠ 1 μL, plasmid / target gene fragment 1~5 μg, CutSmart Buffer 5 μL, and ddH2O is added to 50 μL; the enzyme digestion product is detected by agarose gel electrophoresis and then purified by gel recovery.

3. The method for transient overexpression of exogenous genes in Cyclocarya paliurus leaves according to claim 1, characterized in that, In step (2), the solid culture medium is LB solid culture medium containing 50 μg / mL Kan and 25 μg / mL Rif; in step (3), the liquid culture medium is LB liquid culture medium containing 50 μg / mL Kan and 25 μg / mL Rif.

4. The method for transient overexpression of exogenous genes in Cyclocarya paliurus leaves according to claim 1, characterized in that, In step (3), the propagation temperature is 28℃; after propagation, the cells are collected by centrifugation at 5000 rpm / min for 10 min, the supernatant is discarded, and the cells are resuspended in an infection solution containing 10 mM MgCl2, 10 mM MES and 200 μM acetylsylgenone, and the OD is adjusted. 600 To 0.8; let stand for 3 hours or shake for 1 hour at 28℃.

5. The method for transient overexpression of exogenous genes in Cyclocarya paliurus leaves according to claim 1, characterized in that, It also includes the step (5) of qRT-PCR detection: collecting leaves from infected Cyclocarya paliurus plants, extracting their RNA and reverse transcribing it into cDNA, so as to... Cp18S The gene was used as an internal control, and the overexpression of the target gene was detected by qRT-PCR.

6. An application of the method as described in any one of claims 1 to 5, characterized in that, The exogenous gene is *Cyclocarya paliurus*. CpSnRK1α The gene, whose CDS region sequence is shown in SEQ ID NO.

1.

7. The application according to claim 6, characterized in that, The Qingqianliu CpSnRK1α The CDS region fragment of a gene is amplified in the following ways: Using cDNA reverse transcribed from *Cyclocarya paliurus* leaf RNA as a template, PCR amplification was performed using KOD high-fidelity enzyme. The primers were: CpSnRK1 α -F and CpSnRK1α -R, the sequences are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively; the amplification program is: 95℃ for 3 min; 95℃ for 15 sec, 60℃ for 15 sec, 72℃ for 30 sec, 40 cycles; 72℃ for 5 min.

8. The application according to claim 6, characterized in that, The application is to implement *Cyclocarya paliurus* using the method described in any one of claims 1 to 5. CpSnRK1α The transient overexpression of the gene in Cyclocarya paliurus leaves was used to study the role of the gene in the regulation of sugar metabolism and secondary metabolism in Cyclocarya paliurus.

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