Construction method and application of cyclocarya paliurus root system VIGS instantaneous silencing system

Abstract

The invention provides a construction method of a cyclocarya paliurus root system VIGS instantaneous silencing system. The construction method comprises the following steps: connecting a cyclocarya paliurus CpMYC2 gene with pTRV2 to obtain a recombinant vector pTRV2-CpMYC2; the method comprises the following steps: respectively transforming vectors pTRV2-CpMYC2 and pTRV1 into agrobacterium, so as to obtain agrobacterium containing a recombinant vector pTRV2-CpMYC2 and agrobacterium containing an auxiliary vector pTRV1; the method comprises the following steps: respectively inoculating a pTRV2-CpMYC2-containing agrobacterium tumefaciens and a pTRV1-containing agrobacterium tumefaciens into an LB liquid culture medium; collecting thalli, and resuspending the thalli by using the infection liquid; mixing the two resuspension solutions to obtain a dip dyeing bacterial solution; and infecting the root system of the cyclocarya paliurus with the infection bacterial liquid by adopting a vacuum pumping method or a root injury soaking method. The invention also provides application of the method in verification of cyclocarya paliurus gene functions. The cyclocarya paliurus root system is infected through the method, a target gene silencing system is constructed, and a foundation is laid for cyclocarya paliurus gene function research.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to a method for constructing a transient VIGS silencing system for Cyclocarya paliurus roots and its application. Background Technology

[0002] Qingqianliu [ Cyclocarya paliurus (Batal) Iljinsk *Cyclocarya*, a plant belonging to the genus *Cyclocarya* in the family Juglandaceae, is a monotypic genus endemic to my country. It is characterized by its tall stature, coin-shaped fruits, and long fruiting period, making it highly ornamental. Since the 1980s, research on the chemical composition and pharmacological activities of *Cyclocarya* has become a hot topic. Modern pharmacological studies have also shown that *Cyclocarya* secondary metabolites possess pharmacological activities such as lowering blood sugar, lowering blood lipids, lowering blood pressure, antioxidation, and antitumor effects. With a deeper understanding of the medicinal and health-promoting value of *Cyclocarya*, market demand is constantly increasing. However, its quality formation mechanism remains unclear, and gene function analysis has not been conducted in many studies. Therefore, establishing a rapid and efficient gene function verification technology for *Cyclocarya* is currently a key issue in *Cyclocarya* gene function research.

[0003] In the post-genomic era, virus-induced gene silencing (VIGS) technology plays a crucial role in plant gene function research. VIGS primarily utilizes viral vectors to infect plants carrying target genes. During replication and expression, dsRNA is formed. The dsRNA is cleaved into 21-24 nt siRNA fragments by the specific endonuclease Dicer. The siRNA is then amplified by RNA polymerase, forming single-stranded RNA complexes that bind to certain proteins. These complexes specifically interact with the target gene mRNA, leading to its degradation and thus post-transcriptional gene silencing. Currently, although VIGS is applied in many plants, its application and research in the root system of *Cyclocarya paliurus* are relatively limited. Therefore, this invention attempts to construct a transient VIGS silencing system for *Cyclocarya paliurus* roots, aiming to lay the foundation for further verification of *Cyclocarya paliurus* gene function.

[0004] However, the selection of root infection methods is still needed to construct a transient VIGS silencing system for Cyclocarya paliurus roots. Although there are many infection methods for root VIGS silencing, the silencing efficiency varies among different plants and different plant parts, requiring further investigation. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for constructing a high-efficiency VIGS transient silencing system for Cyclocarya paliurus roots and its application.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A method for constructing a transient VIGS silencing system for Cyclocarya paliurus roots includes the following steps: (1) Qingqianliu CpMYC2 The gene fragment was ligated with the tobacco brittle virus vector pTRV2 to obtain the recombinant vector pTRV2- CpMYC2 ; (2) The recombinant vector was prepared by freeze-thaw method. CpMYC2 The gene and the tobacco brittle virus vector pTRV1 were transformed into Agrobacterium GV3101 to obtain the recombinant vector pTRV2-. CpMYC2 Agrobacterium and Agrobacterium containing the helper vector pTRV1 were used. The transformed Agrobacterium was spread on solid culture medium, and after the colonies grew, single colonies were picked for PCR verification. (3) The pTRV2-- CpMYC2 Single clones of Agrobacterium and Agrobacterium containing the helper vector pTRV1 were inoculated into LB liquid medium for propagation; the bacterial cells were collected by centrifugation, and then resuspended in the infection solution to adjust the OD. 600 To 0.6~1.0; (4) Mix the two resuspensions obtained in step (3) at a volume ratio of 1:1 to obtain the inoculated bacterial solution; (5) Vacuum pressing or root soaking method is used to infect the roots of Cyclocarya paliurus to achieve the silencing of the target gene.

[0007] As one of the preferred embodiments of the present invention, in step (1), the *Cyclocarya paliurus* is used... CpMYC2 The gene and viral vector pTRV2 were double-digested with restriction endonucleases BamHI and XhoI. After double digestion, the digestion products were purified and ligated using a ligase to obtain the recombinant vector pTRV2-. CpMYC2 .

[0008] As one of the preferred embodiments of the present invention, in step (1), *Cyclocarya paliurus* is used. CpMYC2 The nucleotide sequence of the gene fragment is shown in SEQ ID NO.1.

[0009] As one of the preferred embodiments of the present invention, the specific steps of transforming Agrobacterium using the freeze-thaw method in step (2) are as follows: thaw 100 μL of Agrobacterium GV3101 competent cells in an ice bath; add 1 μg of expression vector plasmid, mix thoroughly, and incubate on ice for 5 min; quickly add the centrifuge tube to liquid nitrogen for 5 min; quickly remove it and place it in a 37℃ water bath for 5 min; quickly remove it and place it on ice for 5 min; add 700 μL of antibiotic-free LB liquid medium and culture at 28℃ with shaking for 2-3 h; centrifuge at 6000 rpm / min for 1 min, and resuspend the cells in 100 μL of supernatant for subsequent plating on LB solid medium containing 50 μg / mL Kan and 25 μg / mL Rif.

[0010] As one of the preferred embodiments of the present invention, in step (3), the liquid culture medium is LB liquid culture medium containing 50 μg / mL Kan and 25 μg / mL Rif.

[0011] As one of the preferred embodiments of the present invention, in step (3), the formulation of the infiltration solution is: 10mM MgCl2, 10mM MMES, 200μM acetylsuccinone.

[0012] As one of the preferred methods of the present invention, the specific steps of the vacuum pressure method for infecting the roots of Cyclocarya paliurus in step (5) are as follows: when the Cyclocarya paliurus seeds have grown to 2-3 true leaves, the roots of the seedlings are scratched and the scratched roots are directly immersed in the inoculum solution and connected to the vacuum pressure device. The vacuum pressure is applied for 3 minutes and the pressure is set to 0.06 MPa.

[0013] As one of the preferred embodiments of the present invention, the specific steps of the root-damaged soaking method for infecting the roots of *Cyclocarya paliurus* in step (5) are as follows: a. When the *Cyclocarya paliurus* seeds have grown to 2-3 true leaves, the roots of the seedlings are scratched, and the scratched roots of the seedlings are separated by a black disposable film flowerpot for root separation. Then, the black disposable film flowerpot is filled with soil; b. Another flowerpot is prepared, and the bottom of the flowerpot is filled with cotton soaked in the bacterial solution; c. The roots of the seedlings treated in step a are inserted downward into the flowerpot in step b, so that the roots of the seedlings are soaked in the bacterial solution.

[0014] As one of the preferred embodiments of the present invention, step (6) is also included: Roots of Cyclocarya paliurus plants were collected, RNA was extracted, and reverse transcribed into cDNA as a template for qPCR analysis; and Cyclocarya paliurus was used as a template for analysis. Cp18S Primers were designed using genes as internal reference genes for [the purpose of] [the study ... CpMYC2 Genes were analyzed by relative quantitative qRT-PCR.

[0015] The application of the construction method as described in any one of claims 1 to 9 in verifying the function of the Cyclocarya paliurus gene.

[0016] Design concept and principle of this invention: Tobacco brittle virus-induced gene silencing is currently the most widely used and effective VIGS virus vector in plants, but its infection efficiency on Cyclocarya paliurus roots is still unknown. This invention attempts to conduct VIGS experiments on Cyclocarya paliurus roots based on this vector.

[0017] Studies have shown that the MYC2 transcription factor is involved in multiple secondary metabolic pathways in plants. The MYC2 transcription factor is a member of the Myelocytomatosis proteins (MYCs) family in plants.

[0018] In addition, in order to simultaneously achieve the goals of simple, rapid, and batch processing of Eucommia ulmoides and efficient silencing of the target gene, this invention also requires improvements to the specific infection method of Eucommia ulmoides roots.

[0019] Accordingly, this invention utilizes the *Cyclocarya paliurus* phytorepinephrine dehydrogenase gene. CpMYC2 Using the gene as a positive reporter gene, a VIGS system based on the root system of *Cyclocarya paliurus* induced by tobacco brittle virus was constructed, and the specific infection mode of *Cyclocarya paliurus* root system was optimized and improved.

[0020] The advantages of this invention compared to the prior art are: (1) This invention utilizes Tobacco Crack Virus (TRV)-mediated verification of the gene function of Eucommia ulmoides, involving the use of TRV viral vectors to verify the gene function of Eucommia ulmoides. CpMYC2 The gene marker is a marker gene, which is used in the root system of Eucommia ulmoides through the TRV virus-mediated VIGS system to facilitate subsequent gene function verification and analysis. (2) Through screening and optimization, this invention obtains a method for inoculating the roots of Eucommia ulmoides, thereby achieving the goal of simple, rapid, and batch processing of Eucommia ulmoides and efficient silencing of the target gene; (3) This invention has the advantages of high efficiency, short cycle, simple operation and economic benefits, laying the foundation for the study of gene function of Cyclocarya paliurus. Attached Figure Description

[0021] Figure 1 This is a phenotypic diagram of the plants 10 days after infection in Example 1 (Figure A is "pTRV1+pTRV2-"). CpMYC2 (Figure B shows the phenotype of the infected plants, while Figure B shows the phenotype of the blank control plants). Figure 2 These are the qRT-PCR detection results from Example 1 (in the figure, A, B, and C are the qRT-PCR detection results of root samples on days 2, 16, and 30 after infection, respectively). Figure 3This is a phenotypic diagram of plants 20 days after infection in Example 2 (Figure A is "pTRV1+pTRV2-"). CpMYC2 (Figure B shows the phenotype of the infected plants, while Figure B shows the phenotype of the blank control plants). Figure 4 The results are qRT-PCR detection results in Example 2 (in the figure, A, B, and C are the qRT-PCR detection results of root samples on the 2nd, 16th, and 30th days after infection, respectively). Detailed Implementation

[0022] 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, the reagents, equipment, and methods used in the present invention, unless otherwise specified, are all conventional reagents, equipment, and methods in the art, and will not be described in detail further.

[0023] Example 1 The method for constructing a transient VIGS silencing system for Cyclocarya paliurus roots according to this embodiment includes the following steps: (1) Construction of recombinant carrier For the target plant, Cyclocarya paliurus CpMYC2 The gene-specific fragment (SEQ ID NO.1) was cloned by reverse transcription of RNA extracted from Cyclocarya paliurus roots as a cDNA template, followed by amplification using KOD high-fidelity enzyme; the amplification primers were as follows. CpMYC2 -F (SEQ ID NO.2) CpMYC2 -R (SEQ ID NO.3); the amplification program was: 95℃, 3 min; 95℃ 15 sec, 60℃ 15 sec, 72℃ 30 sec, 40 cycles; 72℃ 5 min for complete extension.

[0024] target gene CpMYC2 The tobacco brittle virus vector pTRV2 was digested with restriction endonucleases BamHI and XhoI. The double digestion system is shown in Table 1.

[0025] Table 1 Double enzyme digestion system

[0026] After double enzyme digestion, the DNA fragments were detected by agarose gel electrophoresis, and the corresponding fragments were recovered from the gel for DNA purification. Then, they were ligated using ligase to obtain the recombinant vector pTRV2- CpMYC2 After sequencing verification confirms correctness, it is ready for use.

[0027] (2) Agrobacterium transformation The recombinant vector pTRV2- was prepared using a freeze-thaw method. CpMYC2The tobacco brittle virus vector PTRV1 was transformed into Agrobacterium GV3101 to obtain the recombinant vector pTRV2-. CpMYC2 Agrobacterium and Agrobacterium containing the helper vector PTRV1, the operation method is as follows: Thaw 100 μL of Agrobacterium GV3101 competent cells in an ice bath; add 1 μg of expression vector plasmid, mix thoroughly, and incubate on ice for 5 min; quickly add the centrifuge tube to liquid nitrogen for 5 min; quickly remove and place in a 37°C water bath for 5 min; quickly remove and place on ice for 5 min; add 700 μL of antibiotic-free LB liquid medium and culture at 28°C with shaking for 2-3 h; centrifuge at 6000 rpm / min for 1 min, resuspend the cells in 100 μL of supernatant, and spread on LB solid medium containing 50 μg / mL Kan and 25 μg / mL Rif.

[0028] After the colonies have grown, single colonies are picked and verified by PCR.

[0029] (3) Obtaining the resuspension The sequenced correctly contains the recombinant vector pTRV2- CpMYC2 Single clones of *Agrobacterium* and *Agrobacterium* strain containing the helper vector pTRV1 were inoculated into LB liquid medium containing 50 μg / mL Kan and 25 μg / mL Rif, respectively, and cultured at 28 °C. The cells were collected by centrifugation at 5000 rpm for 10 min, and resuspended in infection solution (10 mM MgCl2, 10 mM MES, 200 μM acetylsylcholine) to adjust OD. 600 Up to 0.6.

[0030] (4) Preparation of inoculated bacterial solution The two resuspensions obtained in step (3) were mixed at a volume ratio of 1:1 and allowed to stand for 3 hours to obtain "PTRV1+ pTRV2- CpMYC2 "Immersion solution."

[0031] (5) Vacuum compression method to infect the root system of Eucommia ulmoides When the *Cyclocarya paliurus* seeds have grown to 2-3 true leaves, scratch the roots of the seedlings and directly soak the scratched roots in "pTRV1+pTRV2- CpMYC2 "The bacteria were immersed in the bacterial solution and connected to a vacuum pump for 3 minutes at a pressure of 0.06 MPa (using the "pTRV1+pTRV2" infection solution as a blank control). After infection, the Cyclocarya paliurus plants were cultured in the dark for 2 days, and then cultured under a cycle of "24℃, 16h light, 20℃, 8h darkness", with regular watering during the period."

[0032] Figure 1 The phenotype of the plant 10 days after infection.

[0033] (6) qRT-PCR detection of whether the target gene is silenced Roots of *Cyclocarya paliurus* plants were collected on days 2, 16, and 30 post-infection. RNA was extracted and reverse transcribed into cDNA, which was then used as a template for qPCR analysis. Cp18S Genes as internal reference genes for primer design Cp18S -F (SEQ ID NO. 4) Cp18S -R (SEQ ID NO.5), for samples treated with different methods CpMYC2 Genes were analyzed by relative quantitative qRT-PCR. The qRT-PCR reaction program was: 95℃, 2 min; 95℃, 10 sec, 40 cycles; 60℃, 30 s. The results are as follows: Figure 2 As shown.

[0034] Depend on Figure 1 and Figure 2 The results show that the silencing system constructed in this invention can effectively suppress... CpMYC2 Gene expression.

[0035] Example 2 The method for constructing a transient VIGS silencing system for Cyclocarya paliurus roots according to this embodiment includes the following steps: (1) Construction of recombinant carrier Same as Example 1.

[0036] (2) Agrobacterium transformation Same as Example 1.

[0037] (3) Obtaining the resuspension The sequenced correctly contains the recombinant vector pTRV2- CpMYC2 Single clones of *Agrobacterium* and *Agrobacterium* strain containing the helper vector pTRV1 were inoculated into LB liquid medium containing 50 μg / mL Kan and 25 μg / mL Rif, respectively, and cultured at 28 °C. The cells were collected by centrifugation at 5000 rpm for 10 min, and resuspended in infection solution (10 mM MgCl2, 10 mM MES, 200 μM acetylsylcholine) to adjust OD. 600 Up to 0.8.

[0038] (4) Preparation of inoculated bacterial solution The two resuspended solutions obtained in step (3) were mixed in a 1:1 ratio and shaken for 1 hour to obtain "pTRV1+pTRV2- CpMYC2 "Immersion solution."

[0039] (5) Root soaking method to infect the roots of Cyclocarya paliurus a. When the *Cyclocarya paliurus* seeds have grown to 2-3 true leaves, scratch the roots of the seedlings and separate the scratched roots using a black disposable film flowerpot. Then fill the black disposable film flowerpot with soil; b. Prepare another flowerpot, and fill the bottom of the flowerpot with "pTRV1+pTRV2- CpMYC2 "c. Fill cotton with the bacterial solution; insert the seedlings treated in step a downwards into the flowerpots of step b, so that the roots of the seedlings are immersed in the bacterial solution. At this time, part of the root system is immersed in the infection solution, while the other part is planted normally in the soil (using "pTRV1+pTRV2" infection solution as a blank control). After infection, the Cyclocarya paliurus plants are cultured in the dark for 2 days, and then cultured under the cycle of "24℃, 16h light, 20℃, 8h darkness", with regular watering during the period."

[0040] Figure 3 The phenotype of the plant is 20 days after infection.

[0041] (6) qRT-PCR detection of whether the target gene is silenced Same as Example 1, the results are as follows Figure 4 As shown.

[0042] Example 3 The method for constructing a transient VIGS silencing system for Cyclocarya paliurus roots according to this embodiment includes the following steps: (1) Construction of recombinant carrier Same as Example 1.

[0043] (2) Agrobacterium transformation Same as Example 1.

[0044] (3) Obtaining the resuspension The sequenced correctly contains the recombinant vector pTRV2- CpMYC2 Single clones of *Agrobacterium* and *Agrobacterium* strain containing the helper vector pTRV1 were inoculated into LB liquid medium containing 50 μg / mL Kan and 25 μg / mL Rif, respectively, and cultured at 28 °C. The cells were collected by centrifugation at 5000 rpm for 10 min, and resuspended in infection solution (10 mM MgCl2, 10 mM MES, 200 μM acetylsylcholine) to adjust OD. 600 Up to version 1.0.

[0045] (4) Preparation of inoculated bacterial solution The two resuspended solutions obtained in step (3) were mixed in a 1:1 ratio and shaken for 1 hour to obtain "pTRV1+ pTRV2- CpMYC2 "Immersion solution."

[0046] (5) Root soaking method to infect the roots of Cyclocarya paliurus a. When the *Cyclocarya paliurus* seeds have grown to 2-3 true leaves, make a small incision at the base of the seedlings and separate the incisions using a black disposable film pot. Then, fill the black disposable film pot with soil. b. Prepare another pot, filling the bottom with a mixture of pTRV1 and pTRV2. CpMYC2 "c. Fill cotton with the bacterial solution; insert the seedlings treated in step a downwards into the flowerpots of step b, so that the seedling roots are immersed in the bacterial solution for infection (using "pTRV1+pTRV2" infection solution as a blank control). After infection, the Cyclocarya paliurus plants are cultured in the dark for 2 days, and then cultured under the cycle of "24℃, 16h light, 20℃, 8h darkness", with regular watering during the period."

[0047] (6) qRT-PCR detection of whether the target gene is silenced Same as Example 1.

[0048] Experimental Example 1 This experimental example is used to verify different ODs 600 The effects of different infecting bacterial solutions and root immersion methods on the root silencing efficiency of Eucommia ulmoides.

[0049] I. Experimental Methods The method is the same as in Example 1, but the OD of the infection solution is adjusted during the preparation of the bacterial inoculum. 600 The concentrations were adjusted to 0.6, 0.8, and 1.0, respectively, and the roots of *Cyclocarya paliurus* were infected using the vacuum pumping method. The effect of different concentrations of infection solution on the silencing efficiency after 30 days was then studied.

[0050] Meanwhile, referring to the method of Example 2, but during the preparation of the infecting bacterial solution, the OD of the infecting solution is adjusted. 600 The concentrations were adjusted to 0.6, 0.8, and 1.0, respectively, and the roots of *Cephalotaxus fortunei* were infected using the root-damaged immersion method. The effect of different concentrations of infection solution on the silencing efficiency was then studied.

[0051] II. Experimental Results The results are shown in Tables 2 and 3.

[0052] Table 2 Different OD under vacuum pumping method 600 The effect of infecting bacterial solution on plant infection efficiency

[0053] Table 3 Different OD values ​​under root immersion method 600 Effect of infecting bacterial solution on infection efficiency of Cyclocarya paliurus plants

[0054] From the above results, we can conclude that: (1) In the vacuum pressure method, as the OD value of the bacterial culture increases, the silencing efficiency shows a trend of first increasing and then decreasing, indicating that when the OD value increases, the silencing efficiency decreases. 600 The silencing efficiency is highest when the value is 0.8; (2) In the root soaking method, as the OD value of the bacterial solution increased, the silencing efficiency showed a trend of first increasing and then decreasing, indicating that when the OD value increased, the silencing efficiency increased. 600 The silencing efficiency is highest when the value is 0.8; (3) The vacuum pressing method is generally superior to the root soaking method.

[0055] 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 constructing a Cyclobalanopsis glauca root system VIGS transient silencing system, characterized in that, It comprises the following steps: (1) The C. sinensis CpMYC2 The gene fragment and the tobacco rattle virus vector pTRV2 were connected to obtain a recombinant vector pTRV2- CpMYC2 ; (2) using freeze-thaw method, the recombinant vector pTRV2- CpMYC2 and tobacco brittle mosaic virus vector pTRV1 are transformed into Agrobacterium GV3101 respectively, to obtain Agrobacterium containing recombinant vector pTRV2- CpMYC2 , Agrobacterium containing auxiliary vector pTRV1; the transformed Agrobacterium is plated on solid medium, and after the colonies grow, single colonies are picked for PCR verification; (3) the Agrobacterium containing the recombinant vector pTRV2- CpMYC2 , the Agrobacterium strain containing the helper vector pTRV1 are inoculated separately in LB liquid medium for expansion culture; the bacterial bodies are collected by centrifugation, and then the collected bacterial bodies are resuspended with the infection liquid, and the OD 600 is adjusted to 0.6~1.0;​​ (4) The two resuspensions obtained in step (3) are mixed in a volume ratio of 1:1 to obtain a bacterial liquid for dyeing; (5) The root system of Cyclobalanopsis glauca is infected by using a vacuum pumping method or a root injury soaking method to realize the silencing of the target gene.

2. The method for constructing the transient VIGS silencing system of *Cyclocarya paliurus* root system according to claim 1, characterized in that, In step (1), the Rhamnus utilis CpMYC2 The gene and the virus carrier pTRV2 are double-enzymatically cut by restriction endonuclease BamH I and Xho I, the enzyme cutting products are purified, and the purified products are connected by a ligase to obtain a recombinant carrier pTRV2. CpMYC2 .

3. The method according to claim 1, wherein the method is characterized by, In the step (1), the Cyclocarya CpMYC2 The nucleotide sequence of the gene fragment is shown as SEQ ID NO.

1.

4. The method according to claim 1, wherein, In step (2), the specific steps of transforming Agrobacterium by freeze-thaw method are as follows: 100 μL of Agrobacterium GV3101 competent cells are thawed in an ice bath; 1 μg of expression vector plasmid is added and mixed thoroughly, and then the mixture is placed in an ice bath for 5 min; the centrifuge tube is quickly added into liquid nitrogen for 5 min; it is quickly taken out and placed in a 37℃ water bath for 5 min; it is quickly taken out and placed on ice, and then placed in an ice bath for 5 min; 700 μL of LB liquid medium without antibiotics is added, and then the mixture is cultured at 28℃ for 2-3 h; 100 μL of supernatant is reserved by centrifugation at 6000 rpm / min for 1 min, and then the bacterial body is resuspended for subsequent coating on LB solid medium containing 50 μg / mL Kan and 25 μg / mL Rif.

5. The method for constructing the transient VIGS silencing system of *Cyclocarya paliurus* root system according to claim 1, characterized in that, In step (3), the liquid medium is LB liquid medium containing 50 μg / mL Kan and 25 μg / mL Rif.

6. The method according to claim 1, wherein, In step (3), the formula of the infection liquid is as follows: 10 mM MgCl2, 10 mM MES, and 200 μM acetyl-syringone.

7. The method according to claim 1, wherein the method is characterized by, In step (5), the specific steps of the vacuum pumping method for infecting the root system of Cyclobalanopsis glauca are as follows: when the Cyclobalanopsis glauca seed is cultured to 2-3 true leaves, the root of the seedling is scratched, and then the scratched root is directly soaked in the bacterial liquid for dyeing, and then connected to a vacuum pumping device, and then pumped for 3 min with a pressure of 0.06 Mpa.

8. The method according to claim 1, wherein the method is characterized by, In step (5), the specific steps of the root injury soaking method for infecting the root system of Cyclobalanopsis glauca are as follows: a. When the Cyclobalanopsis glauca seed is cultured to 2-3 true leaves, the root of the seedling is scratched, and then the scratched root of the seedling is separated by a black disposable thin film flowerpot, and then the black disposable thin film flowerpot is filled with soil; b. Another flowerpot is prepared, and the bottom of the flowerpot is filled with cotton soaked in the bacterial liquid for dyeing; c. The root of the seedling treated in step a is inserted into the flowerpot in step b, so that the root of the seedling is soaked in the bacterial liquid for dyeing.

9. The method according to claim 1, wherein the method is characterized by, It further comprises step (6): The roots of Cyclocarya paliurus were collected, and RNA was extracted and reversely transcribed into cDNA as a template for qPCR analysis. The primers were designed according to the reference gene of Cyclocarya paliurus and the relative quantitative qRT-PCR analysis was performed on the target gene. Cp18S The primers were designed according to the reference gene of Cyclocarya paliurus and the relative quantitative qRT-PCR analysis was performed on the target gene. CpMYC2 The primers were designed according to the reference gene of Cyclocarya paliurus and the relative quantitative qRT-PCR analysis was performed on the target gene 10. The use of the construction method according to any one of claims 1-9 in verifying the function of Cyclobalanopsis glauca genes.

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