Vacuum permeation transient gene expression method and application of Agrobacterium tumefaciens on loquat leaves

Abstract

This invention provides a method for transient gene expression via vacuum permeation of Agrobacterium tumefaciens in loquat leaves. The method involves: collecting loquat leaves and punching holes on both sides of the midrib parallel to the midrib to obtain leaf discs; preparing an Agrobacterium tumefaciens infection solution; immersing the leaf discs in the Agrobacterium tumefaciens infection solution and performing vacuum treatment to obtain infected leaf discs; rinsing the infected leaf discs with sterile distilled water and then blotting off the surface moisture with sterile filter paper; subsequently transferring the leaf discs flat onto MS solid agar plates and incubating in the dark to obtain loquat leaves with transient gene expression via vacuum permeation of Agrobacterium tumefaciens. The loquat leaves of this invention are available year-round, and transient transformation experiments can be conducted year-round. This method can be used to verify the function of genes related to photosynthetic metabolism, secondary metabolism (flavonoids, anthocyanins, etc.), abiotic stress, and disease resistance in loquat leaves, overcoming the limitation of existing loquat transient transformation methods that can only study fruit development genes.

Description

Technical Field

[0001] This invention belongs to the fields of plant biotechnology and genetic engineering technology, specifically relating to a method for transient gene expression of Agrobacterium tumefaciens in loquat leaves under vacuum and its application. Background Technology

[0002] Loquat is a perennial woody fruit tree of the Rosaceae family. It has a long growth cycle and is difficult to regenerate. There is no stable and efficient transgenic system, so it is impossible to rely on stable transformation to verify the function of homologous genes. In research, Agrobacterium-mediated transient expression technology is generally used to identify gene function.

[0003] Existing loquat transient transformation technology has significant limitations: the existing published patent (CN116837023A) only discloses an in vivo method for transient expression via injection into loquat fruit, which is only applicable to immature fruit materials and has several unavoidable drawbacks: (1) Limited time of use: Only young fruits 55 days and 95 days after full bloom can be used, and experiments can only be carried out during one period of the year (young fruit stage); (2) Limited tissue sites: Experiments can only be conducted in young fruit and cannot verify the function of specific genes in other tissues such as leaves.

[0004] As an evergreen fruit tree, loquat leaves can be used year-round, compensating for the time limitation of fruit injection. However, the underside of common loquat (Eriobotrya japonica) leaves is densely covered with thick hairs, making them naturally hydrophobic and difficult for the infection solution to adhere to. Even after manually wiping away the hairs, the leaf cuticle is dense and hard, preventing the bacterial solution from penetrating the mesophyll cells. This results in extremely low leaf infection efficiency after conventional vacuum infiltration and foliar injection treatments, with no significant upregulation of the target gene, failing to meet the requirements of subsequent experiments. Therefore, there is currently no publicly available technical solution for a highly efficient and instantaneous transformation system using loquat leaves.

[0005] According to plant classification, the genus *Eriobotrya* contains more than 30 species, with *Eriobotrya japonica* being one of them. Compared to model plants such as tobacco (Solanaceae) and Arabidopsis thaliana (Brassicaceae), *Eriobotrya* is more closely related to *Eriobotrya japonica*, and its leaves have no hairs on the underside, making it a more convincing source of evidence for transient expression. However, no studies have been reported on using leaves of *Eriobotrya* plants for transient genetic transformation. Summary of the Invention

[0006] The technical problem to be solved by this invention is to address the shortcomings of the existing technology by providing a method and application of Agrobacterium vacuum infiltration transient gene expression in loquat leaves. This method uses loquat leaves as material, and the leaves are available all year round, without being limited by the phenological period or season of the fruit tree. The transient transformation experiment can be carried out all year round and can be used to verify the function of genes related to photosynthetic metabolism, secondary metabolism (flavonoids, anthocyanins, etc.), abiotic stress, and disease resistance in loquat leaves, thus making up for the shortcoming of existing loquat transient transformation methods that can only study fruit development genes.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for transient gene expression of Agrobacterium tumefaciens through vacuum permeation in loquat leaves, the method being as follows: S1. Preparation of plant materials: Collect healthy functional leaves of the current year from the large-flowered loquat. First, rinse the surface dust with running water, then use sterile filter paper to absorb the surface moisture. Use a punch to make holes on both sides of the main vein of the current year's healthy functional leaves of the large-flowered loquat in a direction parallel to the main vein to obtain leaf discs. S2. Preparation of Agrobacterium tumefaciens infection solution: S201. Agrobacterium tumefaciens containing recombinant plasmids was streaked onto LB solid plates containing the corresponding antibiotics and incubated upside down at 28°C for 2 to 3 days to obtain single colonies. S202. Pick a single colony obtained from S201 and inoculate it into LB liquid medium containing the corresponding antibiotic, and culture with shaking until the bacterial culture reaches OD. 600 After adjusting the concentration to 0.6–0.8, the propagation bacterial solution was obtained. S203. Transfer the expanded bacterial culture obtained in S202 back to LB liquid medium containing the corresponding antibiotic, and continue to culture with shaking until the bacterial culture reaches OD. 600 =0.6~0.8, centrifuge, collect the bacterial cells, resuspend the bacterial cells with transient osmosis buffer, mix well, and let stand in the dark for 2 hours to obtain Agrobacterium tumefaciens infection solution; S3, Vacuum Permeation Infection: The leaf discs obtained in S1 were immersed in the Agrobacterium tumefaciens infection solution obtained in S203, and vacuum treated for 25 min under a pressure of -80 kPa. After discarding the Agrobacterium tumefaciens infection solution, the infected leaf discs were obtained. S4. Cleaning and Co-cultivation: First, rinse the infected leaf discs obtained in S3 with sterile distilled water, then blot dry the surface moisture with sterile filter paper. Transfer the infected leaf discs flat onto MS solid plate culture medium and incubate in the dark to obtain large-flowered loquat leaves with Agrobacterium vacuum permeation transient gene expression.

[0008] Preferably, the healthy functional leaves of the current year of the large-flowered loquat mentioned in S1 are the 2nd to 4th fully unfolded mature leaves from the top of the spring shoot, and the collection criteria are that the leaves are of uniform size, consistent color, free from disease spots, insect bites, mechanical damage, and uniform age.

[0009] Preferably, the diameter of the leaf disc in S1 is 1 cm.

[0010] Preferably, the temperature for shaking culture in S202 and S203 is 28℃, the rotation speed is 200rpm, and the time is 12h to 16h.

[0011] The present invention also provides an application of the above-mentioned method for transient gene expression by Agrobacterium vacuum permeation in loquat leaves, wherein the method is used for functional verification of related genes in loquat leaves.

[0012] Compared with the prior art, the present invention has the following advantages: 1. Loquat is an evergreen fruit tree, and its leaves are available all year round. It is not limited by the phenological period or season of fruit trees, and instantaneous transformation experiments can be carried out all year round. Compared with the existing young fruit injection method, it has a lower threshold for use.

[0013] 2. The method of the present invention can be used to verify the function of genes related to photosynthetic metabolism, secondary metabolism (flavonoids, anthocyanins, etc.), abiotic stress, and disease resistance in loquat leaves, making up for the shortcomings of existing loquat transient transformation methods that can only study fruit development genes.

[0014] 3. Existing methods for transient transformation of loquat only use cultivated loquat fruits. This invention is the first to select leaves of large-flowered loquat as a special material for transient transformation of the loquat genus. Under vacuum permeation conditions, the inoculum can smoothly infiltrate the leaf mesophyll tissue, providing a material with a closer kinship than the model plant for gene verification of common loquat, making the conclusions more reliable.

[0015] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0016] Figure 1 This is a diagram showing the back of the leaf disc before and after vacuum permeation treatment with Agrobacterium infection solution in Example 1 of the present invention.

[0017] Figure 2 This is a diagram showing the state of leaf discs after being infected with Agrobacterium tumefaciens solution and cultured in the dark for 40 hours in Example 1 of the present invention.

[0018] Figure 3 This is a qPCR quantitative diagram of the EjANS gene expression in loquat leaves during vacuum permeation in Example 2 of the present invention. Detailed Implementation

[0019] Example 1

[0020] This embodiment describes a method for transient EjANS gene overexpression by Agrobacterium tumefaciens in loquat leaves under vacuum permeation.

[0021] This embodiment requires strict adherence to aseptic operation procedures throughout the entire process. The transient osmotic buffer consists of the following raw materials at the final concentration (prepared and used immediately, sterilized by a 0.22μm filter membrane): 10mmol / L MES (pH 5.6), 10mmol / L MgCl2, and 150μmol / L acetylsylgenone (AS, which needs to be filtered and sterilized separately and added just before use).

[0022] The preparation methods of the ANS gene recombinant plasmid and Agrobacterium tumefaciens containing the recombinant plasmid in this embodiment are as follows: The common loquat gene annotated as ANS (anthocyanidin synthase) was named EjANS. The nucleotide sequence of its coding region is shown in SEQ ID NO:1. Using common loquat leaf cDNA as a template, the complete coding region sequence (CDS) was obtained by PCR amplification, yielding the gel-recovered PCR product of EjANS. The nucleotide sequences of the upstream primers required for PCR amplification are shown in SEQ ID NO:2, and the nucleotide sequences of the downstream primers are shown in SEQ ID NO:3. Using the Visclone Universal One Step Cloning Kit (CR101-50T) from Coolabion, the reaction solution was prepared according to the standard system: 5 μL Visclone universal Mix, 3 μL of gel-recovered PCR product of EjANS, 1 μL SK vector, and 1 μL sterile water. The reaction was carried out at 50℃ for 15 min. Subsequently, 5 μL of the reaction solution and 50 μL of... DH5α *E. coli* were mixed, placed on ice for 30 min, heat-shocked at 42°C for 60 s, and then 500 μL of kanamycin-resistant LB medium was added. After incubation at 37°C for 1 h, the bacterial suspension was spread onto LB plates containing kanamycin and incubated overnight at 37°C. Bacteria were then picked and sent for testing to obtain the recombinant plasmid pGreen0029SK-EjANS with the correct CDS sequence; the empty vector pGreen0029SK served as a control. The recombinant plasmid and the empty vector were electroporated into *Agrobacterium tumefaciens* GV3101, and positive strains were screened using LB plates at 28°C. The strains were then stored in glycerol at -80°C.

[0023] The method for transient gene expression of Agrobacterium tumefaciens in loquat leaves under vacuum permeation is as follows: S1. Preparation of plant materials: Collect healthy functional leaves of the current year from the large-flowered loquat. First, rinse the surface dust with running water, then use sterile filter paper to absorb the surface moisture. Use a 1cm diameter punch to punch holes on both sides of the main vein of the large-flowered loquat functional leaf (avoiding the main vein and thick lateral veins) to ensure that the leaf vein density and tissue state of each leaf disc are as consistent as possible, reduce systematic errors, and obtain leaf discs. The healthy functional leaves of the current year of the large-flowered loquat are the third fully unfolded mature leaves from the top of the spring shoot. The collection criteria are that the leaves are of uniform size, consistent color, free from disease spots, insect bites, mechanical damage, and uniform age. The collected leaf discs were randomly grouped, with 60 leaf discs in each biological replicate for the treatment group and the control group. S2. Preparation of Agrobacterium tumefaciens infection solution: S201. Agrobacterium tumefaciens containing the pGreen0029SK-EjANS recombinant plasmid and the empty plasmid were streaked onto LB solid plates containing kanamycin, respectively. After incubation at 28℃ for 2 days, single colonies of the treatment group and the control group were obtained. S202. Single colonies from the treatment group and control group obtained in S201 were picked and inoculated into LB liquid medium containing kanamycin. The cultures were then incubated with shaking at 28℃ and 200 rpm for 14 hours until the bacterial culture reached OD500. 600 After the value was 0.7, the propagation bacterial solution of the treatment group and the propagation bacterial solution of the control group were obtained. S203. The bacterial cultures obtained in S202 for the treatment group and the control group were transferred back to LB liquid medium containing kanamycin and cultured with shaking at 28°C and 200 rpm for 14 hours until the bacterial culture OD reached its limit. 600 After reaching a concentration of 0.7, the cells were centrifuged at 7000 rpm for 10 minutes to collect the bacterial cells. The cells were resuspended in transient osmosis buffer, and the OD values ​​of the two bacterial suspensions were adjusted using a spectrophotometer. 600 The same value was obtained, and acetylsuccione was added, mixed well, and allowed to stand at room temperature in the dark for 2 hours to obtain Agrobacterium tumefaciens infection solution of the treatment group and Agrobacterium tumefaciens infection solution of the control group. The volume ratio of the propagation culture medium to LB liquid medium was 1:50; S3, Vacuum Permeation Infection: Beforehand, move the vacuum desiccator and sterile conical flasks into the laminar flow hood. Place the Agrobacterium tumefaciens infection solutions obtained in S203 for the treatment group and control group into sterile conical flasks respectively. Completely immerse the divided leaf discs obtained in S1 into the Agrobacterium tumefaciens infection solutions for the treatment and control groups. Place the open conical flasks into the vacuum desiccator, seal them, and connect the vacuum pump. Slowly evacuate to -80 kPa and maintain the pressure for 25 minutes. During this time, it can be observed that the bacterial solution fully penetrates into the leaf interior and the leaf disc gradually settles. After vacuum treatment, slowly open the vent valve to restore normal pressure, allowing the bacterial solution to fully penetrate into the interstitial spaces of the leaf tissue, completing the infection. Discard the Agrobacterium tumefaciens infection solution to obtain the infected leaf discs (treatment group and control group). The condition of the back of the leaf discs before and after vacuum penetration treatment is as follows. Figure 1 As shown; S4. Cleaning and Co-cultivation: First, rinse the infected leaf discs obtained in S3 three times with sterile distilled water to thoroughly remove the free Agrobacterium bacteria adhering to the leaf surface. Then, gently blot the surface moisture with sterile filter paper to avoid scratching the leaf tissue. Transfer the infected leaf discs flat onto MS solid agar plates, ensuring the discs do not overlap or compress. Seal the plates with sealing film and incubate in the dark at 25°C for 40 hours. This yields leaves of *Agrobacterium tumefaciens* with transient gene expression via vacuum permeation. The leaf discs are in the following condition: Figure 2 As shown.

[0024] This embodiment uses an empty vector negative control, strictly adhering to the single variable principle: The control group was only modified by replacing the plasmid carried by Agrobacterium with an empty vector pGreen0029SK without the target gene inserted. All other operations (bacterial concentration, settling time, vacuum parameters, washing steps, and culture conditions) were completely synchronized with the treatment group. This control was used to exclude the influence of Agrobacterium infection, vector skeleton, and physical operations on leaf gene expression, metabolic level, and physiological state, and to ensure that the differences detected later were caused by overexpression of the target gene.

[0025] In this embodiment, the healthy functional leaves of the current year of loquat in step S1 can also be the second and fourth fully unfolded mature leaves from the top of the spring shoots; in step S201, the culture can be inverted for 3 days; the shaking culture time in steps S202 and S203 can also be 12h, 13h, 15h, and 16h, respectively, and the OD of the bacterial solution... 600 =0.6, 0.8.

[0026] Example 2

[0027] This example is a qPCR quantitative analysis of the ANS gene expression level in loquat leaves obtained from the Agrobacterium vacuum permeation transient gene expression in Example 1.

[0028] RNA was extracted from *Agrobacterium tumefaciens* leaves obtained by liquid nitrogen flash freezing in Example 1 for transient gene expression via vacuum permeation. The RNA quality was shown in Table 1 and met the requirements for downstream reverse transcription cDNA experiments. The RNA was reverse transcribed into cDNA, and qPCR quantification was performed using loquat Actin as an internal reference gene and cDNA as a template. The nucleotide sequences of the upstream primers for Actin internal reference gene qPCR quantification are shown in SEQ ID NO:4, the downstream primers for Actin internal reference gene qPCR quantification are shown in SEQ ID NO:5, the upstream primers for ANS gene qPCR quantification are shown in SEQ ID NO:6, and the downstream primers for ANS gene qPCR quantification are shown in SEQ ID NO:7.

[0029] Table 1. RNA concentration and mass of leaf discs after infection.

[0030] Experimental results are as follows Figure 3 As shown, the relative expression level of EjANS in the empty vector control group (Ev) was 0.53, while the mean relative expression level of EjANS in the overexpression treatment group (Ox) was 1.52, which was significantly higher than that in the control group (P<0.05). This demonstrates that this method can increase the mRNA peak of EjANS and achieve transient overexpression of the gene.

[0031] In summary, the large-flowered loquat used in this invention is an evergreen fruit tree, with leaves available year-round, and is not limited by the phenological period or season of the fruit tree. The instantaneous transformation experiment can be carried out throughout the year. The method of this invention can be used to verify the function of genes related to photosynthetic metabolism, secondary metabolism (flavonoids, anthocyanins, etc.), abiotic stress, and disease resistance in loquat leaves.

[0032] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for transient gene expression of Agrobacterium tumefaciens in the leaves of Loquat macrocarpa under vacuum, characterized in that, The method is as follows: S1. Preparation of plant materials: Collect healthy functional leaves of the current year from the large-flowered loquat. First, rinse the surface dust with running water, then use sterile filter paper to absorb the surface moisture. Use a punch to make holes on both sides of the main vein of the current year's healthy functional leaves of the large-flowered loquat in a direction parallel to the main vein to obtain leaf discs. S2. Preparation of Agrobacterium tumefaciens infection solution: S201. Agrobacterium tumefaciens containing recombinant plasmids was streaked onto LB solid plates containing the corresponding antibiotics and incubated upside down at 28°C for 2 to 3 days to obtain single colonies. S202. Pick a single colony obtained from S201 and inoculate it into LB liquid medium containing the corresponding antibiotic, and culture with shaking until the bacterial culture reaches OD. 600 After adjusting the concentration to 0.6–0.8, the propagation bacterial solution was obtained. S203. Transfer the expanded bacterial culture obtained in S202 back to LB liquid medium containing the corresponding antibiotic, and continue to culture with shaking until the bacterial culture reaches OD. 600 =0.6~0.8, centrifuge, collect the bacterial cells, resuspend the bacterial cells with transient osmosis buffer, mix well, and let stand in the dark for 2 hours to obtain Agrobacterium tumefaciens infection solution; S3, Vacuum Permeation Infection: The leaf discs obtained in S1 were immersed in the Agrobacterium tumefaciens infection solution obtained in S203, and vacuum treated for 25 min under a pressure of -80 kPa. After discarding the Agrobacterium tumefaciens infection solution, the infected leaf discs were obtained. S4. Cleaning and Co-cultivation: First, rinse the infected leaf discs obtained in S3 with sterile distilled water, then blot dry the surface moisture with sterile filter paper. Transfer the infected leaf discs flat onto MS solid plate culture medium and incubate in the dark to obtain large-flowered loquat leaves with Agrobacterium vacuum permeation transient gene expression.

2. The method for transient gene expression of Agrobacterium tumefaciens in loquat leaves under vacuum according to claim 1, characterized in that, The healthy functional leaves of the current year of the large-flowered loquat described in S1 are the 2nd to 4th fully unfolded mature leaves from the top of the spring shoots, and the collection criteria are that the leaves are of uniform size, consistent color, free from disease spots, insect bites, mechanical damage, and uniform age.

3. The method for transient gene expression of Agrobacterium tumefaciens in loquat leaves under vacuum according to claim 1, characterized in that, The diameter of the leaf disc described in S1 is 1 cm.

4. The method for transient gene expression of Agrobacterium tumefaciens in loquat leaves under vacuum according to claim 1, characterized in that, The shaking culture in S202 and S203 was carried out at a temperature of 28℃, a rotation speed of 200 rpm, and a time of 12h to 16h.

5. The method for transient gene expression of Agrobacterium tumefaciens in loquat leaves under vacuum permeation according to claim 1, characterized in that, The centrifugation in S203 was performed at 7000 rpm for 10 min; the transient osmosis buffer consisted of the following raw materials at final concentrations: MES 10 mmol / L, MgCl2 10 mmol / L, and acetylsuccinone 150 μmol / L; the volume ratio of the propagation culture to LB liquid medium was 1:

50.

6. The method for transient gene expression of Agrobacterium tumefaciens in loquat leaves under vacuum according to claim 1, characterized in that, The temperature for dark incubation in S4 is 25℃ and the time is 40h.

7. An application of the method for transient gene expression of Agrobacterium tumefaciens in loquat leaves as described in any one of claims 1-6, characterized in that, The Agrobacterium vacuum permeation transient gene expression method for loquat leaves is used to verify the function of related genes in loquat leaves.

Images