Citrus canker pathogen-mediated citrus leaf transient expression method

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a citrus canker pathogen mediated citrus leaf transient expression method which comprises the following steps: preparing a citrus canker pathogen infection solution which is xanthomonas citrus subspecies; infecting the citrus by using the citrus canker pathogen infection liquid, and culturing to obtain citrus infected by the citrus canker pathogen; preparing an agrobacterium infection solution containing a target gene; and inoculating the citrus infected by the citrus canker pathogen with an agrobacterium infection solution containing a target gene, and carrying out transient expression of the target gene. The transient expression method provided by the invention can realize transient and efficient expression of the target gene in leaves of different citrus varieties. The transient expression method disclosed by the invention can be used for researching gene functions, protein interaction, promoter activity analysis and the like; and the method can also be used for evaluating the citrus canker resistance gene function.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a citrus canker pathogen-mediated transient expression method in citrus leaves. Background Art

[0002] Citrus is one of the internationally traded agricultural products and an important economic fruit tree. Citrus bacterial canker, also known as CBC, is an important bacterial disease of citrus. When it occurs, it will lead to tree decline, leaf and fruit drop, and pustular lesions on the fruit, which seriously affect the quality and yield of citrus. The pathogen of citrus canker is Xanthomonas citri subsp. Citri, referred to as Xcc, which is a Gram-negative bacterium. During the infection and pathogenicity, it releases pathogenic factors into host cells through the type III secretion system. PthA4 is a transcription activator-like effector secreted through the type III secretion system, referred to as TAL. Studies have shown that PthA4 targets the citrus canker susceptibility gene CsLOB1 to induce its expression, leading to susceptibility. The CRISPR / Cas9 technology was used to edit the promoter of the citrus disease-susceptible gene CsLOB1 and the coding region of the CsLOB1 gene, and grapefruit and sweet oranges resistant to ulcer disease were obtained. In addition, there are not many reports and applications of citrus disease resistance or susceptibility genes to ulcer pathogens.

[0003] Plant genetic transformation is widely used in gene function research and crop improvement. At present, the study of the function of citrus canker resistance or susceptibility genes mainly relies on genetic transformation to prepare transgenic citrus materials or virus-induced gene silencing technology VIGS-mediated gene silencing, and then the resistance to canker bacteria is evaluated. The genetic transformation of citrus mainly adopts the Agrobacterium-mediated citrus epicotyl transformation method, which uses citrus explants as transformation materials and requires sterile culture. It is labor-intensive, time-consuming, costly, and has low transformation efficiency. According to statistics, the cycle of transgenic citrus materials is 12 to 18 months, and the transformation efficiency is about 0.1% to 11% due to different variety specificity. VIGS technology can inhibit the expression of endogenous genes in plants, and many viruses have been developed into VIGS tools for the study of plant gene functions. Tobacco rattle virus TRV is a widely used, efficient and durable viral vector, and is one of the most commonly used VIGS vectors. However, there is no evidence that TRV can systemically infect citrus. Citrus leafblotch virus, CLBV for short, can infect most citrus varieties through grafting or seed transmission, and has therefore been developed as a viral vector for citrus VIGS technology. However, the CLBV-VIGS system established on citrus is still unstable and cannot effectively induce VIGS in CLBV-insensitive hosts such as kumquat and grapefruit.

[0004] Transient expression of foreign genes in plant cells or tissues through Agrobacterium-mediated transient transformation technology is widely used in plant gene function research. However, many important economic crops and variants of specific species are highly resistant to Agrobacterium infection, and there are still problems in the transient expression of Agrobacterium in these species. Although the direct expression of the type III effector avrGf1 of ulcer bacteria in grapefruit leaves and the transient expression of the pepper Bs2 gene in lemon leaves have been successfully applied, most citrus varieties have strong resistance to Agrobacterium infection. The method of directly injecting Agrobacterium into citrus leaves for transient gene expression has low expression efficiency and poor reproducibility. Therefore, it is urgent to establish an efficient citrus leaf transient transformation system. Summary of the invention

[0005] The purpose of the present invention is to provide a method for transient expression in citrus leaves mediated by citrus canker bacteria, which solves the problem of low expression efficiency of traditional transient expression methods.

[0006] The technical solution adopted by the present invention is:

[0007] The present invention provides a method for transient expression in citrus leaves mediated by citrus canker bacteria, comprising the following steps:

[0008] preparing an infection solution of citrus canker pathogen, wherein the citrus canker pathogen is Xanthomonas citri subsp. citrus;

[0009] Infecting citrus with an infection solution of citrus canker bacteria, culturing, and obtaining citrus infected with citrus canker bacteria;

[0010] preparing an Agrobacterium infection solution containing the target gene;

[0011] The Agrobacterium infection solution containing the target gene is inoculated into the citrus infected with the citrus canker bacteria to perform transient expression of the target gene.

[0012] Preferably, the citrus varieties applicable to the citrus leaf transient expression method include any one of sweet orange, lemon, trifoliate orange and citron.

[0013] Preferably, the process of preparing the citrus canker bacteria infection solution is:

[0014] The activated X. citri ulcer bacteria were collected and the OD 600 Adjust the value to 0.003-0.3 to obtain the citrus canker bacteria infection solution.

[0015] Preferably, the OD of the citrus canker bacteria is 600 The value is 0.03.

[0016] Preferably, the part of the citrus fruit that is infected by the citrus canker pathogenic bacteria infection liquid is the back of the leaves.

[0017] Preferably, the culture condition is 28° C. for 8 h.

[0018] Preferably, the process of preparing the Agrobacterium infection solution containing the target gene is:

[0019] The Agrobacterium containing the target gene was cultured at 28°C, 200 rpm for 18 h, the cells were collected and resuspended, and the cell concentration was adjusted and then allowed to stand for 2 h.

[0020] Preferably, the bacterial cell concentration is OD 600 is 0.8.

[0021] Preferably, during the resuspension, the formula of the resuspension solution used is:

[0022] Each liter of LB medium contained 0.446 g MS salts, 0.4 g sucrose, 20 mmol (N-morpholino)ethanesulfonic acid, 10 mmol MgCl2 and 200 μmol acetosyringone.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a method for transient expression in citrus leaves mediated by citrus canker bacteria, comprising the following steps: preparing an infection solution of citrus canker bacteria, wherein the citrus canker bacteria is Xanthomonas citrus subspecies citrus; using the infection solution of citrus canker bacteria to infect citrus, culturing, and obtaining citrus infected by citrus canker bacteria; preparing an infection solution of Agrobacterium containing a target gene; inoculating the infection solution of Agrobacterium containing the target gene onto the citrus infected by citrus canker bacteria to perform transient expression of the target gene. The present invention provides a method for transient expression in citrus leaves mediated by Agrobacterium by citrus canker bacteria, and the transient expression method of the present invention can achieve transient and efficient expression of the target gene in leaves of different citrus varieties. The transient expression method of the present invention can be used to study gene function, protein interaction, promoter activity analysis, etc.; it can also be used for functional evaluation of citrus canker resistance genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The fluorescence observation diagrams of Xcc promoting the transient expression of Agrobacterium-mediated GFP on leaves of four different citrus species, namely sweet orange, lemon, trifoliate orange and citron, in Examples 1 and 4 to 6. A: The citrus leaves of different varieties were placed under white light and blue light respectively to take pictures and record the expression of GFP protein; B: The citrus leaves of different varieties were prepared and placed under a laser confocal microscope to observe the expression of GFP protein; 1: Xcc-treated citrus leaves were inoculated with Agrobacterium containing the target gene; 2: Sterile water-treated citrus leaves were inoculated with Agrobacterium containing the target gene; 3: Blank control.

[0026] Figure 2 Different applications of Xcc promoting Agrobacterium-mediated transient expression. A: GUS staining to detect Xcc promoting Agrobacterium-mediated GUS transient expression in citrus leaves; B: Western blot to detect Xcc promoting Agrobacterium-mediated GFP transient expression in citrus leaves.

[0027] Figure 3 Western blot was used to detect the effect of different concentrations of Xcc on Agrobacterium-mediated transient expression.

[0028] Figure 4 is the colonization amount of Xcc in sweet orange leaves after transient expression of CsLOB1 gene, with transient expression of GFP as control. DETAILED DESCRIPTION

[0029] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0030] The inventive concept of the present invention is as follows:

[0031] Transient expression technology is a technology that introduces the target gene into the target cell in a short time and establishes a short and efficient expression system in the cell. Compared with the stable genetic transformation system, plant transient expression technology has the advantages of simple operation, low cost, high efficiency and short cycle. Among them, Agrobacterium-mediated transient expression is the most common. At present, corresponding transient expression systems have been established in various crops such as wheat, rice, and corn, which provides convenience for the research and utilization of their gene functions. Agrobacterium-mediated transient transformation technology has been established and applied in citrus varieties such as grapefruit, Mexican lemon, and kumquat. Studies have shown that transient expression of the Bs2 gene from pepper in lemon can enhance its resistance to citrus canker pathogen Xcc. However, the stability and repeatability of transient transformation experiments on citrus are affected by many factors such as citrus varieties, Agrobacterium strains, expression vectors, temperature and humidity. Therefore, it is very necessary to establish a fast, efficient, and simple method for transient expression of citrus leaves and evaluation of canker resistance gene functions.

[0032] Based on this, the present invention provides a method for transient expression in citrus leaves mediated by citrus canker bacteria, comprising the following steps: preparing a citrus canker bacteria infection fluid, wherein the citrus canker bacteria is Xanthomonas citrus subspecies citrus; using the citrus canker bacteria infection fluid to infect citrus, culturing, and obtaining citrus infected with citrus canker bacteria; preparing an Agrobacterium infection fluid containing a target gene; inoculating the Agrobacterium infection fluid containing the target gene onto the citrus infected with the citrus canker bacteria to transiently express the target gene.

[0033] The purpose of the present invention is to overcome the defects and shortcomings of the Agrobacterium-mediated citrus transient expression method, and to provide a method for transient gene expression in multiple citrus varieties. This method is more efficient and convenient for gene expression, and based on this method, the citrus canker resistance function of the gene can be quickly evaluated.

[0034] In order to enable those skilled in the art to better understand the technical solution of the present invention and implement it, the present invention is further described below in conjunction with specific embodiments. In the description of the present invention, unless otherwise specified, the reagents used are all commercially available, and the methods used are all conventional techniques in the art.

[0035] The abbreviations of the present invention are shown in Table 1.

[0036] Table 1 Abbreviations

[0037] Abbreviation Full journey CBC Citrus bacterial canker Xcc Xanthomonas citri subsp. citri VIGS Gene silencing technology / Virus-induced gene silencing TRV Tobacco rattle virus NA Nutrient Agar MES 2-(N-Morpholino)ethanesulfonic acid AS Acetosyringone

[0038] Example 1

[0039] A method for transient expression in citrus leaves mediated by citrus canker bacteria, comprising the following steps:

[0040] S1. Prepare an infection solution of citrus canker bacteria, wherein the citrus canker bacteria is Xanthomonas citrus subspecies citrus, and use the infection solution of citrus canker bacteria to infect citrus, culture, and obtain citrus infected with citrus canker bacteria, the method is as follows:

[0041] Take the Xcc glycerol stock frozen at -80℃ and streak it on nutrient agar NA medium, and culture it at 28℃ for 2 days. Pick a single colony in 3mL NB liquid medium, and culture it at 28℃ and 200rpm for 16h. Centrifuge at 5000rpm for 5min to collect the bacteria, resuspend them in sterile water, and adjust the OD 600 The value was set to 0.03 to obtain the infection fluid of citrus canker. A microwound was made on the back of the citrus leaves with a needle, but no puncture was done. The infection fluid of citrus canker was absorbed and injected with a needleless syringe. Xcc was inoculated into the sweet orange and cultured at 28℃ for 8h.

[0042] S2. Prepare an Agrobacterium infection solution containing the target gene, inoculate the Agrobacterium infection solution containing the target gene into citrus infected with citrus canker bacteria, and perform transient expression of the target gene, as follows:

[0043] Take the Agrobacterium containing pCAMBIA1380-GFP vector frozen at -80℃ and streak it on LB resistance plate, and culture it at 28℃ for 3 days. Pick a single colony and add it to 3mL LB medium containing antibiotics, and culture it at 28℃, 200rpm shaking for 18h; then centrifuge at 3500rpm for 5min to collect the bacteria and add 3M resuspension solution to resuspend the Agrobacterium, and the final concentration of OD 600 The value was adjusted to 0.8 and allowed to stand for 2 hours. 8 hours after the sweet orange leaves were inoculated with Xcc, the Agrobacterium infection solution containing the target gene was sucked out using a needleless syringe and injected from the back of the sweet orange leaves, which were then bagged to keep moisture and cultured at a constant temperature of 28°C.

[0044] The formula of the resuspension solution: each liter of LB medium contains 0.446 g MS salts, 0.4 g sucrose, 20 mmol MES, 10 mmol MgCl2 and 200 μmol AS.

[0045] In order to verify the transformation effect of this example, the following GUS staining experiment was also performed:

[0046] The promoter sequence of CsHIPP7 was constructed into the pCAMBIA1380-GUS vector and transformed into Agrobacterium to obtain the P CsHIPP7 -GUS vector. CsHIPP7 -GUS vector Agrobacterium was cultured to a concentration of OD 600 =0.8, and then inoculated onto sweet orange leaves infected with Xcc. After 4 days, GUS staining test was performed to analyze the promoter activity of CsHIPP7.

[0047] Example 2

[0048] A method for transient expression in citrus leaves mediated by citrus canker bacteria is as follows:

[0049] In this example, the Xcc concentration in the citrus canker pathogen infection solution was OD 600 =0.3, and the rest of the method is exactly the same as in Example 1.

[0050] Example 3

[0051] A method for transient expression in citrus leaves mediated by citrus canker bacteria is as follows:

[0052] In this example, the Xcc concentration in the citrus canker pathogen infection solution was OD 600 =0.003, and the rest of the method is exactly the same as in Example 1.

[0053] Example 4

[0054] A method for transient expression in citrus leaves mediated by citrus canker bacteria is as follows:

[0055] The citrus variety used in this embodiment is lemon, and the rest of the method is exactly the same as that in Example 1.

[0056] Example 5

[0057] A method for transient expression in citrus leaves mediated by citrus canker bacteria is as follows:

[0058] The citrus variety used in this embodiment is Citrus aurantium, and the rest of the method is exactly the same as that in Example 1.

[0059] Example 6

[0060] A method for transient expression in citrus leaves mediated by citrus canker bacteria is as follows:

[0061] The citrus variety used in this embodiment is citron, and the rest of the method is exactly the same as that in Example 1.

[0062] The results of the citrus leaf transient expression method described in Examples 1 to 6 were analyzed as follows:

[0063] 1. GFP fluorescence observation. The GFP fluorescence observations of Example 1 and Example 4 to Example 6 are as follows:

[0064] The citrus leaves of different varieties were first treated with a concentration of OD 600 =0.03 Xcc treatment, 8 h later inoculated with pCAMBIA1380-GFP vector at a concentration of OD 600= 0.8 Agrobacterium, and fluorescence observation was performed after 6 days. Citrus leaves of different varieties were placed under white light and blue light, and photographed to record the expression of GFP fluorescence. The results are as follows Figure 1 A is shown in FIG. The present invention uses a LUYOR-3415RG dual fluorescent protein observation lamp for observation, with an excitation wavelength of 440 nm and an emission wavelength of 500 nm. Subsequently, the injection area on the citrus leaves of different varieties was cut with a blade and placed in a water droplet on a glass slide, and the slide was carefully pressed with a cover glass to avoid bubbles during the process. A Leica TCS SP8 fluorescence microscope was used for image acquisition, and the fluorescence observation results are shown in FIG. Figure 1 As shown in B. In the present invention, the excitation light wavelength for observing GFP is 488nm, and the emission light wavelength range is 500nm to 530nm. From the above results, it can be seen that GFP signals are detected in leaves of different varieties of citrus, and the pCAMBIA1380-GFP vector is successfully transformed into different varieties of citrus.

[0065] 2. GUS staining analysis, the GUS staining results of Example 1 are as follows:

[0066] The promoter sequence of CsHIPP7 was constructed into the pCAMBIA1380-GUS vector and transformed into Agrobacterium. 600 = 0.03 Xcc for 8 h, and then inoculated at a concentration of OD 600 =0.8 containing P CsHIPP7 -GUS vector was used to generate Agrobacterium. After 4 days, GUS staining test was performed to analyze the promoter activity of CsHIPP7. A leaf disc with a diameter of 9 mm was taken from the injection area using a hole puncher and GUS stain was added. After vacuum treatment for 10 minutes, it was incubated at 37°C for 24 hours. The GUS stain was removed and decolorized with anhydrous ethanol three times until the negative control was white. The GUS staining results were as follows: Figure 2 A, P CsHIPP7 The -GUS vector was successfully transformed into citrus, confirming that the transformation efficiency of the Xcc-mediated citrus leaf transient expression method was higher than that of the traditional transformation method.

[0067] 3. Protein extraction and protein immunoblotting to verify the protein level of Example 1:

[0068] On the sweet orange leaves, first use a concentration of OD 600 = 0.03 Xcc for 8 h, and then inoculated at a concentration of OD 600=0.8 Agrobacterium containing pCAMBIA1380-GFP vector, protein extraction and immunoblotting test were performed after 6 days. Weigh 0.2g sample of sweet orange leaves obtained in Example 1, grind into powder with liquid nitrogen, add 400μLRIPA high-intensity extract, RIPA high-intensity extract purchased from Yazyme, vortex mix, and stand on ice for 20min. Then centrifuge at 15000g for 10min at 4°C to collect the supernatant, add an appropriate amount of protein loading buffer, heat at 98°C for 10min, cool to room temperature and place on ice for standby. Use PAGE gel rapid preparation kit to prepare polyacrylamide gel SDS-PAGE, add protein sample to the spot well, electrophoresis at 150V for 50min, until the protein indicator band reaches the bottom of the gel. Subsequently, the protein was transferred to the PVDF membrane by wet transfer, 250mA constant flow transfer membrane for 1.5h, and then the PVDF membrane was placed in 10% skim milk powder by mass for 3h. Then, the PVDF membrane was incubated with the primary antibody diluted 1:2000 for 1.5 hours, and the membrane was washed with TBST 3 times, each time for 15 minutes. Then, the PVDF membrane was transferred to the diluted secondary antibody and incubated for 1.5 hours, and then the membrane was washed with TBST 3 times, each time for 15 minutes. Finally, the ECL colorimetric solution was covered on the PVDF membrane for luminescence color development, and the membrane was placed in a chemiluminescence instrument to take pictures. The protein detection results are shown in Figure 2. Figure 2 As shown in B, obvious GFP signals were detected in the sweet orange leaves obtained in Example 1.

[0069] 4. Analysis of Xcc disease resistance.

[0070] Since the Xcc inoculation concentration is too high, it may cause more serious leaf ulcer symptoms. The present invention uses Examples 1 to 3 to obtain the Xcc concentrations of OD 600 =0.3, 0.03 and 0.003, the effect on the transient expression mediated by Agrobacterium was tested and it was found that OD 600 = 0.3 still has a good promoting effect on Agrobacterium-mediated transient expression. The results are as follows Figure 3 shown.

[0071] In order to verify the application of the present invention in the evaluation of the function of anti-ulcer gene, the present invention selected the known citrus ulcer susceptibility gene CsLOB1 for verification. The CDS sequence of CsLOB1 was constructed into the pCAMBIA2300-GFP vector and transformed into Agrobacterium. 600 = 0.03 Xcc treatment, 8h later inoculated with a concentration of OD 600 = 0.8 Agrobacterium, and samples were taken after 4 days to analyze the colonization of Xcc in citrus leaves. Use a hole punch to take a leaf disc with a diameter of 6 mm, add 500 μL of sterile water and steel balls to oscillate and grind, and let it settle for 5 minutes. Take 100 μL of the supernatant and perform gradient dilutions, diluting 10 times to 10-6 5 μL of each concentration was spread on NA medium and cultured at 28°C for 2 days. The colonies were counted and the number of colonies per 1 cm was calculated. 2 The number of Xcc colonies on leaves. The colony count was used to evaluate the colonization of Xcc in leaves, thereby evaluating the resistance and sensitivity of the gene to Xcc. Figure 4 As shown, transient expression of CsLOB1 promoted the colonization of Xcc in sweet orange leaves compared with the GFP control, indicating that this transient expression method can be used to evaluate other disease resistance genes against canker pathogens.

[0072] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0073] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for transient expression in citrus leaves mediated by citrus canker bacteria, characterized in that: The following steps are involved: preparing an infection solution of citrus canker pathogen, wherein the citrus canker pathogen is Xanthomonas citri subsp. citrus; Infecting citrus with an infection solution of citrus canker bacteria, culturing, and obtaining citrus infected with citrus canker bacteria; preparing an Agrobacterium infection solution containing the target gene; The Agrobacterium infection solution containing the target gene is inoculated into the citrus infected with the citrus canker bacteria to perform transient expression of the target gene.

2. The method for transient expression of citrus leaves according to claim 1, characterized in that: The citrus varieties applicable to the citrus leaf transient expression method include any one of sweet orange, lemon, trifoliate orange and citron.

3. The method for transient expression of citrus leaves according to claim 1, characterized in that: The process of preparing the citrus canker pathogen infection solution is as follows: The activated X. citri ulcer bacteria were collected and the OD 600 Adjust the value to 0.003-0.3 to obtain the citrus canker bacteria infection solution.

4. The method for transient expression of citrus leaves according to claim 3, characterized in that: The OD of X. citri 600 The value is 0.

03.

5. The method for transient expression in citrus leaves according to claim 1, characterized in that: The part where the citrus canker pathogenic bacteria infects citrus is the back of the leaves.

6. The method for transient expression in citrus leaves according to claim 1, characterized in that: The culture condition is 28° C. for 8 h.

7. The method for transient expression in citrus leaves according to claim 1, characterized in that: The process of preparing the Agrobacterium infection solution containing the target gene is as follows: The Agrobacterium containing the target gene was cultured at 28°C and 200 rpm for 18 h, the cells were collected and resuspended, and the cell concentration was adjusted and then allowed to stand for 2 h.

8. The method for transient expression in citrus leaves according to claim 7, characterized in that: The bacterial concentration is OD 600 is 0.

8.

9. The method for transient expression in citrus leaves according to claim 7, characterized in that: During the resuspension, the formula of the resuspension solution used is: Each liter of LB medium contained 0.446 g MS salts, 0.4 g sucrose, 20 mmol (N-morpholino)ethanesulfonic acid, 10 mmol MgCl2 and 200 μmol acetosyringone.

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