Method for in-situ instantaneous transformation of sweet potato leaves

Abstract

A sweet potato leaf in-situ instantaneous transformation method comprises the following steps that sweet potato stem segments are sheared and cut into a matrix or water, and the sweet potato stem segments are placed in a special incubator to be cultured till rooting; transforming a plasmid carrying a target gene into agrobacterium through a standard transformation program to obtain agrobacterium for infection, and adding the agrobacterium for infection into an infection solution to obtain an agrobacterium infection working solution; the method comprises the following steps of: pricking holes around veins of new leaves of a cultivated sweet potato plant, inversely putting the sweet potato plant into a vacuum flask filled with an agrobacterium infection working solution, completely immersing the leaves into the agrobacterium infection working solution, and treating for 40-50 seconds under the vacuum condition of 0.6-0.8 pa; and immediately transferring the infected plant into a turnover box for dark culture, and transferring the plant into a professional incubator for continuous culture after dark culture is finished, thereby obtaining the target gene introduced sweet potato transformed plant. The method can simplify the operation, shorten the time and improve the expression effect, and can be used for researching gene functions.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology and genetic engineering, and specifically to a method for in situ transient transformation of sweet potato leaves. Background Art

[0002] Plant genetic transformation technology is an essential tool for plant genetic engineering and modern agricultural molecular breeding. Plant genetic transformation mainly includes stable genetic transformation and transient transformation. However, the existing plant stable genetic transformation schemes are complex and inefficient, which restricts the genetic modification of most resource plants or crops and has become a technical barrier to the development and utilization of plant resources. At the same time, transient transformation technology is also mostly expressed in heterologous plants, such as tobacco leaves and onion epidermis, which leads to inaccurate gene function verification or false positives, which seriously hinders plant gene function verification and molecular regulatory network analysis.

[0003] As an important grain crop in my country, sweet potato encounters the same technical obstacles in the process of modern agricultural molecular breeding. At present, the transient transformation of sweet potato is mostly based on tobacco and onion epidermis. Transient heterologous expression of sweet potato genes may lead to silencing of some genes or low expression levels, which hinders the functional verification of sweet potato genes. At the same time, the method of extracting sweet potato protoplasts as transient expression receptors is feasible but difficult to operate and costly. Therefore, there is an urgent need for an economical, simple and suitable method for in situ transient expression of sweet potato leaves. Summary of the invention

[0004] The purpose of the present invention is to provide a method for in situ transient transformation of sweet potato leaves, which can simplify the operation, shorten the time, improve the expression effect, improve the accuracy and homology of sweet potato gene function identification, and can be used for batch gene function identification in sweet potato molecular biology research.

[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0006] A method for in situ transient transformation of sweet potato leaves comprises the following steps:

[0007] (1) Plant culture: Cut the sweet potato stem segments and plant them in a substrate or water, and place them in a professional incubator for culture until they take root; before the infection operation, remove the old leaves on the sweet potato plants and only keep the new leaves as the specific objects for subsequent infection;

[0008] (2) preparing Agrobacterium infection working solution;

[0009] (2-1) Preparing Agrobacterium for infection: The plasmid carrying the target gene is transformed into Agrobacterium through a standard transformation procedure, and the Agrobacterium containing the recombinant plasmid of the target gene is aspirated and added to LB liquid culture medium. SPE antibiotics are then added to the liquid culture medium, which is placed in a constant temperature shaker. After overnight culture, the cells are collected by centrifugation to obtain the Agrobacterium for infection;

[0010] (2-2) adding the Agrobacterium used for infection prepared in step (2-1) to the infection solution, suspending and mixing, and forming an Agrobacterium infection working solution;

[0011] (3) infection step;

[0012] (3-1) introducing the Agrobacterium infection working solution prepared in step (2) into a vacuum bottle;

[0013] (3-2) punching holes around the veins of the new leaves of the sweet potato plants cultured in step (1), and then placing the sweet potato plants upside down in a vacuum bottle containing an Agrobacterium infection working solution, so that the punched leaves are completely immersed in the Agrobacterium infection working solution;

[0014] (3-3) Turn on the negative pressure vacuum pump, adjust the pressure in the vacuum bottle to 0.6-0.8 Pa, and maintain it for 40-50 seconds, then quickly turn off the vacuum pump, keep the leaves in the negative pressure environment for 25-35 seconds, then take out the sweet potato plants, and let the excess Agrobacterium infection working solution on the leaves dry naturally in a well-ventilated environment;

[0015] (4) Immediately transfer the infected plants in step (3) to a turnover box for dark culture. After the dark culture is completed, transfer the plants to a professional culture box for further culture to obtain sweet potato transformants into which the target gene has been introduced, and use RT-qPCR to detect the transient expression of the target gene in the plant.

[0016] Preferably, in step (2-1), when the OD600 of the bacterial solution is 0.8-1.0, the bacteria are collected by centrifugation to obtain Agrobacterium used for infection.

[0017] In the further step (2), the preparation process of the infection solution is: accurately add acetosyringone and SILWET-77 surfactant into 1 / 4MS culture medium, mix thoroughly, and prepare an infection solution with specific active ingredients; the working concentration of acetosyringone in the infection solution is 30 mg / l, and the working concentration of SILWET-77 surfactant in the infection solution is 0.02wt%.

[0018] Preferably, in step (2), the original vector of the plasmid carrying the target gene is 35S-RUBY or pCAMBIA1300-EGFP.

[0019] Preferably, in step (2), the Agrobacterium is strain EHA105.

[0020] Preferably, in step (1), the length of the sweet potato stem segment is 20 cm; the culture conditions set in the professional incubator are: the temperature is 28° C., and the light-dark culture cycle is set to alternate 16 h of light and 8 h of darkness.

[0021] Preferably, in step (1) and step (4), the culture conditions set in the turnover box are: humidity of 85-95%, and dark culture for 24 hours in a dark environment; the culture conditions set in the professional incubator are: temperature is stable at 28°C, and the light-dark culture cycle is set to 16 hours of light and 8 hours of darkness for 3 days.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention utilizes a vacuum infiltration infection method to allow the Agrobacterium bacteria liquid carrying the target gene to efficiently infiltrate the sweet potato leaf tissue, and achieve high expression of the target gene, which can be used for the study of gene function. The method is applicable to sweet potato seedlings of any variety, is simple to operate and takes a short time, has low requirements on the leaf state, and the Agrobacterium infection liquid infiltrates evenly, and can achieve infection and penetration of the entire sweet potato leaf at one time.

[0024] (2) The method for transient gene expression established by the present invention is simple and easy to operate and has a good expression effect. It solves the problem of gene expression silencing or specific expression caused by heterologous transformation of sweet potato functional genes to a certain extent, improves the accuracy and homology of sweet potato gene function identification, and can be used for batch gene function identification in sweet potato molecular biology research. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the infection process of sweet potato leaves;

[0026] Figure 2 This is a comparative diagram of the expression of the RUBY gene in sweet potato leaves;

[0027] Figure 3 To show the expression of RUBY gene in leaves of different sweet potato varieties;

[0028] Figure 4 This is a comparison chart of the expression levels of RUBY in different sweet potato leaves detected by RT-qPCR;

[0029] Figure 5 The expression diagram of the EGFP gene in sweet potato leaves; (a) phenotype of uninfected leaves and local magnification, (b) phenotype of infected leaves and local magnification, (c) comparison of EGFP gene expression levels in leaves before and after infection, (d) EGFP expression in leaf cells after infection. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0033] The original vectors 35S-RUBY and pCAMBIA1300-EGFP of the plasmid carrying the target gene in this example were purchased from Zhili Zhongxin (Wuhan) Biotechnology Co., Ltd.

[0034] Example 1

[0035] like Figure 1 As shown, a method for in situ transient transformation of sweet potato leaves comprises the following steps:

[0036] (1) Plant culture: Cut sweet potato stem segments with a length of 20 cm and insert them into a substrate or water, and place them in a professional incubator with a strictly controlled temperature of 28°C and a light-dark cycle of 16 h light and 8 h dark alternating for cultivation until they take root; before the infection operation, remove the old leaves on the sweet potato plants and keep only the new leaves as the specific objects for subsequent infection to ensure the infection effect;

[0037] (2) preparing Agrobacterium infection working solution;

[0038] (2-1) Preparation of Agrobacterium for infection: The plasmid carrying the RUBY gene (SEQ ID No. 1) was transformed into the Agrobacterium EHA105 strain by a standard transformation procedure. The original vector used was 35S-RUBY. Then, 10 μL of the Agrobacterium containing the RUBY gene recombinant plasmid was aspirated and added to 100 ml of LB liquid culture medium. Then, 50 μL of 50 mg / ml SPE antibiotics was added to the liquid culture medium. The culture was placed in a 28°C constant temperature shaker (200 rap / min) and cultured overnight. When the bacterial solution OD600 = 0.8-1.0, the bacterial cells were collected by centrifugation at 5000 rap / min to obtain the Agrobacterium for infection.

[0039] (2-2) adding the Agrobacterium prepared in step (2-1) to 100 ml of infection solution, suspending and mixing, and preparing an infection solution working solution with specific active ingredients to enhance the infection ability of Agrobacterium on sweet potato plants;

[0040] The preparation process of the infection solution is as follows: accurately add 153 μL of 100 μmol / L acetosyringone to 100 ml of 1 / 4 MS culture medium (weigh: 0.1185 g MS powder dissolved in 100 ml distilled water, MS powder purchased from Qingdao Haibo Biotechnology Co., Ltd., item number: HB8469) to make its working concentration 30 mg / l, and add 2 μL of SILWET-77 surfactant to make its working concentration 0.02 wt%.

[0041] (3) infection step;

[0042] (3-1) introducing the Agrobacterium infection working solution prepared in step (2) into a specially made vacuum bottle with good sealing and negative pressure resistance to ensure that the bacterial solution will not leak or be contaminated during subsequent operations;

[0043] (3-2) using a syringe needle that has been strictly sterilized, puncture holes around the veins of the new leaves of the sweet potato plants cultured in step (1), and then invert the sweet potato plants into a vacuum bottle containing Agrobacterium infection working solution, so that the punctured leaves are completely immersed in the Agrobacterium infection working solution to ensure that the bacterial solution is in full contact with the leaves;

[0044] (3-3) Turn on the negative pressure vacuum pump, adjust the pressure in the vacuum bottle to 0.6-0.8 Pa, and maintain it for 45 seconds. During this period, the Agrobacterium infection working solution can more efficiently enter the leaf tissue under the action of negative pressure; then quickly turn off the vacuum pump, keep the leaves in the negative pressure environment for 30 seconds, and then carefully remove the sweet potato plants, and let the excess Agrobacterium infection working solution on the leaves dry naturally in a well-ventilated environment to avoid contamination or uneven infection caused by the flow of the Agrobacterium infection working solution;

[0045] (4) Immediately transfer the infected plants in step (3) to a turnover box with a humidity of 85%-95% and a dark environment for 24 hours of dark culture to provide ideal conditions for the initial integration and stabilization of the bacterial solution in the plant body; finally, transfer the plants to a professional incubator with a temperature stabilized at 28°C and a light-dark culture cycle set to alternate 16 hours of light and 8 hours of darkness, and continue to culture for 3 days. After 3 days, observe the expression of the target gene, and use RT-qPCR to detect the transient expression of the target gene in the plant body to obtain the sweet potato transformed strain with the target gene introduced.

[0046] like Figure 2 As shown in the figure, this method can be used to instantly transform the vector plasmid containing the RUBY gene into sweet potato leaves, and it can express normally, synthesize betacyanin, and turn the leaves red. At the same time, the experiment was repeated using 6 different sweet potato varieties, such as Figure 3As shown in the figure, the method can instantly transform the vector plasmid containing the RUBY gene into different sweet potato leaves, showing different degrees of red. At the same time, q-RTPCR was used to detect the expression of RUBY in sweet potato leaves, and it was found that the relative expression of RUBY in the transformed leaves was significantly higher than that in the control leaves. The relative expression of RUBY in the transformed leaves of Qining 26 was 1032.46 times that of the control (as shown in the figure). Figure 4 ). This further indicated that the RUBY gene could be normally expressed in sweet potato leaves, proving that this method could integrate the target gene into leaves for transient expression.

[0047] Example 2

[0048] like Figure 1 As shown, a method for in situ instantaneous transformation of sweet potato leaves is provided. Steps (1) and steps (3)-(4) involved in the method are consistent with those in Example 1, except for step (2):

[0049] (2) preparing Agrobacterium infection working solution;

[0050] (2-1) Preparation of Agrobacterium for infection: The plasmid carrying the EGFP gene (SEQ ID No. 2) was transformed into the Agrobacterium EHA105 strain by a standard transformation procedure. The original vector used was pCAMBIA1300-EGFP. Then, 10 μL of the Agrobacterium containing the EGFP gene recombinant plasmid was aspirated and added to 100 ml of LB liquid culture medium. Then, 50 μL of 50 mg / ml SPE antibiotics was added to the liquid culture medium. The culture was placed in a 28°C constant temperature shaker (200 rap / min) and cultured overnight. When the bacterial solution OD600 = 0.8-1.0, the bacterial cells were collected by centrifugation at 5000 rap / min to obtain the Agrobacterium for infection.

[0051] (2-2) adding the Agrobacterium prepared in step (2-1) to 100 ml of infection solution, suspending and mixing, and preparing an infection solution working solution with specific active ingredients to enhance the infection ability of Agrobacterium on sweet potato plants;

[0052] The preparation process of the infection solution remains consistent with that of Example 1.

[0053] like Figure 5 As shown, green fluorescent spots can be observed in the sweet potato leaves after 3 days of culture under 488nm fluorescent light, while there are no obvious fluorescent spots in the control, indicating that the EGFP protein is expressed in the sweet potato leaves and is excited to emit green fluorescence under the fluorescent light ( Figure 5 a and 5b). At the same time, RT-qPCR detected the expression of EGFP gene, and it was also found that the relative expression of EGFP gene in transformed leaves was significantly higher than that in the control ( Figure 5c). Observation under a fluorescence microscope also revealed that the transformed cell walls and nuclei appeared distinctly green, consistent with literature reports ( Figure 5 d) The experimental results show that EGFP can be integrated into sweet potato leaves through this method and can be expressed normally.

[0054] The above two examples show that this method can effectively integrate the target gene into sweet potato leaves for expression, provide technical support for sweet potato gene in situ expression experiments, such as subcellular localization, BiFC, LUC, etc., and provide a methodological basis for sweet potato molecular assisted breeding.

Claims

1. A method for in situ transient transformation of sweet potato leaves, characterized in that: The following steps are involved: (1) Plant culture: Cut the sweet potato stem segments and plant them in a substrate or water, and place them in a professional incubator for culture until they take root; before the infection operation, remove the old leaves on the sweet potato plants and only keep the new leaves as the specific objects for subsequent infection; (2) preparing Agrobacterium infection working solution; (2-1) Preparing Agrobacterium for infection: The plasmid carrying the target gene is transformed into Agrobacterium through a standard transformation procedure, and the Agrobacterium containing the recombinant plasmid of the target gene is aspirated and added to LB liquid culture medium. SPE antibiotics are then added to the liquid culture medium, which is placed in a constant temperature shaker. After overnight culture, the cells are collected by centrifugation to obtain the Agrobacterium for infection; (2-2) adding the Agrobacterium used for infection prepared in step (2-1) to the infection solution, suspending and mixing, and forming an Agrobacterium infection working solution; (3) infection step; (3-1) introducing the Agrobacterium infection working solution prepared in step (2) into a vacuum bottle; (3-2) punching holes around the veins of the new leaves of the sweet potato plants cultured in step (1), and then placing the sweet potato plants upside down in a vacuum bottle containing an Agrobacterium infection working solution, so that the punched leaves are completely immersed in the Agrobacterium infection working solution; (3-3) Turn on the negative pressure vacuum pump, adjust the pressure in the vacuum bottle to 0.6-0.8 Pa, and maintain it for 40-50 seconds, then quickly turn off the vacuum pump, keep the leaves in the negative pressure environment for 25-35 seconds, then take out the sweet potato plants, and let the excess Agrobacterium infection working solution on the leaves dry naturally in a well-ventilated environment; (4) Immediately transfer the infected plants in step (3) to a turnover box for dark culture. After the dark culture is completed, transfer the plants to a professional culture box for further culture to obtain sweet potato transformants into which the target gene has been introduced, and use RT-qPCR to detect the transient expression of the target gene in the plant.

2. The method for in situ transient transformation of sweet potato leaves according to claim 1, characterized in that: In step (2-1), when the OD600 of the bacterial solution is 0.8-1.0, the bacteria are collected by centrifugation to obtain the Agrobacterium used for infection.

3. The method for in situ transient transformation of sweet potato leaves according to claim 1, characterized in that: In step (2), the preparation process of the infection solution is: accurately add acetosyringone and SILWET-77 surfactant into 1 / 4MS culture medium, mix thoroughly, and prepare an infection solution with specific active ingredients; the working concentration of acetosyringone in the infection solution is 30 mg / l, and the working concentration of SILWET-77 surfactant in the infection solution is 0.02wt%.

4. The method for in situ transient transformation of sweet potato leaves according to claim 1, characterized in that: In step (2), the original vector of the plasmid carrying the target gene is 35S-RUBY, pCAMBIA1300-EGFP.

5. The method for in situ transient transformation of sweet potato leaves according to claim 1, characterized in that: In step (2), the Agrobacterium is strain EHA105.

6. The method for in situ transient transformation of sweet potato leaves according to claim 1, characterized in that: In step (1), the length of the sweet potato stem segment is 20 cm; the culture conditions set in the professional incubator are: the temperature is 28° C., and the light-dark culture cycle is set to alternate 16 h of light and 8 h of darkness.

7. The method for in situ transient transformation of sweet potato leaves according to claim 1, characterized in that: In step (1) and step (4), the culture conditions set in the turnover box are: humidity of 85-95%, dark culture for 24 hours in a dark environment; the culture conditions set in the professional incubator are: temperature is stable at 28°C, and the light-dark culture cycle is set to 16 hours of light and 8 hours of darkness for 3 days.

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