Novel agrobacterium tumefaciens-mediated injection method applied to instantaneous transformation of strawberry fruits
The novel strawberry fruit injection method addresses inefficiencies in Agrobacterium-mediated transformation by targeting the fruit stalk for uniform gene delivery, enhancing dye diffusion and transformation efficiency in strawberry fruits.
Patent Information
- Application Number
- CN202411528365.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-30
- Publication Date
- 2024-12-13
AI Technical Summary
Existing Agrobacterium-mediated transient transformation methods for strawberry fruit face challenges in transformation efficiency and expression stability, necessitating an improved method for efficient gene delivery.
A novel injection method targeting the fruit stalk of strawberry fruits, using Agrobacterium strain GV3101, with controlled injection into the central pith through the fruit stalk, optimizing the selection of strawberry variety 'Jian De Hong' in the ginkgo stage, and evaluating transformation efficiency by injecting myb10-vigs and empty vectors.
Enhances the dye diffusion and injection effect, improving the positive rate of transient transgenic strawberry fruit by ensuring uniform gene distribution and higher transformation efficiency.
Smart Images
Figure CN119120567A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of genetic engineering, in particular to a novel injection method for instantaneous transformation of strawberry fruit mediated by Agrobacterium. Background Art
[0002] Strawberry is a perennial herbaceous plant of the genus Fragaria in the Rosaceae family. It is native to Europe and is now widely cultivated around the world. Strawberry is one of the most important economic fruits in the world and is loved by people for its rich nutritional value and unique flavor. Strawberry fruits are rich in vitamin C, folic acid, potassium, and antioxidants such as anthocyanins and flavonoids, which have many benefits for human health.
[0003] Strawberry transient injection is a technique for studying gene function in strawberry fruit that allows researchers to quickly express foreign genes in strawberry cells without the need for stable transformation. This method typically uses Agrobacterium-mediated transient transformation, where Agrobacterium is used as a vector to deliver the target gene into strawberry cells. The application of transient injection technology in strawberry research includes but is not limited to the following aspects:
[0004] 1) Gene function analysis: By transiently expressing foreign genes, researchers can quickly evaluate the role of these genes in strawberry fruit development, defense, and nutritional characteristics. For example, through transient injection technology, it is possible to study how specific transcription factors regulate malic acid synthesis in strawberry fruit.
[0005] 2) Research on promoter function: Transient injection technology can be used to analyze the activity of specific promoters in strawberry fruit, which is crucial for understanding the regulatory mechanism of gene expression. Previous studies have analyzed the function of strawberry fruit-specific expression promoters through transient expression.
[0006] 3) Signal transduction research: Strawberry transient injection technology has also been used to study cell signal transduction pathways, which is of great significance for revealing the molecular basis of strawberry fruit ripening regulation. For example, the research team of China Agricultural University has established an "effect gene-reporter gene analysis technology" based on the efficient transient gene transformation system of strawberry fruit.
[0007] 4) Disease resistance protein research: Transient injection technology can also be used to study the expression of disease resistance proteins in strawberry leaves, which has potential value for developing resistant plants and understanding plant defense mechanisms.
[0008] 5) Gene silencing research: Through the transient injection technology, researchers can also achieve silencing of specific genes in strawberries, which helps to study the loss-of-function effects of genes. The advantage of transient injection technology is its rapidity and flexibility, which allows researchers to obtain experimental results in a shorter period of time, which is of great significance for the study of gene function and crop improvement. However, this technology also faces some challenges, such as optimization of transformation efficiency and improvement of expression stability. With the advancement of technology, strawberry transient injection technology is expected to play a greater role in future strawberry research and breeding.
[0009] The "Jian De Hong" strawberry variety was selected and bred by the Zhejiang Academy of Agricultural Sciences. It is a hybrid of the "06-3-6" strain, which has large fruits, good firmness, high yield and strong disease resistance, and the "Yue Xin" strain, which is early-maturing, has strong continuous fruiting, good taste and strong disease resistance. The "Jian De Hong" strawberry is rich in anthocyanins and turns red in layers during the color change period. The boundary between the red top and the white bottom is obvious. This feature makes it a standard variety for strawberry injection.
[0010] Therefore, a new Agrobacterium-mediated injection method for transient transformation of strawberry fruit is urgently needed to improve the efficiency of strawberry injection. Summary of the invention
[0011] In view of the shortcomings of the prior art, the present invention discloses a novel injection method for Agrobacterium-mediated transient transformation of strawberry fruit to solve the problems raised in the above background technology.
[0012] To achieve the above object, the present invention provides the following technical solution: a novel injection method for Agrobacterium-mediated transient transformation of strawberry fruit, comprising the following steps:
[0013] S1. Select Agrobacterium strain;
[0014] S2. Prepare bacterial solution, culture the Agrobacterium carrying the target gene to a certain concentration, then centrifuge and collect the bacterial precipitate, and resuspend it in an appropriate culture medium;
[0015] S3, selecting strawberry fruit varieties to be injected;
[0016] S4, select the fruit stalk of the strawberry fruit as the injection point for injection;
[0017] S5. Finally, the transient transformation efficiency of strawberry fruits injected with Agrobacterium strains was evaluated.
[0018] Preferably, in step S1, the Agrobacterium strain selected is GV3101.
[0019] Preferably, in step S3, the selected strawberry variety is "Jian De Red" strawberry fruit in the ginkgo stage.
[0020] Preferably, in step S4, the injection position is selected at the fruit stalk, and the injection at the fruit stalk is consistent with the direction of the strawberry vascular bundle under normal cultivation conditions; the fruit stalk is close to the central pith, and the gene carried by Agrobacterium is injected into the central pith of the fruit through a syringe, thereby spreading throughout the fruit.
[0021] Preferably, in step S4, during the injection, the injection volume is controlled until the injected bacterial solution overflows the fruit stalk, ensuring that enough Agrobacterium completely enters the fruit tissue.
[0022] Preferably, step S5 evaluates the positive rate of transgenic instantaneous strawberry fruits, specifically comprising selecting several groups of strawberry fruits in the white fruit stage, with two strawberry fruits in each group, and injecting Agrobacterium carrying myb10-vigs and Agrobacterium not carrying myb10-vigs as empty vectors into the fruit stalks of the strawberry fruits in each group, respectively, and finally performing statistics on the injection effects, making effect statistical graphs, and obtaining the injection positive rate.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] 1. The present invention significantly improves the dye diffusion effect and injection effect of the injected fruit by optimizing the strawberry Agrobacterium injection method and the selection of the injected variety, and significantly improves the positive rate of transient transgenic strawberry fruit.
[0025] 2. In the present invention, the fruit stalk of the strawberry fruit is selected as the injection point. Compared with the traditional injection method, this injection method has the advantage that there is a thicker layer of flesh between the fruit tip and the central pith. Injection from the fruit tip is easy to deviate from the central pith, while the fruit stalk is close to the central pith. The method of the present invention makes it easier to inject the gene carried by Agrobacterium into the central pith of the fruit through a syringe, so that it spreads throughout the fruit. At the same time, injection at the fruit stalk is consistent with the direction of the strawberry vascular bundle under normal cultivation conditions, which is more conducive to the diffusion of the injection liquid, thereby enhancing the effect of instantaneous injection and improving the efficiency of strawberry injection.
[0026] 3. In the present invention, Jiande red strawberry fruits were selected. Compared with red strawberries, they maintain the white fruit stage longer, have more obvious characteristics, are easier to distinguish from large green fruits in color, and are easier to select suitable injection materials, which is convenient for evaluating the conversion efficiency in the later stage and improves the experimental efficiency. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.
[0028] In the attached picture:
[0029] Figure 1This is a schematic diagram of a novel injection method for Agrobacterium-mediated transient transformation of strawberry fruit;
[0030] Figure 2 This is the effect diagram of the strawberry "Jian De Hong" and "Hong Yan" of the present invention during the ginkgo period;
[0031] Figure 3 This is a comparison of the dye effects of the fruit stalk injection (left) and the fruit tip injection (right) of the present invention;
[0032] Figure 4 The effect diagram of the fruit of "Jian De Hong" of the present invention injected with myb10-vigs (left) and without injection (right);
[0033] Figure 5 This is a statistical diagram of the effect of injecting myb10-vigs into the "Jian De Hong" fruit of the present invention. DETAILED DESCRIPTION
[0034] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.
[0035] like Figure 1 As shown, a novel injection method for Agrobacterium-mediated transient transformation of strawberry fruit comprises the following steps:
[0036] S1. Select an Agrobacterium strain; the selected Agrobacterium strain is GV3101.
[0037] S2. Prepare bacterial solution, culture the Agrobacterium carrying the target gene to a certain concentration, then centrifuge and collect the bacterial precipitate, and resuspend it in appropriate culture medium.
[0038] S3. Select the strawberry fruit variety to be injected; the selected strawberry fruit is in the ginkgo stage; and the selection of strawberry fruit in this embodiment: "Jian De Hong" strawberry fruit maintains the ginkgo stage longer than "Hong Yan", has more obvious characteristics, is easier to distinguish from large green fruits in color, and is easier to select suitable injection materials. Therefore, "Jian De Hong" strawberry fruit in the ginkgo stage (18-21 days after flowering) is selected as the injection material.
[0039] S4. Select the fruit stalk of the strawberry fruit as the injection point for injection; the injection position is selected at the fruit stalk, and the injection at the fruit stalk is consistent with the direction of the strawberry vascular bundle under normal cultivation conditions; the fruit stalk is close to the central pith, and the gene carried by Agrobacterium is injected into the central pith of the fruit through a syringe, so as to spread throughout the fruit. During the injection, the injection volume is controlled until the injected bacterial liquid overflows the fruit stalk to ensure that enough Agrobacterium completely enters the fruit tissue. Compared with the traditional injection method, the advantage of this injection method is that there is a thicker layer of flesh between the fruit tip and the central pith, and injection from the fruit tip is easy to deviate from the central pith.
[0040] S5. Finally, the transient transformation efficiency of strawberry fruits injected with Agrobacterium strains was evaluated, and the positive rate of transgenic transient strawberry fruits was evaluated. Specifically, 9 groups of strawberry fruits in the white fruit stage were selected, with two strawberry fruits in each group, and the strawberry fruits in each group were injected with Agrobacterium carrying myb10-vigs and Agrobacterium not carrying myb10-vigs as empty vectors at the fruit stalks. Finally, the injection effects were statistically analyzed, an effect statistical graph was made, and the injection positive rate was obtained. Specific embodiment:
[0042] Example 1: Select the "Jian De Hong" strawberry fruit in the ginkgo stage (18-21 days after flowering) as the injection material;
[0043] like Figure 2 Shown is a comparison between "Jian De Red" ginkgo and "Hong Yan" ginkgo. The characteristics of "Jian De Red" ginkgo are more obvious and easier to select.
[0044] Example 2: Select the "Jian De Hong" fruits in the ginkgo stage, and inject easy-to-observe red dye at the fruit tip and fruit stalk respectively;
[0045] like Figure 3 The figure shows the dye effect of the fruit tip injection (control group) and the fruit stalk injection treatment group (experimental group). It can be seen that the effect of dye injection from the fruit stalk is more uniform.
[0046] Example 3: Select the "Jian De Hong" and "Hong Yan" fruits in the ginkgo stage, and inject the Agrobacterium carrying myb10-vigs (to inhibit fruit coloring) and the Agrobacterium without myb10-vigs into the fruit stalk respectively;
[0047] like Figure 4 The figure shows the coloring effect of myb10-vigs injection and empty injection in the "Jian De Hong" treatment group. The upper layer of the figure shows the selected ginkgo fruits with the same growth status. When one of them is colored first, the colored fruit is marked as 1 and the uncolored one is marked as 0; if they are colored at the same time, they are all marked as 0;
[0048] Among the 9 groups of "Jian De Hong" ginkgo fruits injected, 7 groups injected with myb10-vigs produced positive results, that is, the injection of myb10-vigs inhibited coloring, and the other 2 groups had non-positive results and there was no change in coloring.
[0049] like Figure 5 Shown is a statistical chart of the injection effect of "Jian De Hong" myb10-vigs. Among the 9 groups of "Jian De Hong" ginkgo injected, 7 groups produced positive results, 2 groups were non-positive results, and the injection positive rate was 67%.
[0050] It can be seen that the novel injection method for Agrobacterium-mediated transient transformation of strawberry fruit proposed in the present invention significantly improves the staining effect and injection effect of the injected fruit by optimizing the Agrobacterium injection method of strawberry and the selection of injection varieties, and significantly improves the positive rate of transient transgenic strawberry fruit.
[0051] Finally, it should be noted that the above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A novel injection method for transient transformation of strawberry fruit mediated by Agrobacterium, characterized in that: The following steps are involved: S1. Select Agrobacterium strain; S2. Prepare bacterial solution, culture the Agrobacterium carrying the target gene to a certain concentration, then centrifuge and collect the bacterial precipitate, and resuspend it in an appropriate culture medium; S3, selecting strawberry fruit varieties to be injected; S4, select the fruit stalk of the strawberry fruit as the injection point for injection; S5. Finally, the transient transformation efficiency of strawberry fruits injected with Agrobacterium strains was evaluated.
2. The novel injection method for the instantaneous transformation of strawberry fruit mediated by Agrobacterium according to claim 1, characterized in that: In step S1, the selected Agrobacterium strain is GV3101.
3. The novel injection method for transient transformation of strawberry fruit mediated by Agrobacterium according to claim 1, characterized in that: In step S3, the strawberry variety selected is Jiande red strawberry fruit in the ginkgo stage.
4. The novel injection method for transient transformation of strawberry fruit mediated by Agrobacterium according to claim 1, characterized in that: In step S4, the injection position is selected at the fruit stalk, and the injection at the fruit stalk is consistent with the direction of the strawberry vascular bundle under normal cultivation conditions; the fruit stalk is close to the central pith, and the gene carried by Agrobacterium is injected into the central pith of the fruit through a syringe, thereby spreading throughout the fruit.
5. The novel injection method for transient transformation of strawberry fruit mediated by Agrobacterium according to claim 1, characterized in that: In step S4, during the injection, the injection volume is controlled until the injected bacterial solution overflows the fruit stalk, ensuring that the Agrobacterium completely enters the fruit tissue.
6. The novel injection method for transient transformation of strawberry fruit mediated by Agrobacterium according to claim 1, characterized in that: In step S5, the positive rate of transgenic instantaneous strawberry fruits is evaluated, which specifically includes selecting several groups of strawberry fruits in the white fruit stage, with two strawberry fruits in each group, and injecting Agrobacterium carrying myb10-vigs and Agrobacterium not carrying myb10-vigs as empty vectors into the fruit stalks of the strawberry fruits in each group, respectively. Finally, the injection effect is statistically analyzed, an effect statistical graph is made, and the injection positive rate is obtained.