阅读说明：本技术 一种利用发根农杆菌转化获得整株转基因木本植株的方法 (Method for obtaining whole transgenic woody plant by utilizing agrobacterium rhizogenes transformation ) 是由 张世忠 刘琳 郑成超 马晓君 于 2020-05-20 设计创作，主要内容包括：本发明公开了一种利用发根农杆菌转化获得整株转基因木本植株的方法。本发明所述的方法是先将携带目的基因的普通或特定载体转入发根农杆菌中,再用活化的发根农杆菌菌液侵染预培养的木本植物组培苗,培养至植株长出转基因毛状根后,转至添加地塞米松或MdWUSCHEL1蛋白质的根芽转化预培养基中诱导根生芽,再经过扩繁生根从而获得整株转基因木本植株。通过本发明的方法得到转基因植株的转化效率高,不仅充分利用了木本植物自身的细胞全能性,促进转基因根向整株转基因植株的分化,并且成本低,该方法适用性广,可以用于各种木本植物的转基因培育,具有广阔应用前景。(The invention discloses a method for obtaining a whole transgenic woody plant by transforming agrobacterium rhizogenes. The method comprises the steps of firstly transferring a common or specific carrier carrying a target gene into agrobacterium rhizogenes, then infecting a pre-cultured woody plant tissue culture seedling with activated agrobacterium rhizogenes bacterial liquid, culturing until a plant grows a transgenic hairy root, transferring the plant to a root bud transformation pre-culture medium added with dexamethasone or MdWUSCHEL1 protein to induce root germination, and then propagating and rooting to obtain the whole transgenic woody plant. The method has the advantages of high transformation efficiency of the transgenic plant obtained by the method, low cost, wide applicability, wide application range, wide application prospect and capability of promoting the differentiation of the transgenic root to the whole transgenic plant by fully utilizing the cell totipotency of the woody plant.)

1. A method for obtaining a whole transgenic woody plant by utilizing agrobacterium rhizogenes transformation, which is characterized by comprising the following steps:

(1) transferring the expression vector containing the target gene into agrobacterium rhizogenes and activating to obtain activated agrobacterium rhizogenes bacterial liquid containing the target gene;

(2) putting the cultured seedlings of woody plant groups into a subculture medium for subculture, cutting off callus masses, and transferring the cut callus masses into a rooting medium for preculture to obtain precultured tissue culture seedlings;

(3) taking out the pre-cultured tissue culture seedlings, cutting off callus, dipping the activated agrobacterium rhizogenes bacterial liquid containing target genes at the cut, and then sucking off redundant bacterial liquid to obtain infected tissue culture seedlings;

(4) putting the infected tissue culture seedlings back to a rooting culture medium for co-culture, and then inserting the infected tissue culture seedlings into an MS culture medium containing antibiotics for culture until transgenic hairy roots grow out to obtain transgenic root systems;

(5) when the root of the transgenic root system grows to 5cm, cutting off the transgenic root system, and sequentially transferring the transgenic root system into a root bud transformation pre-culture medium twice to culture until buds grow to obtain transgenic overground part transgenic buds;

(6) and transferring the transgenic bud into a subculture medium, and then transferring the transgenic bud into a rooting medium for culturing until the transgenic bud roots to obtain the whole transgenic woody plant.

2. The method for obtaining the whole transgenic woody plant by utilizing the agrobacterium rhizogenes transformation as the claim 1, wherein the expression vector in the step (1) comprises a common expression vector and a specific expression vector, and the specific expression vector is a dexamethasone inducible promoter vector PTA7002 inserted withCamV35S WUSCHEL1-CamV35S target geneAnd (4) sequencing.

3. The method for obtaining whole transgenic woody plant by utilizing the agrobacterium rhizogenes transformation according to claim 1, wherein the subculture time in the step (2) is 28-32 days, and the preculture time is 4-6 days.

4. The method for obtaining whole transgenic woody plant by utilizing the agrobacterium rhizogenes transformation as claimed in claim 1, wherein the rooting medium in the step (2) contains 0-0.7mg/L IBA, 0-0.1mg/L LIAA, 200mM/L cPTIO, 20-25g/L sucrose and 8.5g/L agar per liter of MS liquid medium, and the pH is 5.80-5.85.

5. The method for obtaining whole transgenic woody plant by utilizing the agrobacterium rhizogenes transformation according to claim 1, wherein the co-culture time in the step (4) is 1-2 days, and the culture condition is 28 ℃ and 24h in the dark.

6. The method for obtaining whole transgenic woody plant by utilizing Agrobacterium rhizogenes transformation according to claim 1, wherein dexamethasone or MdWUSCHEL1 protein is added to the root bud transformation preculture medium transformed in the second time in the step (5).

7. The method for obtaining whole transgenic woody plants by utilizing the agrobacterium rhizogenes transformation according to claim 6, wherein the addition amount of dexamethasone is 5-10 μm/L.

8. The method for obtaining whole transgenic woody plant by utilizing Agrobacterium rhizogenes transformation according to claim 6, wherein the concentration of MdWUSCHEL1 protein is 5 μ M/L.

9. The method for obtaining the whole transgenic woody plant by the transformation of Agrobacterium rhizogenes as claimed in claim 1, wherein the pre-culture medium for root bud transformation is MS liquid culture medium containing 0-1.0mg/L TDZ, 0-1.0mg/L NAA, 500mg/L timentin, 30g/L sucrose and 8.5g/L agar, and the pH is 5.80-5.85.

10. The method for obtaining whole transgenic woody plant by utilizing agrobacterium rhizogenes transformation according to claim 1, wherein the woody plant comprises apple, kiwi, cherry, peach and osmanthus.

Technical Field

The invention belongs to the technical field of plant transgenosis, and particularly relates to a method for obtaining a whole transgenic woody plant by transforming agrobacterium rhizogenes.

Background

Transformation of plants with Agrobacterium rhizogenes is a traditional method of plant transgenesis. The agrobacterium rhizogenes contains Ri plasmid, LB end of Ri plasmid T-DNA contains four rooting related genes of RolA, RolB, RolC and RolD, and RB end contains three synthetic genes of auxin, cytokinin and opine. The existence of the genes related to rooting and hormone enables the agrobacterium rhizogenes to infect plants, and then the plants grow adventitious roots at the successful infection parts, and the adventitious roots have the characteristics of in vitro growth, high growth speed, no geotropism and the like.

Agrobacterium rhizogenes strains vary in their ability to transform different plants. Most of the plants successfully transformed by the existing agrobacterium rhizogenes are herbaceous plants, such as corn, peanut, spinach, ginseng, alfalfa, chrysanthemum, saussurea involucrate and the like, and many of the herbaceous plants are easily transformed into the whole transgenic plants from transgenic root systems. However, for woody plants, not only transformation efficiency is low, but also the types of plants successfully transformed are few, and at present, only a few woody plants such as apple, orange, camptotheca acuminate and berberis aristata are successfully transformed, and only orange is successfully induced into the whole plant by transgenic roots. The main reasons are that the dedifferentiation efficiency of woody plants is lower than that of herbaceous plants, the totipotency of cells is poor, and the differentiation difficulty from roots to buds is high.

The bud is differentiated from the apical meristem, and the WUSCHEL1 gene is a determining factor for the differentiation of the apical meristem, and it promotes the differentiation of the apical meristem, and thus the formation of the bud. The WUSCHEL1 gene also enables certain tissues or organs to induce somatic embryogenesis without the addition of any exogenous hormones. Although the prior art mentions that the method of injecting the bacterial solution of agrobacterium rhizogenes with target genes enables the rooted seedlings to grow new transgenic hairy roots, the invention still stays in the stage of obtaining transgenic roots finally, and does not obtain whole transgenic plants, and the characteristics of WUSCHEL1 genes have important significance for obtaining whole woody plants.

Disclosure of Invention

The invention discloses a method for obtaining a whole transgenic woody plant by utilizing agrobacterium rhizogenes transformation, which overcomes the defects of low efficiency and high cost of the traditional plant transgenic method, transfers WUCCHEL 1 gene into a transgenic plant, improves the totipotency of woody plant cells, promotes the differentiation of transgenic roots into the whole transgenic plant, successfully obtains the whole transgenic woody plant containing WUCCHEL 1 gene, and improves the transformation efficiency of the woody plant.

In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:

the invention provides a method for obtaining a whole transgenic woody plant by utilizing agrobacterium rhizogenes transformation, which is characterized by comprising the following steps:

(1) transferring the expression vector containing the target gene into agrobacterium rhizogenes and activating to obtain activated agrobacterium rhizogenes bacterial liquid containing the target gene;

(2) placing the tissue culture seedling of the woody plant into a subculture medium, carrying out subculture for 28-32 days under the conditions of 25 ℃, 16h of illumination/8 h of darkness, cutting off a callus, transferring the callus into a rooting culture medium, and carrying out pre-culture for 4-6 days under the conditions of 25 ℃, 16h of illumination/8 h of darkness to obtain a pre-culture tissue culture seedling;

(3) taking out the pre-cultured tissue culture seedling, cutting off callus, dipping the activated agrobacterium rhizogenes bacterial liquid containing the target gene at the cut, and then sucking off redundant bacterial liquid to obtain an infected tissue culture seedling;

(4) putting the infected tissue culture seedlings back into a rooting culture medium, culturing for 1-2 days at 28 ℃ under the condition of 24h in the dark, and then inserting into an MS culture medium containing 500mg/L timentin for culturing until transgenic hairy roots grow out, so as to obtain transgenic root systems;

(5) when the root of the transgenic root system grows to 5cm, cutting off the transgenic root system, firstly transferring the transgenic root system into a root bud transformation pre-culture medium, culturing for 5 days at 25 ℃ under 16h of illumination/8 h of darkness, and then transferring the transgenic root system into a root bud transformation pre-culture medium added with dexamethasone or MdWUSHEL 1 protein to culture until buds grow to obtain transgenic overground part transgenic buds;

(6) and transferring the transgenic bud into a subculture medium, and then transferring the transgenic bud into a rooting medium for culturing until the transgenic bud roots to obtain the whole transgenic woody plant.

Further, the expression vector in the step (1) comprises a common expression vector and a specific expression vector.

Further, the specific expression vector is prepared by inserting CamV35S into dexamethasone inducible promoter vector PTA 7002: : WUSCHEL1-CamV35S: : the sequence of the target gene.

Further, the addition amount of the dexamethasone is 5-10 mu m/L.

Further, the concentration of the MdWUSCHEL1 protein is 5 mu M/L.

Further, the root bud transformation pre-culture medium is MS liquid culture medium containing 0-1.0mg/L TDZ, 0-1.0mg/L NAA, 500mg/L timentin, 30g/L sucrose and 8.5g/L agar, and the pH value is 5.80-5.85.

Furthermore, the rooting culture medium is 20-25g/L of sucrose and 8.5g/L of agar, the pH value is 5.80-5.85, the MS liquid culture medium contains 0-0.7mg/L of IBA, 0-0.1mg/L of IAA, 200mM/L of cPTIO, 20-25g/L of sucrose and 8.5g/L of agar, and the pH value is 5.80-5.85.

Further, the woody plant comprises apple, kiwi, cherry, peach and sweet osmanthus.

Further, the detection primer sequence of the whole transgenic woody plant is as follows:

Forward：ATGGTGAGCAAGGGCGAGGAG；

Reward：TTACTTGTACAGCTCGTCCATG。

compared with the prior art, the invention has the following advantages and beneficial effects:

the WUSCHEL1 gene and the target gene which promote the growth of plant buds are transferred into the root system of the woody plant by utilizing the advantage of high transformation efficiency of the agrobacterium rhizogenes, and the cell totipotency of the woody plant is further utilized to dedifferentiate the transgenic root system to form the whole transgenic plant. The method utilizes the traditional method of combining transgenosis and a molecular mechanism, has wide applicability, can be used for the transgenic cultivation of various woody plants, not only well improves the transformation efficiency of the transgenic plants, but also promotes the differentiation of transgenic roots to the whole transgenic plants, has simple operation and low cost, and has wide application prospect in scientific research and practical agricultural planting.

Drawings

FIG. 1 is a map of a transgenic vector.

FIG. 2 shows transgenic buds of transgenic apples obtained in example 4 of the present invention.

FIG. 3 shows the transgenic shoots of transgenic kiwi fruit obtained in example 5 of the present invention.

FIG. 4 shows the transgenic bud of the transgenic cherry obtained in example 6 of the present invention.

FIG. 5 shows the results of protein assays in examples 4-6 of the present invention, in which 1: GFP in apple, 2: GFP in kiwi, 3: GFP in cherries.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to specific examples.

The test methods described in the following examples, unless otherwise specified, were carried out according to conventional or manufacturer's recommended procedures and conditions; the reagents and materials, unless otherwise indicated, are commercially available or may be prepared using conventional methods.