Genetic transformation method of brassica plant and application thereof
Through recombinant Agrobacterium infection and matrix culture, the genetic transformation process of Brassica plants is simplified, the problems of complex operation and long cycle are solved, and the hairy roots are rapidly induced and experimental cycles are shortened, providing efficient methods for research .
Patent Information
- Application Number
- CN202411514741.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-29
- Publication Date
- 2025-01-03
AI Technical Summary
The prior art is complex in the genetic transformation process of Brassica plants, has high cost and long cycles, making it difficult to effectively induce hairy roots, affecting research efficiency.
Recombinant Agrobacterium infects Brassica plant explants and performs dark culture and hairy root induced culture in the matrix to simplify the process, omit sterile operation and complex conditions, and promote root growth with vermiculite and Hogland nutrient solution .
It reduces the complexity and cost of the experiment, shortens the experimental cycle, can quickly obtain positive hairy roots, successfully inoculate root swelling pathogens, and provides a method to quickly verify the disease resistance of transgenic plants.
Smart Images
Figure CN119242696A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a genetic transformation method of Brassica plants and an application thereof. Background Art
[0002] Brassicae root knot fungus is an obligate parasite on cruciferous crops such as rapeseed and cabbage. The root cells of diseased plants divide abnormally and swell, which can inhibit plant growth at best and kill plants at worst. It is one of the biggest threats to the growth of cruciferous plants and one of the most economically significant diseases in the world. Therefore, studying the interaction mechanism between root knot fungus and its host is particularly important for the prevention and treatment of root knot disease. However, root knot fungus is an obligate living parasite that relies on a host to complete its life cycle. It cannot be isolated and cultured in vitro using a prepared culture medium, and it is difficult to knock out the genes of the root knot fungus itself.
[0003] Existing technologies usually use Agrobacterium-mediated genetic transformation technology to transfer a gene into Brassica plants to verify the function of the gene. However, traditional Agrobacterium-mediated genetic transformation methods usually require explants to be placed in different culture media for co-cultivation, screening and cultivation, etc. The process is cumbersome, the experimental operation is complex, the conditions are harsh, the cost is high, and the experiment and subsequent research can only be carried out after the T0 generation seeds are identified and harvested. The whole process takes a long time. Summary of the invention
[0004] The object of the present invention is to provide a genetic transformation method of Brassica plants and its application, simplify the culture process, culture the explants in a matrix to induce hairy roots, do not require complex aseptic operations and culture conditions, and can reduce the complexity and cost of the experiment.
[0005] The invention provides a genetic transformation method of Brassica plants, comprising the following steps: using recombinant Agrobacterium to infect Brassica plant explants to obtain infected Brassica plant explants; placing the infected Brassica plant explants in a substrate for dark culture and hairy root induction culture to obtain positive Brassica plants;
[0006] The recombinant Agrobacterium includes a basic Agrobacterium and a target gene transferred into the basic Agrobacterium;
[0007] The basic Agrobacterium includes MSU440 and / or LBA1334;
[0008] The matrix includes vermiculite.
[0009] As a preferred solution, the substrate comprises a substrate soaked with plant nutrient solution.
[0010] As a preferred solution, the plant nutrient solution includes Hoagland nutrient solution.
[0011] As a preferred embodiment, the explant includes a hypocotyl.
[0012] As a preferred solution, the hypocotyl includes the hypocotyl of a Brassica plant when two cotyledons are unfolded.
[0013] As a preferred solution, after the hairy root induction culture, the method further comprises: cutting off the initially grown non-positive hairy roots.
[0014] As a preferred embodiment, the Brassica plant includes rapeseed.
[0015] As a preferred solution, the infection method includes: dipping the Brassica plant explant into the recombinant Agrobacterium.
[0016] As a preferred solution, the dark culture time is 1 to 2 days, and the temperature is 20 to 24°C; the hairy root induction culture time is 7 to 14 days, and the temperature is 20 to 24°C.
[0017] The present invention also provides the application of the genetic transformation method described in the above scheme in the verification of gene function of Brassica plants and / or the cultivation of transgenic plants.
[0018] Beneficial effects:
[0019] The present invention provides a method for genetic transformation of Brassica plants, comprising the following steps: using recombinant Agrobacterium to infect Brassica plant explants to obtain infected Brassica plant explants; placing the infected Brassica plant explants in a matrix for dark culture and hairy root induction culture to obtain positive Brassica plants, and limiting the type of Agrobacterium and the type of matrix. The method of the present invention can simplify the culture process, and the hairy roots can be induced by culturing the explants in the matrix. The whole process does not require complicated aseptic operation and culture conditions, thereby reducing the complexity and cost of the experiment; and there is no need to harvest T0 generation seeds, and the identified positive hairy roots can be transferred to nutrient-rich soil for subculture, and the experiment can be directly carried out for subsequent research, which greatly shortens the experimental cycle. The results of the embodiment show that the positive hairy roots obtained by the method of the present invention can be successfully inoculated with the pathogenic root root fungus of rapeseed root root disease, and the whole cycle is shortened to two and a half months, which provides a fast and effective method for studying the interaction between protozoan root root fungus and rapeseed in the future, and identifying the disease resistance of transgenic plants. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.
[0021] Figure 1 The pictures of positive hairy roots in Example 1 and Example 2;
[0022] Figure 2 The pictures of positive hairy roots in Example 3 and Example 4 are shown;
[0023] Figure 3 This is a picture of the hairy roots after inoculation with root knot fungi in Application Example 1. Scale bar = 2 cm. DETAILED DESCRIPTION
[0024] The invention provides a genetic transformation method of Brassica plants, comprising the following steps: using recombinant Agrobacterium to infect Brassica plant explants to obtain infected Brassica plant explants; placing the infected Brassica plant explants in a substrate for dark culture and hairy root induction culture to obtain positive Brassica plants;
[0025] The recombinant Agrobacterium includes a basic Agrobacterium and a target gene transferred into the basic Agrobacterium;
[0026] The basic Agrobacterium includes MSU440 and / or LBA1334;
[0027] The matrix includes vermiculite.
[0028] The present invention transfers the target gene into the basic Agrobacterium to obtain a recombinant Agrobacterium. As an embodiment, the present invention transfers the recombinant vector containing the target gene into the basic Agrobacterium to obtain a recombinant Agrobacterium. As an embodiment, the target gene includes any gene that needs functional verification. As an embodiment, the basic vector of the recombinant vector of the present invention includes a vector containing a GFP tag. In a specific embodiment, the basic vector can be a plant binary expression vector. The present invention utilizes a recombinant vector containing a GFP tag to transform the basic Agrobacterium, which is beneficial to the subsequent use of fluorescence screening to obtain positive hairy roots.
[0029] As an embodiment, the basic Agrobacterium of the present invention includes MSU440 and / or LBA1334. In a specific embodiment, the basic Agrobacterium may be MSU440 or LBA1334. The present invention utilizes a specific Agrobacterium rhizogenes to transform rapeseed, which can improve the transformation efficiency.
[0030] After obtaining the recombinant Agrobacterium, the present invention uses the recombinant Agrobacterium to infect Brassica plant explants to obtain infected Brassica plant explants. As an embodiment, the Brassica plant of the present invention includes rapeseed; as an embodiment, the rapeseed includes Brassica rapa and / or Westar. In a specific embodiment, the rapeseed can be Brassica rapa or Westar.
[0031] As an embodiment, the explant of the present invention includes a hypocotyl. As an embodiment, the hypocotyl includes a hypocotyl of a Brassica plant when two cotyledons are unfolded. As an embodiment, the preparation of the explant of the present invention includes: cutting 1 cm upward from the root with a blade inclined at 30 to 60 degrees to obtain a hypocotyl. The hypocotyl of a Brassica plant when two cotyledons are unfolded is healthier and more tender than that at other stages, which is conducive to improving the infection efficiency.
[0032] As an embodiment, the infection method of the present invention includes dipping the Brassica plant explant into the recombinant Agrobacterium.
[0033] In the present invention, the infected Brassica plant explants are placed in a matrix for dark culture to obtain co-cultured explants. In the present invention, the dark culture is co-culture. As an embodiment, the present invention inserts the infected Brassica plant explants into a matrix for dark culture. As an embodiment, the dark culture time is 1 to 2 days, and the temperature is 22°C. As an embodiment, the matrix of the present invention includes vermiculite. As an embodiment, the matrix includes a matrix soaked in a plant nutrient solution. As an embodiment, the plant nutrient solution includes Hoagland nutrient solution. Hoagland nutrient solution can provide plants with balanced macroelements and trace elements, and reasonable nutrients can promote plant root growth and increase growth rate. The present invention uses Hoagland nutrient solution to soak the matrix, which can provide nutrition for rapeseed explants and promote rooting of the explants. The present invention places the infected Brassica plant explants in a matrix for culture, which can omit the steps of medium preparation, sterilization and the like in the traditional genetic transformation method, simplify the experimental steps and shorten the experimental period; and co-cultivation in the matrix is conducive to the subsequent operation of cutting off non-positive hairy roots.
[0034] In the present invention, the explant after the co-cultivation is subjected to hairy root induction culture to obtain hairy roots. As an embodiment, the hairy root induction culture of the present invention can be carried out in the original matrix. As an embodiment, the time of the hairy root induction culture of the present invention is 7 to 14 days, and the temperature is 22° C. As an embodiment, the time of the hairy root induction culture can be 7, 8, 9, 10, 11, 12, 13 or 14 days.
[0035] After obtaining the hairy roots, as an embodiment, the present invention identifies the hairy roots, cuts off non-positive hairy roots, and obtains positive hairy roots. Cutting off non-positive hairy roots can reduce the impact on the growth of positive hairy roots and improve transformation efficiency.
[0036] After obtaining the positive hairy roots, as an embodiment, the present invention transplants the positive hairy roots into a culture matrix for subculture to obtain positive Brassica plants. As an embodiment, the temperature of the subculture in the present invention is 22° C. In a specific embodiment of the present invention, the culture matrix can be soil, or vermiculite and soil.
[0037] The present invention also provides the use of the genetic transformation method described in the above scheme in the verification of gene function of Brassica plants and / or the cultivation of transgenic plants. The genetic transformation method described in the present invention can be used to transfer the target gene into Brassica plants to cultivate transgenic plants. The positive hairy roots obtained by the genetic transformation method described in the present invention can be used for subsequent root knot fungus infection experiments to quickly verify the resistance of Brassica plants to root knot disease after the introduced target gene is overexpressed or silenced. On the one hand, the present invention can utilize the above-mentioned transformation method to clone the gene to be verified into a plasmid containing a tag to obtain a recombinant plasmid, utilize Agrobacterium rhizogenes to infect Brassica plants, induce positive hairy roots, and then inoculate root-knot fungi, and count the disease index after a few days, so as to quickly determine the resistance of the gene to be verified to root-knot fungi after overexpression in Brassica; on the other hand, a plasmid vector that can be used to induce host gene silencing is introduced with a relevant tag, and the plasmid carrying the tag after modification is transferred into Agrobacterium rhizogenes, and then the positive hairy roots mediated by Agrobacterium rhizogenes are inoculated with root-knot fungi, and the disease index is counted after a few days, so as to quickly determine the influence of the gene on the virulence of root-knot fungi.
[0038] To further illustrate the present invention, a genetic transformation method for Brassica plants and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.
[0039] Example 1
[0040] (1) Select full, intact, and uniform-sized Brassica napus seeds, sow the selected seeds in nutrient soil, with 20 seeds in each box, and culture them in a constant temperature culture room at 22°C for 5 days; when the rapeseed grows to the point where two cotyledons unfold, select well-growing rapeseed seedlings, cut the rapeseed hypocotyls with a new blade tilted at 45°, and quickly soak them in ddH2O to prevent water loss and wilting, thereby obtaining the rapeseed hypocotyls.
[0041] (2) The plasmid Pub-GFP (published in the literature: Haixiang Yu, Ai Fang Xiao, et, al. GmNAC039 and GmNAC018 activate the expression of cysteine protease genes to promote soybean nodule senescence, The Plant Cell, 2023.) was transformed into Agrobacterium rhizogenes LBA1334 by electroporation to obtain positive bacteria, and the positive bacteria were inoculated onto a Kana+Rif double-antibody LB plate, placed in a 28°C incubator, and inverted for culture; the working concentrations of Kana and Rif in the double-antibody LB plate were both 50 μg / mL; when the LB plate was covered with colonies, the bacteria were gently scraped on the LB plate with a 1 mL blue pipette tip to avoid puncturing the culture medium to obtain positive Agrobacterium rhizogenes.
[0042] (3) Take the rape hypocotyl obtained in step (1), dip the wound of the rape hypocotyl into the positive Agrobacterium rhizogenes obtained in step (2); quickly insert the rape hypocotyl dipped into a black 10×10 cm small square box filled with vermiculite, insert 16 rape hypocotyls into each box, and co-cultivate at 22° C. in the dark for 1 to 2 days; the vermiculite is soaked with Hoagland's nutrient solution.
[0043] (4) After the co-cultivation is completed, hairy root induction culture is carried out under normal light conditions, and the temperature of the hairy root induction culture is 22°C; after the hairy roots appear, the rapeseed is pulled out of the vermiculite with tweezers, placed in ddH2O for cleaning, and the positive transgenic roots are observed with a fluorescent stereomicroscope, and the non-positive hairy roots that are initially grown are cut off with small scissors; the pruned rapeseed roots are placed in a sterilized Hoagland nutrient solution, placed in a sealed box, and cultured in a 22°C culture room to maintain moisture. Thereafter, the seedling status is observed, water is replenished in time every day, and the nutrient solution is replaced every 7 days if necessary to prevent and control the occurrence of diseases and insect pests.
[0044] (5) After 7 to 14 days of induction culture of hairy roots, positive hairy roots were identified using a fluorescent stereo microscope. Positive hairy roots showed obvious green fluorescence under the fluorescent stereo microscope. The positive seedlings were counted and the positive conversion rate was calculated. The results are shown in Table 1. Representative pictures of positive hairy roots were taken under white light and green fluorescence, respectively. The results are shown in Table 1. Figure 1 As shown in A, Figure 1 White means the picture was taken under white light, and GFP means the picture was taken under green fluorescence.
[0045] Example 2
[0046] A rapeseed genetic transformation method similar to Example 1, except that in step (2), the plasmid Pub-GFP3 is transferred into Agrobacterium rhizogenes MSU440 to obtain positive Agrobacterium rhizogenes cells.
[0047] The positive seedlings were counted, the positive transformation rate was calculated, and pictures of representative positive hairy roots were taken under white light and green fluorescence, respectively. The results are shown in Figure 1 As shown in B and Table 1.
[0048] Example 3
[0049] A rapeseed genetic transformation method similar to Example 1, except that in step (1), rapeseed Westar seeds are selected and cultured to obtain rapeseed Westar hypocotyls.
[0050] The positive seedlings were counted, the positive transformation rate was calculated, and pictures of representative positive hairy roots were taken under white light and green fluorescence, respectively. The results are shown in Figure 2 As shown in A and Table 1, Figure 2 White means the picture was taken under white light, and GFP means the picture was taken under green fluorescence.
[0051] Example 4
[0052] A rapeseed genetic transformation method similar to Example 2, except that in step (1), rapeseed Westar seeds are selected and cultured to obtain rapeseed Westar hypocotyls.
[0053] The positive seedlings were counted, the positive transformation rate was calculated, and pictures of representative positive hairy roots were taken under white light and green fluorescence, respectively. The results are shown in Figure 2 As shown in B and Table 1.
[0054] Comparative Example 1
[0055] A rapeseed genetic transformation method similar to Example 1, except that in step (2), the plasmid Pub-GFP3 is transferred into Agrobacterium rhizogenes K599 to obtain positive Agrobacterium rhizogenes cells.
[0056] The positive seedlings were counted and the positive transformation rate was calculated. The results are shown in Table 1.
[0057] Comparative Example 2
[0058] A rapeseed genetic transformation method similar to Example 3, except that in step (2), the plasmid Pub-GFP3 is transferred into Agrobacterium rhizogenes K599 to obtain positive Agrobacterium rhizogenes cells.
[0059] The positive seedlings were counted and the positive transformation rate was calculated. The results are shown in Table 1.
[0060] Table 1 Results of transformation rate of rapeseed positive seedlings
[0061] Group Rapeseed (plant) Positive seedlings (trees) Conversion rate Example 1 32 6 18.75％ Example 2 30 13 43.33％ Example 3 30 2 6.66％ Example 4 30 1 3.33％ Comparative Example 1 30 0 0％ Comparative Example 2 31 0 0％
[0062] According to Table 1, it can be seen that in Example 1, 32 hairy root induced plants were obtained by transforming Xiaoyun with LBA1334 Agrobacterium, 6 positive seedlings were identified, and the transformation efficiency was 18.75%; in Example 2, 30 hairy root induced plants were obtained by transforming Xiaoyun with MSU440 Agrobacterium, 13 positive seedlings were identified, and the transformation efficiency was 43.33%; in Comparative Example 1, 30 hairy root induced plants were obtained by transforming Xiaoyun with K599 Agrobacterium, 0 positive seedlings were identified, and the transformation efficiency was 0%.
[0063] In Example 3, 30 hairy root induced plants were obtained by transforming Westar with LBA1334 Agrobacterium, 2 positive seedlings were identified, and the transformation efficiency was 6.66%; in Example 4, 30 hairy root induced plants were obtained by transforming Westar with MSU440 Agrobacterium, 1 positive seedling was identified, and the transformation efficiency was 3.33%; in Comparative Example 2, 31 hairy root induced plants were obtained by transforming Westar with K599 Agrobacterium, 0 positive seedlings were identified, and the transformation efficiency was 0%.
[0064] It can be seen that both Agrobacterium rhizogenes MSU440 and LBA1334 can transform rapeseed to obtain positive plants, among which, the efficiency of transforming rapeseed with Agrobacterium rhizogenes MSU440 is the highest.
[0065] Application Example 1
[0066] (1) The GFP green fluorescence signal was strong and healthy positive hairy roots and non-positive hairy roots in Example 2 were transplanted into matrix soil, placed in a 22°C constant temperature culture room for subculture, and recorded as the experimental group and the control group (CK) respectively; the matrix soil was purchased from Jiangsu Peilei Matrix Technology Development Co., Ltd.
[0067] (2) Preparation of crude extract of dormant spores of Plasmodiophora brassicae: Take out the swollen rape root sample stored in a -30°C refrigerator (published in the literature: Ying Zhao, et al, Transcriptome Analysis of Arabidopsis thaliana in Response to Plasmodiophora brassicae during Early Infection, Frontiers in Microbiology, 2017), wash it with water, cut the root sample into pieces with a kitchen knife, put it into a juicer, add an appropriate amount of water to grind it, and after it is fully ground, filter it with 8 layers of gauze to obtain the crude extract of dormant spores of Plasmodiophora brassicae; then take 100 μL of the crude extract and dilute it 1000 times with ddH2O, count it with a hemocytometer under a microscope, and calculate the concentration of the spore suspension to obtain a concentration of 1×107 spores / mL of crude spore extract.
[0068] (3) Inoculation of root hairs with root knot fungi: After the rapeseed root hairs were subcultured for 5 days in step (1), each root hair was inoculated with 1 mL of the fungi in step (2) at a concentration of 1×10 7 spores / mL of crude spore extract for routine culture.
[0069] (4) After 40 days of inoculation, the rapeseed plants were observed for disease. The results showed that all positive hairy roots in the experimental group were successfully inoculated with root-knot fungi. Obvious swelling was observed on the roots, and the roots showed strong fluorescence under a fluorescence microscope, indicating that the transgenic hairy roots could be successfully inoculated with root-knot fungi. Figure 3 Middle A), Figure 3 White means the picture was taken under white light, and GFP means the picture was taken under green fluorescence. The non-positive hairy roots in the control group failed to be successfully inoculated with root-knot bacteria ( Figure 3 (B).
[0070] In summary, the method of the present invention can simplify the culture process, and hairy roots can be induced by culturing explants in vermiculite. The whole process does not require complicated aseptic operation and culture conditions, thereby reducing the complexity and cost of the experiment; and there is no need to harvest T0 generation seeds, and the identified positive hairy roots can be transferred to nutrient-rich soil for subculture, and the experiment can be directly carried out for subsequent research, and the root-knot fungus, the pathogen of rapeseed root root disease, can be successfully inoculated, and the whole cycle is shortened to two and a half months, which greatly shortens the experimental cycle, and provides a fast and effective method for studying the interaction between root-knot fungus and rapeseed in the future, and identifying the disease resistance of transgenic plants.
[0071] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for genetic transformation of Brassica plants, characterized in that: The method comprises the following steps: using recombinant Agrobacterium to infect Brassica plant explants to obtain infected Brassica plant explants; placing the infected Brassica plant explants in a substrate for dark culture and hairy root induction culture to obtain positive Brassica plants; The recombinant Agrobacterium includes a basic Agrobacterium and a target gene transferred into the basic Agrobacterium; The basic Agrobacterium includes MSU440 and / or LBA1334; The matrix includes vermiculite.
2. The genetic transformation method according to claim 1, characterized in that The substrate comprises a substrate soaked with a plant nutrient solution.
3. The genetic transformation method according to claim 2, characterized in that The plant nutrient solution includes Hoagland nutrient solution.
4. The genetic transformation method according to claim 1, characterized in that The explant includes a hypocotyl.
5. The genetic transformation method according to claim 4, characterized in that The hypocotyl includes the hypocotyl of the Brassica plant when two cotyledons are unfolded.
6. The genetic transformation method according to claim 1, characterized in that After the hairy root induction culture, the method further comprises: cutting off the initially grown non-positive hairy roots.
7. The genetic transformation method according to claim 1, characterized in that The Brassica plants include rapeseed.
8. The genetic transformation method according to claim 1, characterized in that The infection method includes: dipping the Brassica plant explant into the recombinant Agrobacterium.
9. The genetic transformation method according to claim 1, characterized in that The dark culture time is 1 to 2 days, and the temperature is 20 to 24°C; the hairy root induction culture time is 7 to 14 days, and the temperature is 20 to 24°C.
10. Use of the genetic transformation method according to any one of claims 1 to 9 in verifying gene function in Brassica plants and / or cultivating transgenic plants.