Agrobacterium rhizogenes-mediated cork oak genetic transformation method
Through Agrobacter rhizobium-mediated genetic transformation method, the young stem segments of the oak cork seedlings were invaded and the transgenic hairy roots were induced, which solved the cumbersome and inefficient problems of traditional breeding methods, and achieved efficient oak cork breeding and genes. edit.
Patent Information
- Application Number
- CN202411703894.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-26
- Publication Date
- 2025-02-14
AI Technical Summary
Traditional corn oak breeding methods such as grafting and breeding operations are complicated, low survival rate, difficult to manage, and poor breeding effect, making it difficult to meet the needs of forest breeding.
Using Agrobacter rhizobium mediated genetic transformation method, a new method for gene editing and breeding is provided by infecting Agrobacter rhizobium rhizobium rhizobium rhizobium rhizobium rhizobium rhizobium rhizobium rhizobium rhizobium rhizobium rhizobium rhizobium rhizobium rhizobium rhizobium seedlings to induce transgenic hairy roots.
It has achieved efficient genetic transformation of Corpse, promoted rooting and the cultivation of transgenic plants, provided new breeding and gene editing research channels, and improved breeding efficiency and survival rate.
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Figure CN119432905A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant genetic engineering, in particular to a method for genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes. Background Art
[0002] Cork oak (Quercus variabilis BI.) is a deciduous tree belonging to the genus Quercus in the Fagaceae family. Its bark has a well-developed cork layer, which is light, soft and insulated, and is an important raw material for the light industry and defense industry. Its cupules are rich in tannins and are often used to extract tannins. Its fruit shell can be used as medicine, and it is an important economic tree in my country. Cork oak has few fruits, the quality of seedlings varies, and the traditional breeding effect is not good, so grafting is often used in production. However, grafting has the disadvantages of cumbersome operation, low survival rate, and difficult management, which have become major problems in breeding work. With the rapid development of science and technology, gene editing is the theoretical frontier and research hotspot in the current field of life sciences. The emergence of genetic transformation technology provides new ideas for forest breeding. Summary of the invention
[0003] The purpose of the present invention is to provide a method for genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes in order to solve the above problems.
[0004] The present invention achieves the above-mentioned purpose through the following technical solutions:
[0005] A method for genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes comprises the following steps:
[0006] S1. First, Quercus variabilis seedlings are cultivated from Quercus variabilis seeds, and different parts of the Quercus variabilis seedlings are cut out as infection explants;
[0007] S2, resuscitating the Agrobacterium rhizogenes, and expanding the culture of the resuscitated Agrobacterium rhizogenes to obtain an infection solution, wherein the Agrobacterium rhizogenes is K599, and the vector has a 35S::Ruby reporter gene;
[0008] S3, explants from different parts are infected at the incision with Agrobacterium rhizogenes and planted in vermiculite matrix;
[0009] S4, culturing the infected explants in dark and light conditions;
[0010] S5. Perform morphological observation and sampling tests on the cultured explants to determine the explants with the best cultivation status and the corresponding cutting position from the Quercus variabilis seedlings.
[0011] Preferably, the method for cultivating Quercus variabilis seedlings in step S1 comprises sowing the Quercus variabilis seeds after germination, and cutting the explants to be infected after culturing for 30-40 days.
[0012] Preferably, in step S1, the different parts of the Quercus variabilis seedlings include a first leafy young stem segment G1, a second leafy young stem segment G2, a third leafy young stem segment G3, a fourth leafy young stem segment G4 and a hypocotyl.
[0013] Preferably, the method for resuscitating Agrobacterium rhizogenes in step S2 comprises streaking the expression vector K599 Agrobacterium rhizogenes containing the 35S::RUBY reporter gene on a TY solid culture medium containing 30 mg / L kanamycin and 30 mg / L streptomycin, and culturing overnight to obtain a single colony, picking a single colony on a TY liquid culture medium containing 30 mg / L kanamycin and 30 mg / L streptomycin, inoculating the single colony on the TY liquid culture medium, and culturing in the dark at 28°C and 220 rpm in a shaker for 12-16 hours until the bacteria enter logarithmic growth, and then expanding the bacteria that have undergone the primary culture.
[0014] Preferably, the method for expanding the culture of Agrobacterium rhizogenes comprises: taking the initial culture to reach OD 600 = 1.0 was inoculated into TY solid culture medium with 100 μl of bacterial solution, spread, and continued to be cultured in the dark at 28°C, 0 rpm in a shaker for 12-16 h until the surface of the culture medium was evenly covered with solid bacteria for solid infection.
[0015] Preferably, the method for expanding the culture of Agrobacterium rhizogenes comprises: taking the initial culture to reach OD 600 = 1.0, 200 μl of the bacterial solution was inoculated into 200 mL of TY liquid medium, and the culture was continued in a dark incubator at 28°C and 220 rpm for 12-16 h until the bacterial solution concentration OD 600 =1.0, for liquid infection.
[0016] Preferably, the explant infection method in step S3 includes solid smearing method, liquid immersion method, liquid vacuum method or liquid injection method.
[0017] Preferably, the light cultivation in step S4 includes culturing in a constant temperature incubator at 28° C., with 16 h of light and 8 h of darkness, and watering once every 3 days.
[0018] Preferably, the morphological observation and sampling detection in step S5 include morphological observation, stereomicroscope photography and recording, and sampling PCR detection.
[0019] The beneficial effects of the present invention are:
[0020] 1. The present invention uses Agrobacterium rhizogenes as a medium and infects young stem segments of cork oak seedlings as explants to obtain transgenic hairy roots, which provides a basis for later functional gene mining and gene editing research of cork oak, solves problems in cork oak breeding, and accelerates the process of functional gene mining and gene editing research of cork oak, providing a new method for genetic improvement of cork oak forests.
[0021] 2. The genetic transformation method mediated by Agrobacterium rhizogenes used in the present invention has the characteristics of easy operation, fast growth of genetically transformed roots, stable genetic traits, etc., and plays an important role in promoting rooting, obtaining transgenic plants, and cultivating new varieties.
[0022] 3. The present invention uses the young stem segments of Quercus variabilis seedlings as explants, optimizes the infection time and infection method, and has a higher transgenic hairy induction rate. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.
[0024] Figure 1 The invention relates to Quercus variabilis seedlings of four different developmental stages of Quercus variabilis.
[0025] Figure 2 It is a schematic diagram of explants taken from different parts of Quercus variabilis in the present invention.
[0026] Figure 3 The invention relates to the influence of different infection modes on the genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes.
[0027] Figure 4 It is a schematic diagram of different forms of wounds formed by infection observed under a stereo microscope in the present invention (A. bacterial death; B. browning death; C. callus; D. callus rooting; E. direct rooting).
[0028] Figure 5 It is the PCR identification band of the genetically transformed root in the present invention.
[0029] Figure 6 It is a map of the plasmid 35S::RUBY of the present invention. DETAILED DESCRIPTION
[0030] The following is combined with Figure 1-6 , the technical solution of the present invention is further explained:
[0031] A method for genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes comprises the following steps:
[0032] S1, such as Figure 1 As shown, first, Quercus variabilis seedlings are cultivated by Quercus variabilis seeds. That is, first, the Quercus variabilis seeds are sown after germination, and the explants to be infected are cut after culturing for 30-40 days; that is, the selected glossy, plump and large good seeds are subjected to germination treatment, soaked in 40℃ warm water, cooled naturally, repeated twice, soaked for a total of 48 hours, and then the seeds floating on the surface are removed, covered with a wet towel to keep moisture, placed at 25℃ room temperature for germination, and sown in 3-6mm vermiculite soaked with water when the chest breaks and the white is exposed, watered once every 3 days, and placed in a greenhouse at 25℃, 16h light, 8h dark for 30-40 days, until the Quercus variabilis seedlings grow to four leaf stem segments, providing explants for subsequent infection.
[0033] like Figure 2 As shown, after the Quercus variabilis seedlings grow to four stem segments with leaves, they can be cut and different parts of the Quercus variabilis seedlings can be cut separately as infection explants.
[0034] That is to say, in this embodiment, different parts of the cork oak seedlings include the first leafy young stem segment G1, the second leafy young stem segment G2, the third leafy young stem segment G3, the fourth leafy young stem segment G4 and the hypocotyl, wherein the first leafy stem segment (the top bud part, G1), the second leafy stem segment (G2), the third leafy stem segment (G3), and the fourth leafy stem segment (G4) are all cut, and the hypocotyl is a stem segment that is not cut and only has a cut wound at the junction of the root and stem, and then is smeared with bacterial solution for infection.
[0035] In the study of the induction process of positive callus and transgenic hairy roots, it was found that the stem segments of Quercus variabilis seedlings at different developmental stages (10 days, 20 days, 30 days, and 40 days after whitening) showed different growth results. As can be seen from Table 1, at 10 days and 20 days, the explants of Quercus variabilis seedlings were relatively young and tender, and the cutting treatment would cause them to die after infection with bacteria. After 30 days, the explants gradually became lignified, which was conducive to the explants being infected with Agrobacterium rhizogenes without affecting their own in vitro growth to form positive callus or transgenic hairy roots. However, the first leafy stem segment (S1) was too young and tender, and the cutting treatment would cause them to die after infection with bacteria. The third leafy stem segment (S3) and the fourth leafy stem segment (S4) were severely lignified and could not be infected by Agrobacterium rhizogenes. After in vitro, the cut bevel showed browning and died. The hypocotyl was severely lignified, and Agrobacterium rhizogenes could not infect and enter. However, since the main root was not cut, it did not affect nutrient absorption, and they grew normally but did not produce transgenic hairy roots. Therefore, the second leafy stem segment (S2) of Quercus variabilis seedlings grown in the greenhouse for 30-40 days after whitening is the most suitable explant material for Agrobacterium rhizogenes-mediated genetic transformation of Quercus variabilis.
[0036] Table 1 Effects of different developmental stages on genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes
[0037]
[0038]
[0039] S2. Resuscitating the Agrobacterium rhizogenes strain. That is, the method for resuscitating the Agrobacterium rhizogenes strain includes streaking the expression vector K599 Agrobacterium rhizogenes containing the 35S::RUBY reporter gene on a TY solid medium containing 30 mg / L kanamycin and 30 mg / L streptomycin, culturing overnight to obtain a single colony, picking a single colony on a TY liquid medium containing 30 mg / L kanamycin and 30 mg / L streptomycin, inoculating the single colony on the TY liquid medium, culturing in the dark at 28°C and 220 rpm in a shaker for 12-16 hours until the bacteria enter logarithmic growth, and then expanding the bacteria that have undergone primary culture.
[0040] It should be noted that the TY liquid culture medium in this embodiment is TY liquid culture medium (TY Medium) purchased on the market, adjusted to 9.11 g / L by adding water.
[0041] TY solid medium: TY liquid medium (TY Medium) purchased from the market, add water to adjust to 9.11g / L, and add 15g agar powder per liter.
[0042] The main reagents include:
[0043] Stock solution: kanamycin (kana) 30 mg / mL, streptomycin (strep) 30 mg / mL.
[0044] Working solution: kanamycin (kana) 30mg / mL, streptomycin (strep) 30mg / mL.
[0045] It should be noted that the hormone involved in the present invention has water as solvent and needs to be filtered with a 0.22 μm sterile filter membrane after preparation and then packaged in 1.5 mL centrifuge tubes and stored at -20°C.
[0046] Specifically, the revived Agrobacterium rhizogenes is expanded and cultured to obtain an infection solution, wherein the Agrobacterium rhizogenes is K599 and the vector has a 35S:: Ruby reporter gene. That is, there are two ways to expand the primary cultured Agrobacterium rhizogenes, namely solid culture and liquid culture.
[0047] It should be noted that if Figure 6As shown, in the embodiment of the present invention, the target plasmid is 35S::RUBY. The target plasmid refers to a plasmid that can express a functional gene. In some embodiments, the method for expanding the culture of Agrobacterium rhizogenes includes taking the initial culture to reach OD 600 = 1.0, 100 μl of bacterial solution was inoculated into TY solid medium, spread, and continued to be cultured in the dark at 28°C, 0 rpm in a shaker for 12-16 hours until the surface of the medium was evenly covered with solid bacteria, and then used for solid infection. In other words, this expansion culture method uses TY solid medium for expansion culture.
[0048] In some embodiments, the method for expanding the culture of Agrobacterium rhizogenes comprises taking a preliminary culture to reach OD 600 = 1.0, 200 μl of the bacterial solution was inoculated into 200 mL of TY liquid medium, and the culture was continued in a dark incubator at 28°C and 220 rpm for 12-16 h until the bacterial solution concentration OD 600 =1.0, for liquid infection. That is, this expansion culture method uses TY liquid culture medium for expansion culture.
[0049] S3. Infect the explants at different positions with Agrobacterium rhizogenes at the incisions and plant them in the vermiculite matrix; that is, in this embodiment, four infection methods can be selected for the explant incisions, specifically, solid smearing method, liquid immersion method, liquid vacuum method or liquid injection method.
[0050] The solid smearing method includes cutting off the first leafy stem segment (top bud, G1), the second leafy stem segment (G2), the third leafy stem segment (G3), and the fourth leafy stem segment (G4), while the hypocotyl (hypocotyl) is not cut and only the wound is cut and then smeared with solid bacteria for infection. The infected explants are planted in vermiculite that has been irrigated with water.
[0051] The liquid injection method involves injecting 1 mL of bacterial solution into the phloem of the oblique section of the explant stem segment, and then planting the infected explant in vermiculite that has been irrigated with water.
[0052] The liquid immersion method involves immersing the oblique cut surface of the explant stem segment in liquid bacterial solution for 10 minutes, and then planting the infected explant in vermiculite that has been irrigated with water.
[0053] The liquid vacuum method is to immerse the oblique cut wound of the explant stem segment in liquid bacterial solution and place it in a vacuum dryer. The vacuum degree in the box reaches -0.1M Pa and vacuum is drawn for 5 minutes. The infected explant is planted in vermiculite that has been irrigated with water.
[0054] like Figure 3As shown in the figure, the second leafy stem segment (S2) of Quercus variabilis seedlings grown in the greenhouse for 30-40 days after the whitening was observed was infected by solid smearing method, liquid immersion method, liquid vacuum method and liquid injection method, and the growth of the stem segments was observed, and the number of stem segments with different growth conditions was counted ( Figure 3 Four growth conditions were observed in the experiment ( Figure 4 AE). In terms of the number of stem segments with rooting, callus, browning and bacterial death, the liquid injection method had the best effect on the genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes, with the most roots, while the solid smear method had a poor effect on the genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes.
[0055] It should be noted that Figure 3 The data are the mean ± standard error of three replicates; different lowercase letters indicate significant differences in growth under the same treatment method (P<0.05).
[0056] S4, the infected explants were cultured in dark and light, respectively. That is, the infected explants were cultured in a constant temperature incubator at 28°C, shaded and dark for 48 hours, and then cultured in a constant temperature incubator at 28°C, with 16 hours of light and 8 hours of darkness, and watered every 3 days.
[0057] S5. Perform morphological observation and sampling of the cultured explants to determine the explants with the best culture status and the corresponding cutting position from the Quercus variabilis seedlings. Figure 5 As shown, the morphological observation and sampling detection include morphological observation, stereomicroscope photography and sampling PCR detection.
[0058] It should be noted that Figure 5 In the figure, M: 5000 bp DNA Marker; WT: sterile water control; NC: negative control; PC: positive control; N1-N5: transformed roots.
[0059] Specifically, the second leaf stem segment (G2) of Quercus variabilis seedlings grown in the greenhouse for 30-40 days after the whitening was observed, and the morphological changes of the wound on the cut surface of the stem segment were observed. The results showed that callus was formed on the wound after 20 days of in vitro moisturizing dark culture. Figure 4 C), transgenic hairy roots are produced in 30-45 days, including callus rooting ( Figure 4 D) and direct rooting ( Figure 4 E).
[0060] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.
Claims
1. A method for genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes, comprising the following steps: S1. First, Quercus variabilis seedlings are cultivated from Quercus variabilis seeds, and different parts of the Quercus variabilis seedlings are cut out as infection explants; S2, resuscitating the Agrobacterium rhizogenes, and expanding the culture of the resuscitated Agrobacterium rhizogenes to obtain an infection solution, wherein the Agrobacterium rhizogenes is K599, and the vector has a 35S::Ruby reporter gene; S3, explants from different parts are infected at the incision with Agrobacterium rhizogenes and planted in vermiculite matrix; S4, culturing the infected explants in dark and light conditions respectively; S5. Perform morphological observation and sampling tests on the cultured explants to determine the explants with the best cultivation status and the corresponding cutting position from the Quercus variabilis seedlings.
2. The method for genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The method for cultivating Quercus variabilis seedlings in step S1 comprises sowing Quercus variabilis seeds after germination, and cutting explants to be infected after culturing for 30-40 days.
3. The method for genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: In step S1, the different parts of the Quercus variabilis seedlings include the first young and tender stem segment with leaves G1, the second young and tender stem segment with leaves G2, the third young and tender stem segment with leaves G3, the fourth young and tender stem segment with leaves G4 and the hypocotyl.
4. The method for genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The method for resuscitating the rhizogenes Agrobacterium in step S2 includes streaking the expression vector K599 rhizogenes containing the 35S::RUBY reporter gene on a TY solid culture medium containing 30 mg / L kanamycin and 30 mg / L streptomycin, culturing overnight to obtain a single colony, picking a single colony on a TY liquid culture medium containing 30 mg / L kanamycin and 30 mg / L streptomycin, inoculating the single colony on the TY liquid culture medium, culturing in the dark at 28° C. and 220 rpm in a shaker for 12-16 hours until the bacteria enter logarithmic growth, and then expanding the bacteria that have undergone the primary culture.
5. The method for genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes according to claim 4, characterized in that: The method for expanding the culture of Agrobacterium rhizogenes comprises the steps of sucking the initial culture to reach OD 600 = 1.0 was inoculated into TY solid culture medium with 100 μl of bacterial solution, spread, and continued to be cultured in the dark at 28°C, 0 rpm in a shaker for 12-16 h until the surface of the culture medium was evenly covered with solid bacteria for solid infection.
6. The method for genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes according to claim 4, characterized in that: The method for expanding the culture of Agrobacterium rhizogenes comprises the steps of sucking the initial culture to reach OD 600 = 1.0, 200 μl of the bacterial solution was inoculated into 200 mL of TY liquid medium, and the culture was continued in a dark incubator at 28°C and 220 rpm for 12-16 h until the bacterial solution concentration OD 600 =1.0, for liquid infection.
7. The method for genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The explant infection method in step S3 includes solid smearing method, liquid immersion method, liquid vacuum method or liquid injection method.
8. The method for genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The dark culture in step S4 includes culturing in a 28° C. constant temperature incubator under light shielding for 48 hours.
9. The method for genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The light culture in step S4 includes culturing in a 28° C. constant temperature incubator with 16 h of light and 8 h of darkness, and watering once every 3 days.
10. The method for genetic transformation of Quercus variabilis mediated by Agrobacterium rhizogenes according to claim 1, characterized in that: The morphological observation and sampling detection in step S5 include morphological observation, stereomicroscope photography and recording, and sampling PCR detection.