Application of Agrobacterium in promoting in situ transient expression of foreign genes in bamboo

Abstract

The application belongs to the technical field of plant transgenic engineering, and particularly relates to application of Agrobacterium GV3101 or Agrobacterium AGL1 in promoting in-situ transient expression of exogenous genes in bamboo. The method can directly perform in-situ transient expression of exogenous genes, does not need a tissue culture dedifferentiation process (about 1 year), and only needs 2-3 weeks for the whole process, is convenient and short in cycle; and the method breaks through the limitations of complex and time-consuming traditional bamboo transgenic process, and avoids the problem of low bamboo regeneration efficiency, and provides an effective way for future use of the transient gene expression technology to quickly verify gene functions in bamboo.

Description

Technical Field

[0001] This invention belongs to the field of plant transgenic engineering technology, specifically relating to the application of Agrobacterium GV3101 or Agrobacterium AGL1 in promoting the in situ transient expression of exogenous genes in bamboo. Background Technology

[0002] Bamboo is one of the earliest non-timber natural resources developed and utilized by humans, and it is one of my country's most distinctive forestry resources. Bamboo has a short growth cycle, maturing in 4-5 years, and once planted, it can be used sustainably. Both its growth and utilization are environmentally friendly, possessing both ecological and economic functions, making it a green and renewable biomass resource with the greatest advantages and potential for developing ecological and livelihood-oriented forestry. Moreover, with the rapid development of the bamboo industry, the demand for multifunctional, multi-purpose, high-performance, and high-value-added high-quality bamboo suitable for industrial use is increasing daily. However, bamboo's characteristics—thin-walled and hollow, highly variable laterally, with a large taper and uneven internode length, as well as its preference for moisture and inability to withstand cold—severely restrict the progress of bamboo's industrial utilization.

[0003] Existing technologies can cultivate bamboo through hybridization or transgenic technology. However, bamboo has an uncertain flowering period, dies after flowering, and has a complex genetic background with chromosome numbers ranging from 48 to 186. Hybridization is difficult and the fruit set rate is low, severely restricting the cultivation of new bamboo varieties with high yield through hybridization. Existing transgenic technology for cultivating bamboo also has the following drawbacks: (1) It requires cultivation through explants, but bamboo explants are difficult to obtain and tissue culture takes a long time. Moreover, this cultivation technology also has limitations such as low genetic transformation efficiency and difficulty in purifying transgenic chimeras; (2) It is a stable genetic transformation technology that integrates plasmid fragments into bamboo. The chromosomes of the bamboo itself are difficult to remove in the short term through hybridization and other methods, which has the defect of genetically modified safety risks and is not conducive to the cultivation of bamboo species for shoots; (3) The regeneration rate of the genetically modified shoots mediated by Agrobacterium is very low. For example, in the reports of editing PSY1 of Bambusa textilis and PDS of Bambusa pubescens using CRISPR / Cas9 gene editing technology, the efficiency of genetically modified shoots mediated by Agrobacterium is between 68% and 70%, but the regeneration rate of the shoots is less than 3%; (4) Agrobacterium-mediated genetic modification technology based on bamboo dedifferentiation regeneration has a long cycle and requires a complex dedifferentiation and redifferentiation process, which is time-consuming and labor-intensive.

[0004] Therefore, it is necessary to develop a simple and efficient method for expressing exogenous genes in bamboo that avoids the drawback of low regeneration rate. Summary of the Invention

[0005] To address the above problems, one of the objectives of this invention is to provide a method for promoting the expression of exogenous genes in bamboo through Agrobacterium GV3101 or Agrobacterium AGL1. This method not only overcomes the limitations of the traditional bamboo transgenic process being complex and time-consuming, but also avoids the problem of low bamboo regeneration efficiency, providing an effective way to rapidly verify gene function in bamboo using transient gene expression technology in the future.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0007] In one aspect, this invention provides the application of Agrobacterium GV3101 or Agrobacterium AGL1 in promoting the in situ transient expression of exogenous genes in bamboo.

[0008] Another aspect of the present invention provides a method for in situ transient expression of exogenous genes in bamboo, comprising: using Agrobacterium GV3101 or Agrobacterium AGL1 to mediate the infection of exogenous genes into bamboo species to be transgenic.

[0009] Another aspect of the present invention provides the application of Agrobacterium GV3101 or Agrobacterium AGL1 in the targeted cultivation of bamboo varieties.

[0010] The beneficial effects of this invention include at least the following:

[0011] (1) The method of the present invention can directly express exogenous genes in situ transiently without the need for tissue culture dedifferentiation process (about 1 year). The whole process only takes 2-3 weeks. The method is convenient and has a short cycle.

[0012] (2) The method of the present invention uses Agrobacterium-mediated transient expression technology to establish an efficient in situ transient gene expression method for bamboo for the first time. This method not only breaks through the limitations of the traditional bamboo transgenic process being complex and time-consuming, but also avoids the problem of low bamboo regeneration rate, providing an effective way to quickly verify gene function in bamboo using transient gene expression technology in the future. Attached Figure Description

[0013] Figure 1 The following is an example of RUBY gene expression in bamboo leaves in Example 1; where A: bamboo seedlings infected with Agrobacterium; BC: RUBY gene expression in bamboo leaves (note: triangles indicate expression locations); D: RUBY gene expression in bamboo leaves mediated by different types of Agrobacterium.

[0014] Figure 2Example 2 shows the in situ transient expression of the Agrobacterium-mediated RUBY gene in bamboo shoots of Phyllostachys aureosulcata 'Spectabilis'; where A: bamboo shoots used for Agrobacterium infection; BC: expression of the RUBY gene in bamboo shoot sheaths and lateral branches (Note: the triangles indicate the expression locations).

[0015] Figure 3 The image shows the expression of the RUBY gene in bamboo shoots of P. aureosulcata 'Spectabilis' in Example 3, and the accumulation of betalains. A: Control group; B: Experimental group: RUBY gene expression in the bamboo shoot sheaths and lateral branches (black box area is a magnified view).

[0016] Figure 4 The image shows the expression of the RUBY gene in bamboo shoots of *Phyllostachys aureosulcata* 'Aureocarlis' in Example 4, and the accumulation of betalains. A: Control group; B: Experimental group: RUBY gene expression in the bamboo shoot sheaths and lateral branches (black box area is a magnified view).

[0017] It should be noted that the highlighted areas in the image are red after the RUBY gene is expressed. To meet the requirements of the attached image, the image has been processed to be darker (i.e., the highlighted areas, which are also the triangle indicators). Detailed Implementation

[0018] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0019] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0020] This invention provides an application of Agrobacterium GV3101 or Agrobacterium AGL1 in promoting the in situ transient expression of exogenous genes in bamboo.

[0021] It should be noted that in the above applications, the applicable bamboo varieties can be all known bamboo varieties, such as moso bamboo, golden-striped bamboo, or yellow-stemmed bamboo; when infecting bamboo, bamboo shoots or bamboo seedlings can be selected; regarding the selection of Agrobacterium, Agrobacterium GV3101 or Agrobacterium AGL1-mediated exogenous gene, when infecting bamboo shoots or seedlings, causes less damage to bamboo compared to other Agrobacterium species, and has a higher exogenous gene expression level; in particular, for infecting bamboo seedlings with a height ≤5cm, the expression efficiency of exogenous gene mediated by Agrobacterium GV3101 can reach 8. The expression efficiency of exogenous genes mediated by Agrobacterium AGL1 reached 76.9% (5.2%). Furthermore, the expression efficiency of exogenous genes mediated by Agrobacterium GV3101 was also high (91.7%) when infecting bamboo seedlings with a height >5cm. This means that Agrobacterium GV3101-mediated exogenous gene expression is high in both bamboo seedlings with a height ≤5cm and >5cm. Moreover, when bamboo shoots are used as the infection target, Agrobacterium GV3101-mediated infection can also lead to a large expression of exogenous genes.

[0022] Another embodiment of the present invention provides a method for in situ transient expression of a foreign gene in bamboo. This method may include: using Agrobacterium GV3101 or Agrobacterium AGL1 to mediate the infection of the foreign gene into the bamboo species to be transgenic. It should be noted that, as described above, the object of the foreign gene infection mediated by Agrobacterium GV3101 or Agrobacterium AGL1 can be bamboo seedlings, bamboo shoots, or other infectable objects.

[0023] It should be noted that Agrobacterium-mediated transient expression technology is widely used for gene activity and function verification in dicotyledonous tobacco, but there are no reports of Agrobacterium-mediated transient expression in monocotyledonous plants; at the same time, transient expression technology in dicotyledonous plants is not applicable to monocotyledonous plants such as rice and bamboo.

[0024] In some specific embodiments, the method for in situ transient expression of exogenous genes in bamboo can use Agrobacterium GV3101 to mediate the infection of exogenous genes in bamboo shoots or seedlings of the bamboo species to be transgenic. As mentioned above, Agrobacterium GV3101-mediated exogenous gene infection, regardless of whether the infected object is bamboo seedlings or bamboo shoots, can promote the expression of exogenous genes in the infected object in large quantities, and the expression level is significantly higher than that of other Agrobacterium species, while causing less damage to the infected object.

[0025] In some specific embodiments, the above-mentioned method for in situ transient expression of exogenous genes in bamboo can use Agrobacterium AGL1 to mediate the infection of exogenous genes into bamboo seedlings to be transgenic. As mentioned above, Agrobacterium AGL1-mediated exogenous gene infection, when using bamboo seedlings as the infection target, can also promote the expression of exogenous genes in the infected target, especially for bamboo seedlings ≤5cm in height, where the exogenous gene expression efficiency is higher; moreover, Agrobacterium AGL1 has a higher infection efficiency than some other Agrobacterium-mediated infections, such as Agrobacterium LBA4404 and EHA105.

[0026] In some specific embodiments, the method for in situ transient expression of the exogenous gene in bamboo may include: transforming the exogenous gene into Agrobacterium GV3101 or Agrobacterium AGL1 and then culturing it in a culture medium until OD. 600 Reach 0.6-0.8; centrifuge and collect the bacterial cells, wash with infection solution and resuspend to OD. 600 The target concentration is 0.4-0.6. The bamboo to be transgenic is then infected with an inoculum containing the exogenous gene, either Agrobacterium GV3101 or Agrobacterium AGL1. It should be noted that the culture medium used in the above method can be any medium known in the art, such as YEP medium, YEB medium, or LB medium; the inoculum can be any inoculum known in the art, such as an inoculum composed of MES and MgCl2, or an inoculum composed of redistilled water, MS medium, and sucrose.

[0027] In some specific embodiments, the method of infecting the transgenic bamboo with an infusion solution containing Agrobacterium GV3101 or Agrobacterium AGL1 containing exogenous genes can be achieved by pricking the apical meristem of the bamboo seedling or the top of the bamboo shoot with a sharp needle (such as a syringe needle). In other specific embodiments, when using bamboo seedlings as the target of infection, after pricking the bamboo seedlings, the above-ground parts of the bamboo seedlings can be quickly immersed upside down in the Agrobacterium GV3101 or Agrobacterium AGL1 infusion solution, and then placed in a vacuum chamber for negative pressure vacuuming. This helps the Agrobacterium infusion solution to fully contact the unopened tender leaves of the bamboo seedlings, thereby improving the infection efficiency.

[0028] In some specific embodiments, the method for in situ transient expression of exogenous genes in bamboo can also use a RUBY reporter gene marker to label the exogenous gene. Specifically, when transgenic technology is applied to plant breeding, antibiotics and herbicides can be selected as commonly used resistance selection markers, but they can affect the normal growth of plants. Betalains are a class of plant natural products that can be synthesized from tyrosine as a substrate through the catalysis of three enzymes (cytochrome P450 enzyme (CYP76AD1), 3,4-dihydroxy-L-phenylalanine 4,5-dioxygenase (DODA), and glycosyltransferase (GT)). The expression of three genes, CYP76AD1, DODA, and GT, promotes the accumulation of betalains (ruby red color). The RUBY reporter system, which is visually accessible in plants such as Arabidopsis and rice, has been established. In this invention, the RUBY reporter system has been successfully applied to bamboo. The expression of the RUBY reporter gene in bamboo can be clearly observed with the naked eye without affecting plant growth. Thus, the RUBY reporter gene can be used to label exogenous genes, allowing for real-time tracking and observation of their expression in bamboo—a simple and convenient method.

[0029] It should be noted that the exogenous gene can also be a CRISPR-based gene editing tool (such as a CRISPR / Cas9 vector), which also requires Agrobacterium-mediated transmission. After obtaining stable editing material using a CRISPR-based gene editing tool, to eliminate CRISPR interference, the CRISPR T-DNA needs to be isolated using antibiotics. However, as mentioned above, using antibiotic resistance selection markers can affect the normal growth of plants, and under antibiotic selection pressure, the regeneration rate of transgenic bamboo shoots is very low. Therefore, in this invention, the RUBY reporter tag can be combined with the exogenous gene. When isolating T-DNA, the expression status of the exogenous gene can be clearly understood and screened without resistance selection, which is simple and efficient.

[0030] It should also be noted that in the above-mentioned method for in situ transient expression of exogenous genes in bamboo, the bamboo species to be transgenic can be bamboo species known in the art, such as moso bamboo, golden bamboo, or yellow-stemmed bamboo.

[0031] Another embodiment of the present invention provides the application of Agrobacterium GV3101 or Agrobacterium AGL1 in the targeted cultivation of bamboo varieties. Specifically, based on the ability of Agrobacterium GV3101 or Agrobacterium AGL1 to mediate the in situ transient expression of exogenous genes in bamboo, Agrobacterium GV3101 or Agrobacterium AGL1 can be used for the targeted cultivation of bamboo varieties. It should be understood that the targeted cultivation of bamboo varieties using Agrobacterium GV3101 or Agrobacterium AGL1 also includes other steps known in the art in plant cultivation processes, such as seed culture, fertilization management, and field management of seedlings.

[0032] It should be noted that, in the above applications and methods, the use of Agrobacterium GV3101 or Agrobacterium AGL1 can achieve in situ transient expression of exogenous genes in bamboo. This can overcome the shortcomings of bamboo, such as thin and hollow walls, large lateral variability, large taper, uneven internode length, and its preference for moisture and intolerant of cold, which seriously restrict the industrial utilization of bamboo. It can also directionally cultivate bamboo varieties with high quality and diverse growth environments. Especially for bamboo species used for shoots, as mentioned above, due to the long and unstable flowering cycle of bamboo, plasmid fragments are easily integrated into the bamboo's own chromosomes, which are difficult to remove in the short term through hybridization and other methods. Bamboo species used for shoots have transgenic safety risks. This invention is applicable to transient expression, which can avoid the transgenic safety risks of bamboo species used for shoots. In addition, this invention can use bamboo shoots and bamboo seedlings for in situ expression, avoiding the limitations of existing transgenic technologies such as the difficulty in obtaining explants, long tissue culture time, and difficulty in purifying transgenic chimeras. This reduces the cycle and difficulty of cultivating new bamboo varieties and improves efficiency.

[0033] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0034] In the following examples, the YEP culture medium consisted of 1 g beef extract, 1 g yeast extract, and 0.5 g NaCl per 100 mL of distilled water.

[0035] In the following examples, the inoculum was: 10 mmol MES (pH 6.0) and 10 mmol MgCl2 per 1000 mL of distilled water.

[0036] Example 1: Agrobacterium infection and transformation of bamboo seedlings

[0037] (1) The published RUBY reporter gene (pHDE-35S::RUBY vector) was transformed into four Agrobacterium tumefaciens strains: AGL1, LBA4404, EHA105, and GV3101, and cultured in YEP medium until OD200. 600Reaching 0.6-0.8;

[0038] (2) Centrifuge and collect the bacterial cells, wash once with the infection solution, and resuspend in the infection solution until OD200 is reached. 600 Reaching 0.4-0.6;

[0039] (3) Sow the seeds of moso bamboo (Phyllostachysedulis) in a seedling tray. Use one-month-old seedlings as material. Remove the moso bamboo seedlings from the seedling tray and wrap them with aluminum foil. Figure 1 A) Use a sharp needle (such as a syringe needle) to puncture the meristem near the top of the bamboo seedling;

[0040] (4) Quickly invert the above-ground part of the bamboo seedling and immerse it in the Agrobacterium infection solution, then place it in a vacuum chamber for negative pressure vacuuming, 25 inches-27 inches Hg negative pressure treatment for 2 minutes;

[0041] (5) Remove the infected bamboo seedlings from the vacuum chamber, remove the tin foil and transfer them to a seedling tray for cultivation. Cover them with a transparent film on the first day to maintain humidity (relative humidity of about 80%). Remove the film on the second day and continue cultivation.

[0042] (6) On the 3rd day, red (betalain) appeared on the unopened tender leaves of the bamboo seedlings. Figure 1 B); On the 5th day, the leaves unfolded, and the red color became more pronounced. Figure 1 C);

[0043] The results of the above embodiments are as follows: Figure 1 As shown in Table D and Table 1, Agrobacterium AGL1 and GV3101 strains caused less damage to bamboo leaves and had higher expression levels; among them, GV3101 had the highest transformation efficiency in both seedlings less than 5 cm tall and seedlings between 5 cm and 10 cm tall, reaching 85.2% and 91.7% respectively.

[0044] Table 1 shows the expression efficiency of the RUBY gene mediated by different types of Agrobacterium in bamboo leaves.

[0045]

[0046] Example 2: Agrobacterium infection and transformation of bamboo shoots

[0047] (1) The pHDE-35S::RUBY vector was transformed into Agrobacterium GV3101 strain and cultured in YEP medium until OD. 600 Reaching 0.6-0.8;

[0048] (2) Centrifuge and collect the bacterial cells, wash once with the infection solution, and resuspend in the infection solution until OD200 is reached. 600 Reaching 0.4-0.6;

[0049] (3) Using young shoots (approximately 40cm) of outdoor-grown golden bamboo (P. aureosulcata 'Spectabilis') and yellow-stemmed bamboo (P. aureosulcata 'Aureocarlis') as materials; puncturing the top of the shoot with a sharp needle (such as a syringe needle), and injecting an infection solution containing Agrobacterium into the shoot cavity and the gap between the shoot and the sheath using a syringe. Figure 2 A); and bamboo shoots injected with an infection solution free of Agrobacterium tumefaciens served as the control group.

[0050] (4) 15 days later, in the bamboo shoot shell ( Figure 2 B, 3B, 4B), lateral branches ( Figure 2 C, 3B, 4B), stem ( Figure 3 The presence of red at point B indicates that RUBY gene expression promotes the synthesis and accumulation of betaine.

[0051] The results of the above examples show that the exogenous RUBY gene can be expressed in large quantities in moso bamboo leaves, and Agrobacterium GV3101 is a suitable strain for in situ gene expression in moso bamboo leaves and bamboo shoots.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for in situ transient expression of a foreign gene in bamboo, characterized in that, The method comprises using Agrobacterium GV3101 or Agrobacterium AGL1 to mediate foreign gene infection of a to-be-transgenic bamboo species, and the to-be-transgenic bamboo species is bamboo shoots or seedlings; the method comprises the following steps: after the foreign gene is transformed into Agrobacterium GV3101 or Agrobacterium AGL1, the Agrobacterium GV3101 or Agrobacterium AGL1 is cultured in a culture medium to OD 600 0.6-0.8; centrifugation is performed and bacterial cells are collected, the bacterial cells are resuspended to OD 600 0.4-0.6 by using an infection solution; the infection solution containing the Agrobacterium GV3101 or Agrobacterium AGL1 with the foreign gene is used to infect a to-be-transgenic bamboo; the to-be-transgenic bamboo species is Phyllostachys edulis; when the Agrobacterium AGL1 is used to mediate foreign gene infection of the to-be-transgenic bamboo species, the height of the to-be-transgenic bamboo seedling is less than or equal to 5 cm.

2. The method for in situ transient expression of a foreign gene in bamboo according to claim 1, wherein, When using Agrobacterium GV3101 to mediate the infection of transgenic bamboo species with exogenous genes, the height of the transgenic bamboo seedlings should be 5cm-10cm.

3. The method for in situ transient expression of a foreign gene in bamboo according to claim 1 or 2, characterized in that, Use the RUBY reporter gene marker to label foreign genes.

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