Method for introducing genes into pollen based on needle-free injection propeller and low-pressure penetration

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a method for introducing genes into pollen based on a needle-free injection propeller and low-pressure osmosis. The method comprises: (1) washing the collected pollen to obtain the washed pollen; (2) preparing a plasmid containing a target gene, loading a solution containing the plasmid into a needle-free injection propeller, and aiming the nozzle of the needle-free injection propeller at the washed pollen for repeated spraying; (3) subjecting the pollen treated in step (2) to low-pressure osmosis treatment, culturing in the dark after the low-pressure treatment, and obtaining transformed pollen containing the target gene. The method of the present invention for introducing genes into pear pollen using a needle-free injection propeller and low-pressure osmosis can not only successfully introduce exogenous genes into pollen, but also does not require complex instruments, does not require high costs, is easy to operate, does not affect the vitality of pollen, and has a wide range of applications.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for introducing genes into pollen based on a needle-free injection propeller and low-pressure penetration. Background Art

[0002] Regardless of the development trend of transgenic technology, transformation protocols should be developed in the direction of simplicity, ease of operation and convenience for breeding (Cunningham et al., 2018; Huang et al., 2023). However, most technologies developed based on Agrobacterium-mediated and direct DNA transfer require time-consuming, labor-intensive and expensive procedures, such as long-term in vitro culture to create the necessary conditions for regenerating protoplasts or explants (Mini et al., 2018), and the use of expensive instruments such as gene guns (O'Brien and Lummis, 2006). Pollen grains are ideal carriers for introducing foreign genes into germ lines and are very abundant (Eapen 2011, Gad et al., 2020). However, the success rate of using pollen as a carrier to produce transgenic plants is very low, especially for trees (Yin et al., 2021), mainly because two layers of outer wall are formed during pollen development (Codjoe JM et al., 2021, Jaffri and MacAlister, 2021), preventing foreign genes from entering pollen (Zhao et al., 2017). This is the first and most difficult step in using pollen as a vector for transgenic plants. At present, the highly efficient transformation system using pollen as a vector has not been successfully established for many tree species. Summary of the invention

[0003] In view of the above technical problems, the present invention provides a method for introducing genes into pollen based on a needle-free injection propeller and low-pressure penetration. The method provided by the present invention is simple, rapid, and can effectively introduce exogenous genes into pollen.

[0004] The present invention is achieved through the following technical solutions:

[0005] A method for introducing genes into pollen based on a needle-free injection propeller and low-pressure penetration, the method comprising:

[0006] (1) washing the collected pollen to obtain washed pollen;

[0007] (2) preparing a solution containing the exogenous gene, loading the solution containing the exogenous gene into a needle-free injection propeller, and aiming the nozzle of the needle-free injection propeller at the cleaned pollen to repeatedly spray;

[0008] (3) The pollen treated in step (2) is subjected to low-pressure osmosis treatment, and then cultured in the dark to obtain transformed pollen containing the exogenous gene.

[0009] Furthermore, in step (1), the method for cleaning the pollen is:

[0010] (1) Weigh 0.01-0.03 g of pollen and place it in a 10 ml centrifuge tube;

[0011] (2) Add 3-5 ml of BK medium to a 10 ml centrifuge tube, shake and mix, then centrifuge and discard the supernatant;

[0012] (3) Repeat step (2) 2-3 times, add 5-8 ml BK medium to a 10 ml centrifuge tube, shake and mix, and then dispense the pollen solution into 1.5 ml centrifuge tubes;

[0013] (4) Place the pollen solution in 1.5 ml centrifuge tubes on ice, and then shake the pollen solution on ice on a shaker; centrifuge in a low-temperature high-speed centrifuge, remove the supernatant, and obtain the cleaned pollen; place the cleaned pollen on ice for later use.

[0014] Furthermore, in step (2), the exogenous gene is in the form of plasmid DNA.

[0015] Furthermore, step (2) is specifically as follows:

[0016] 1) Using distilled water as a solvent, a solution containing an exogenous gene is prepared; the concentration of the exogenous gene in the solution containing the exogenous gene is 10-15 ng / µL;

[0017] 2) Add the solution containing the exogenous gene into the needle-free injection pusher;

[0018] 3) Aim the nozzle of the needle-free injection propeller at the pollen and spray it. Repeat the spraying 6-8 times, keeping the distance between the nozzle and the pollen at 30-40 mm.

[0019] Furthermore, in step 3), the pollen is placed at the bottom of a 1.5 mL centrifuge tube, the mouth of the centrifuge tube is perpendicular to the ground, and then the nozzle of the needle-free injection propeller is aligned with the mouth of the centrifuge tube for injection.

[0020] Furthermore, step (3) is specifically as follows:

[0021] 1) placing the pollen treated in step (2) on ice, and placing the pollen on ice in a vacuum drying oven for low-pressure infiltration. The conditions for low-pressure infiltration are: pressure -80 Pa, treatment time 4-6 min;

[0022] 2) After low-pressure infiltration, let stand for 2-3 minutes, then centrifuge at 4°C, 10,000-12,000 rpm for 4-5 minutes, and remove the supernatant;

[0023] 3) Add the pollen treated in step 2) to the BK medium and shake to mix well. Incubate in a shaker at 28°C in the dark for 2-3 h.

[0024] Furthermore, the pollen is pear pollen or apple pollen.

[0025] Furthermore, the transformed pollen is used for pollination to cultivate seeds with exogenous genes.

[0026] Beneficial technical effects of the present invention:

[0027] The method of using a needle-free injection propeller and low-pressure penetration in the present invention is an indirect transformation technology that uses pollen to transform the object; the present invention uses a needle-free injection propeller and low-pressure penetration technology to quickly and effectively introduce a plasmid containing a target gene into pollen, and the introduction rate reaches 43%, which is a relatively high introduction rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1A This is an electron micrograph of pollen that has not been treated with a needle-free injection propeller;

[0029] Figure 1B This is an electron microscope image of pollen after being sprayed by a needle-free injection propeller;

[0030] Figure 1C It is a statistical chart of the density of holes on the surface of pollen in the control group and pollen treated by needle-free injection propeller;

[0031] Figure 2 This is a microscopic image of a GFP-labeled plasmid introduced into pear pollen in an embodiment of the present invention, wherein A is the Bright-field field, B is the GFP field, and C is the Merge field;

[0032] Figure 3 This is a diagram showing the results of quantitative gene expression in seeds according to an embodiment of the present invention;

[0033] Figure 4 This is the microscopic imaging after the plasmid marked with GFP was introduced into apple pollen in the embodiment of the present invention, wherein A is the Bright-field field, B is the GFP field, and C is the Merge field. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] On the contrary, the present invention covers any substitution, modification, equivalent method and scheme made on the essence and scope of the present invention as defined by the claims. Further, in order to make the public have a better understanding of the present invention, some specific details are described in detail in the detailed description of the present invention below. Those skilled in the art can fully understand the present invention without the description of these details.

[0036] Example 1: A method for introducing genes into pollen based on a needle-free injection propeller and low-pressure penetration, the method comprising:

[0037] (1) washing the collected pollen to obtain washed pollen;

[0038] (2) preparing a solution containing the exogenous gene, loading the solution containing the exogenous gene into a needle-free injection propeller, and aiming the nozzle of the needle-free injection propeller at the cleaned pollen to repeatedly spray;

[0039] (3) The pollen treated in step (2) is subjected to low-pressure infiltration treatment, and then cultured in the dark to obtain transformed pollen containing the exogenous gene.

[0040] In this embodiment, the pollen collection steps are specifically as follows: picking flowers and taking anthers in the early flowering period (i.e., when the flower buds are in the shape of balloons); when manually collecting pollen, take a flower in each hand, rub the flower centers against each other, and rub all the anthers and filaments onto the paper. Do not damage the style during the rubbing process, use a fine mesh sieve to separate the anthers and filaments, and after screening, spread the anthers thinly on the paper, and place them in a room at room temperature (about 25°C) to dry. Generally, the anthers will crack in two days and nights, and when a large amount of pollen is scattered, use sulfuric acid paper (100mm×100mm) to divide and install them in a silica gel box, and put them into a -20°C refrigerator for storage. When using, take out the pollen from the -20°C refrigerator, thaw it for 1 hour, and then perform the cleaning step.

[0041] In this embodiment, the method for cleaning pollen is:

[0042] (1) Weigh 0.01-0.03 g of pollen and place it in a 10 ml centrifuge tube;

[0043] (2) Add 3-5 ml of BK medium to a 10 ml centrifuge tube, shake and mix, then centrifuge and discard the supernatant;

[0044] (3) Repeat step (2) 2-3 times, add 5-8 ml BK medium to a 10 ml centrifuge tube, shake and mix, and then dispense the pollen solution into 1.5 ml centrifuge tubes;

[0045] (4) Allow the pollen solution in 1.5 ml centrifuge tubes to stand on ice for a certain period of time, then shake the pollen solution on ice on a shaker; centrifuge in a low-temperature high-speed centrifuge, remove the supernatant, and obtain the cleaned pollen; place the cleaned pollen on ice for later use.

[0046] Specifically, in this embodiment, the pollen solution in 1.5 ml centrifuge tubes is placed on ice for 4-6 minutes, and then the pollen solution placed on ice is shaken on a shaker at 25-30 (preferably 28°C)°C for 5-10 minutes, with the shaker rotating at 60-80 rpm; then centrifuged at 4°C and 10000-12000 rpm for 6-10 minutes, the supernatant is removed, and the washed pollen is obtained; the washed pollen is placed on ice for standby use. The above-mentioned process of washing pollen is used to remove some impurities in the outer wall of the pollen. It can be seen that the method provided by the present invention does not require fresh pollen, and can use low-temperature dried and long-term preserved, vigorous pollen.

[0047] In step (2) of this embodiment, the exogenous gene is in the form of plasmid DNA.

[0048] In this embodiment, step (2) is specifically as follows:

[0049] 1) Using distilled water as a solvent, a solution containing an exogenous gene is prepared; the concentration of the exogenous gene in the solution containing the exogenous gene is 10-15 ng / µL;

[0050] 2) Add the solution containing the exogenous gene into the needle-free injection pusher;

[0051] 3) Aim the nozzle of the needle-free injection propeller at the pollen and spray, repeat the spraying 6-8 times, and control the distance between the nozzle and the pollen to be 30-40 mm; specifically, in this step, place the pollen in a 1.5 mL centrifuge tube, place the mouth of the centrifuge tube vertically to the ground, and then aim the nozzle of the needle-free injection propeller at the mouth of the centrifuge tube and spray; at this time, the distance between the nozzle and the pollen is the height distance of the centrifuge tube, which is 30-40 mm.

[0052] Step (3) of this embodiment is specifically as follows:

[0053] 1) placing the pollen treated in step (2) on ice, and placing the pollen on ice in a vacuum drying oven for low-pressure infiltration. The conditions for low-pressure infiltration are: pressure -80 Pa, treatment time 4-6 min;

[0054] 2) After low-pressure infiltration, let stand for 2-3 minutes, then centrifuge at 4°C, 10,000-12,000 rpm for 4-5 minutes, and remove the supernatant;

[0055] 3) Add the pollen treated in step 2) to the BK medium and shake to mix well. Incubate in a shaker at 28°C in the dark for 2-3 h.

[0056] In this embodiment, the raw materials of the BK culture medium include sucrose, Ca(NO3)2, H3BO4, MgSO4, and KNO3; wherein the concentration of sucrose is 15% (mass volume ratio, W / V, unit: g / mL), the concentration of Ca(NO3)2 is 5.5mM, the concentration of H3BO4 is 16mM, the concentration of MgSO4 is 16mM, and the concentration of KNO3 is 16mM; and the BK culture medium is prepared using water as a solvent, and the pH is 5.8-6.0, and the pH is adjusted using KOH.

[0057] Wherein, the pollen is pear pollen or apple pollen.

[0058] The transformed pollen is used for pollination to cultivate seeds with foreign genes and to achieve the expression of related genes.

[0059] Example 2: This example takes pear pollen as an example and provides a method for introducing genes into pear pollen based on a needle-free injection propeller and low-pressure penetration, the method comprising:

[0060] Collection of pear pollen: Collect flowers and take anthers when they are in the early flowering stage, i.e. when they are in the shape of balloons. When collecting pollen manually, hold a flower in each hand, rub the flower centers against each other, and rub all the anthers and filaments onto paper. Do not damage the style during the rubbing process. Use a fine mesh sieve to separate the anthers and filaments. After screening, spread the anthers thinly on paper and dry them indoors at about 25°C. Generally, the anthers will crack in two days and nights. When a large amount of pollen is released, use sulfuric acid paper (100mm*100mm) to divide the devices into silica gel boxes and store them in a -20°C refrigerator for use.

[0061] Pollen cleaning:

[0062] ① Take out the pollen from the -20℃ refrigerator, thaw it for 1 hour, weigh 0.1-0.3g of pollen and put it in a 10ml centrifuge tube, add an appropriate amount of BK to shake and mix it, and then divide it into 1.5ml centrifuge tubes. Repeat each treatment three times.

[0063] ② Place the pollen solution in 1.5 ml centrifuge tubes on ice for 4 minutes, shake on ice at 28°C for 10 minutes, centrifuge at 4°C, 12,000 rpm for 10 minutes, and remove the supernatant.

[0064] Processing of pear pollen:

[0065] ①Put the above-treated pollen on ice for later use

[0066] ② Pipette 1-2 ml of distilled water, place it in the glass tube of the syringe, add an appropriate amount of PRI-101-GFP plasmid (the vector is PIR-101, the promoter is CaMV 35S promoter), the optimal concentration is 10 ng / ul, and mix well with a pipette.

[0067] ③ Turn the switch of the syringe, aim the nozzle at the mouth of the 1.5m centrifuge tube vertically to the ground, press the switch to spray the pollen, and repeat the spraying 6-8 times.

[0068] Figure 1A The pollen that was not treated by the needle-free injection propeller was the control group (of which, Figure 1A The small picture in the lower right corner is a picture of the complete pollen grains of the control group under a scanning electron microscope); Figure 1B It is pollen that has been sprayed by a needle-free injection propeller (among which, Figure 1B The small picture in the lower left corner is a picture of pollen grains after spraying under a scanning electron microscope); Figure 1C The density of pores on the surface of pollen from the control group and the pollen treated by needleless injection propeller is shown in Table 1. *** indicates a difference of P < 0.001. Figure 1A-Figure 1C It can be seen that the density of pores on the pollen surface after being sprayed by the needle-free injection propeller is significantly increased, which is conducive to the plasmid carrying exogenous genes entering the interior of the pollen.

[0069] ④ The treated pollen was opened and placed on ice, placed in a vacuum drying oven, infiltrated at -80 Pa low pressure for 4 min, opened and left for 2 min, centrifuged at 4°C, 12000 rpm for 5 min, and the supernatant was removed.

[0070] ⑤ Add 500ul of BK culture medium to the pollen treated above, shake and mix evenly, and place in a shaker at 28℃ in the dark for 2.5h.

[0071] like Figure 2 As shown, 10ul of the cultured pollen was pipetted onto a glass slide and covered with a coverslip. Pollen fluorescence was observed in the GFP field under the fully automatic intelligent living cell fluorescence microscopy imaging system. Green fluorescence was seen at the tip of the pollen tube under the GFP field. When observing, it was necessary to add a scale bar and shoot three fields: Bright-field, GFP, and Merge.

[0072] Under a 20X microscope, select GFP and Bright-field, randomly take 3-5 pictures, repeat each treatment three times, and calculate the pollen germination rate, pollen tube length, GFP gene introduction rate, and fluorescence intensity.

[0073] Pollen germination rate (%) = (number of pollen germination / total number of pollen) × 100%;

[0074] Pollen tube length: Image-Pro Plus software was used to count the length of pollen tubes;

[0075] GFP gene introduction rate (%) = (total number of pollen tubes / pollen tube fluorescence) × 100%;

[0076] Table 1 is a comparison of the germination rate and pollen tube length after the pollen was treated with the needle-free injection propeller and the control. As shown in Table 1, the pollen treatment method provided by the present invention does not affect the pollen germination rate, does not affect the pollen vitality, and has no significant effect on the pollen tube length.

[0077] Table 1 Pollen germination rate and pollen tube length

[0078]

[0079] Note: Lowercase letters represent variance analysis in the same column. The same letters indicate no difference, and different letters indicate significant difference (p<0.05).

[0080] Using a needleless syringe and low-pressure penetration, the GFP-labeled plasmid was introduced into the pollen. After 2.5 hours of culture, the tip of the pollen tube had green fluorescence, indicating that the GFP-labeled vector was introduced into the pollen and successfully expressed. According to statistics, the introduction rate reached 43%

[0081] In the present invention, the experiment is designed according to factors affecting the conversion rate of gene introduction into pollen, such as the spraying distance and spraying times that produce micropores on the pollen surface, the germination rate of cells, and the introduction rate of GFP.

[0082] Spraying distance: Through relevant experiments, 1.5ml centrifuge tubes (the distance between the bottom of the tube and the tube mouth is 40mm) are used to load pollen. This height is most suitable for pollen spraying, and there is no significant difference in pollen germination rate compared with the control.

[0083] Number of spraying: Select the spraying distance and design the spraying times as 3, 5, 6, 8, and 10. Through multiple experiments, it was found that when the spraying times were 6-8, the number of micropores produced on the pollen surface was more than that of the control, and the pollen germination rate was not significantly different from that of the control.

[0084] After selecting the injection distance and injection times, the GFP plasmid was dissolved in distilled water, placed in the glass tube of the syringe, and injected according to the above parameters. Low-pressure infiltration was performed at -80Pa for 4 minutes. After culturing for 2.5 hours, the fluorescence was observed under the fully automatic intelligent living cell fluorescence microscopy system, and the introduction rate was calculated to be 43%. The method provided by the present invention for introducing genes into pear pollen using a needle-free injection propeller and low-pressure infiltration can not only successfully introduce exogenous genes into pollen, but also the instrument has a simple structure, does not require a high cost, is easy to operate, does not cause serious mechanical damage to plant cells, has a wide range of applications, and can be used theoretically for any flowering plant, saving time and manpower, does not require lengthy tissue culture, and has high transformation efficiency.

[0085] Using the above method, the apple gene (mdgh3.1) was transferred into the pollen of the 'Qiuyue' pear. On April 10, 20 flowers of the 'Xuehua' pear were pollinated. The fruits were harvested on September 2 of the same year, and 9 fruits were finally obtained. The seeds were taken out and the gene was quantitatively expressed in the seeds, such as Figure 3 As shown in the figure, the mdgh3.1 in transgenic seeds was significantly higher (p<0.001) than that in the control and empty vector. This result shows that the gene can be successfully transferred into seeds using pollen as a vector.

[0086] Example 3: This example takes apple pollen as an example. The method of Example 1 is used to introduce a plasmid with a GFP marker into apple pollen. After culturing for 3 hours, Figure 4 As shown in Figures AC, the tip of the pollen tube has green fluorescence, indicating that the GFP-labeled vector is successfully introduced into the pollen and expressed successfully using the method provided by the present invention.

[0087] The present invention uses a needle-free injection propeller and a low-pressure osmosis method to introduce genes into pollen. Not only can the foreign gene be successfully introduced into the pollen, but also it does not require complex instruments, does not require high costs, is easy to operate, does not cause serious mechanical damage to plant cells, has a wide range of applications, and can be used in theory for any flowering plant, saves time and manpower, does not require lengthy tissue culture, and has high conversion efficiency. According to experimental statistics, the introduction rate can reach 43%. The present invention also achieves the introduction of foreign genes into pollen using pear and apple pollen as carriers.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for introducing genes into pollen based on a needle-free injection propeller and low-pressure penetration, characterized in that: The method comprises: (1) washing the collected pollen to obtain washed pollen; (2) preparing a solution containing the exogenous gene, loading the solution containing the exogenous gene into a needle-free injection propeller, and aiming the nozzle of the needle-free injection propeller at the cleaned pollen to repeatedly spray; (3) subjecting the pollen treated in step (2) to low-pressure osmosis treatment, and then culturing in the dark to obtain transformed pollen containing the exogenous gene; In step (1), the method for cleaning the pollen is: (1.1) Weigh 0.01-0.03 g of pollen and place it in a 10 ml centrifuge tube; (1.2) Add 3-5 ml of BK medium to a 10 ml centrifuge tube, shake and mix, then centrifuge and discard the supernatant; (1.3) Repeat step (1.2) 2-3 times, add 5-8 ml BK medium to a 10 ml centrifuge tube, shake and mix, and then dispense the pollen solution into 1.5 ml centrifuge tubes; (1.4) Place the pollen solution in 1.5 ml centrifuge tubes on ice, and then shake the pollen solution on ice on a shaker; centrifuge in a low-temperature high-speed centrifuge, remove the supernatant, and obtain the cleaned pollen; place the cleaned pollen on ice for later use; Step (2) is as follows: (2.1) Using distilled water as a solvent, preparing a solution containing an exogenous gene; the concentration of the exogenous gene in the solution containing the exogenous gene is 10-15 ng / µL; (2.2) Adding the solution containing the exogenous gene into the needle-free injection pusher; (2.3) Aim the nozzle of the needle-free injection propeller at the pollen and spray it. Repeat the spraying 6-8 times, keeping the distance between the nozzle and the pollen at 30-40 mm. Step (3) is as follows: (3.1) Place the pollen treated in step (2) on ice, and place the pollen on ice in a vacuum drying oven for low-pressure infiltration. The conditions for low-pressure infiltration are: pressure -80 Pa, treatment time 4-6 min; (3.2) After low-pressure infiltration, let stand for 2-3 minutes, then centrifuge at 4°C, 10,000-12,000 rpm for 4-5 minutes, and discard the supernatant; (3.3) Add the pollen treated in step (3.2) to BK medium and shake to mix thoroughly. Incubate in a shaker at 28°C in the dark for 2-3 h.

2. The method for introducing genes into pollen based on a needle-free injection propeller and low-pressure penetration according to claim 1, characterized in that: In step (2), the exogenous gene is in the form of plasmid DNA.

3. The method for introducing genes into pollen based on a needle-free injection propeller and low-pressure penetration according to claim 1, characterized in that: In step (2.3), the pollen is placed at the bottom of a 1.5 mL centrifuge tube, the mouth of the centrifuge tube is vertical to the ground, and then the nozzle of the needleless injection propeller is aligned with the mouth of the centrifuge tube for injection.

4. The method for introducing genes into pollen based on a needle-free injection propeller and low-pressure penetration according to claim 1, characterized in that: The pollen is pear pollen or apple pollen.

5. The method for introducing genes into pollen based on a needle-free injection propeller and low-pressure penetration according to claim 1, characterized in that: The transformed pollen is used for pollination to cultivate seeds with foreign genes.

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