A method for improving the synthesis efficiency of exogenous products in Nicotiana benthamiana and its application

Abstract

The present invention discloses a method for improving the synthesis efficiency of exogenous products in Nicotiana benthamiana and its application, belonging to the technical field of plant bioengineering. The method is to cultivate Nicotiana benthamiana seedlings in a light environment with a ratio of red light to far-infrared light. The method of the present invention has the advantages of improving the uniformity of chassis tobacco, the synthesis efficiency of heterologous products and the stability of output, and has great economic value when applied to the production of heterologous products, and has higher accuracy and sensitivity when applied to gene function verification.

Description

Technical Field

[0001] The present invention relates to a method for improving the synthesis efficiency of exogenous products in Nicotiana benthamiana and its application, belonging to the technical field of plant bioengineering. Background Art

[0002] Synthetic biology realizes the heterologous synthesis of various types of substances through various chassis such as plants, microorganisms, and cells. Among them, the plant chassis has the advantages of rich precursors, no need for sugar sources or carbon sources, wide applicability, and high codon adaptability for plant-derived genes. Nicotiana benthamiana is a commonly used chassis plant for heterologous synthesis. The transient transformation synthesis system mediated by Agrobacterium and based on tobacco as the chassis has the advantages of low planting cost, short growth cycle, high genetic transformation efficiency, etc., and can ensure the correct post-translational modification of proteins for the synthesis of natural products. It is widely used in metabolic pathway analysis and heterologous reconstruction. Improving the synthesis efficiency of the transient transformation synthesis system of the plant chassis is an important research content in heterologous production of synthetic biology.

[0003] At present, the optimization research on plant synthesis chassis mainly focuses on the transformation of plasmid elements and the systematic optimization of key catalytic enzymes in the product synthesis pathway. However, as the synthesis workshop for heterologous products, the growth state of plants will affect the heterologous synthesis process. The light environment has been widely reported to regulate plant morphogenesis and its endogenous metabolic processes, and systematically regulate the physiological growth of plants. Currently, there are two types of cultivation methods for Agrobacterium-mediated transient transformation of Nicotiana benthamiana, namely natural environment cultivation and artificial controllable environment cultivation. Among them, in natural environment cultivation, the cultivation environment of plants is not controlled, and the growth states of chassis tobacco in different batches are affected by the natural environment. The instability of the natural environment leads to poor uniformity of plant growth and unstable synthesis efficiency of heterologous products. Artificial controllable environment cultivation is generally growth chamber cultivation, using fluorescent lamps or new full-spectrum LEDs. The cultivation environment in the growth chamber improves the uniformity of plant growth, but does not specifically improve the synthesis efficiency of heterologous products. Therefore, the chassis plants cultivated by the existing cultivation methods have defects such as low synthesis efficiency and poor stability between batches, and there is an urgent need to develop a stable and efficient cultivation technology for heterologous synthesis plant chassis.

[0004] In the prior art, the patent application with publication number CN112522306A discloses a method for significantly improving the transient transformation efficiency of peach leaves by weak light treatment. After weak light cultivation of peach seedlings, transient transformation is carried out to improve the transformation efficiency of peach leaves. However, peach plants are not commonly used plant chassis in synthetic engineering, and weak light treatment of peach plants will also affect the subsequent growth and biomass accumulation of the plants, and is not conducive to the synthesis of heterologous products while improving the transgenic efficiency, and cannot effectively improve the synthesis efficiency of heterologous products. Summary of the Invention

[0005] The object of the present invention is to overcome the disadvantages that the chassis plants cultivated in the natural light environment have poor batch quality due to the change of the light environment, and thus the good production of products cannot be guaranteed, and the deficiency that the optimization of the light quality environment in the current artificial light environment cultivation still takes plant growth as the premise, resulting in low heterologous synthesis efficiency of the plant chassis. The present invention provides a method for improving the synthesis efficiency of exogenous products in Nicotiana benthamiana and its application.

[0006] The first aspect of the present invention provides a method for improving the synthesis efficiency of exogenous products in Nicotiana benthamiana, and the method is to cultivate Nicotiana benthamiana seedlings in a light environment with a red light ratio to far-infrared light.

[0007] In one embodiment of the present invention, the method includes the following steps:

[0008] S1. Sow and raise seedlings of Nicotiana benthamiana. The seeds of Nicotiana benthamiana can be sown in a seedling rock wool plug tray and raised under ordinary fluorescent lamps. The seedling raising conditions adopt the usual cultivation conditions for tobacco.

[0009] In one embodiment of the present invention, the light intensity in S1 is 180±15 μmol m -2 s -1 , the light cycle is 16 hours of light, and the temperature is 19-23 °C. Preferably, the seedling raising time is 14 days.

[0010] S2. Cultivate the seedlings cultivated in S1 in a light environment of red light and far-red light. Preferably, the ratio of red light to far-red light is 7:3, and the total light intensity is 180±15 μmol m -2 s -1 . Preferably, the cultivation time is 7 days.

[0011] In one embodiment of the present invention, the wavelength of the red light is 600-700 nm, and the wavelength of the far-red light is 700-750 nm.

[0012] In one embodiment of the present invention, the light cycle in S2 is 16 hours of light.

[0013] S3. Infect the leaves of the plants cultivated in S2 with Agrobacterium carrying a plasmid encoding a heterologous target protein gene, and perform dark adaptation. In one embodiment of the present invention, the dark adaptation time is 24 hours.

[0014] In one embodiment of the present invention, the expression of the heterologous target gene includes encoding a target protein or encoding a protein that catalyzes a target product.

[0015] S4. Transfer the plants after dark adaptation in S3 back to the light environment with a red light ratio to far-red light to continue growing. After 3-5 days, the content of the protein encoded by the transiently transformed heterologous gene or the product catalyzed by its encoded protein can be detected.

[0016] The second aspect of the present invention provides an application of the above method for improving the synthesis efficiency of exogenous products in Nicotiana benthamiana in the biosynthesis industry, agriculture, and the expression of exogenous proteins in bioreactors for industrial production.

[0017] The third aspect of the present invention provides an application of the above method for improving the synthesis efficiency of exogenous products in Nicotiana benthamiana in gene function verification.

[0018] The beneficial effects of the present invention are as follows:

[0019] By cultivating Nicotiana benthamiana with a combination of red light and far-red light, it is beneficial to improve the synthesis efficiency of heterologous products in the leaves of the chassis Nicotiana benthamiana. At the same time, the chassis uniformity and the stability of heterologous product synthesis output are also significantly improved, with great economic benefits. In addition, as a widely used heterologous expression tool for gene function verification, the verification effect of the Nicotiana benthamiana heterologous expression system is affected by the heterologous product expression efficiency. By using this method to establish a heterologous expression system, the efficient cultivation light quality is beneficial to improving the product synthesis efficiency, and thus significantly improves the accuracy and sensitivity of gene function verification. The method of the present invention optimizes the cultivation environment of the chassis plant from the perspective of light quality, provides a new direction for improving the heterologous synthesis efficiency of the chassis plant through agricultural environment regulation technology, and provides development potential for improving the heterologous synthesis efficiency of the chassis plant. The mutual optimization of the chassis plant and the cultivation environment will become an important research content in the development of synthetic biology plant chassis, and has great market and economic value in both synthetic biology and agricultural environmental engineering. Description of the Drawings

[0020] Figure 1 The above-ground parts of the cultivated tobacco in each group with different light quality treatments in Example 1 of the present invention.

[0021] Figure 2 The leaf areas of the tobacco in each group with different light quality treatments in Example 1 of the present invention.

[0022] Figure 3 The dry weights of the above-ground parts of the tobacco in each group with different light quality treatments in Example 1 of the present invention.

[0023] Figure 4 The fluorescence excited by the GFP protein in the leaves of the tobacco in each group with different light quality treatments in Example 1 of the present invention.

[0024] Figure 5 The relative expression levels of the GFP protein in the tobacco in each group with different light quality treatments in Example 1 of the present invention.

[0025] Figure 6 The yields of taxadiene per unit leaf weight of the tobacco in each group with different light quality treatments in Example 2 of the present invention.

[0026] Figure 7The yield of taxadiene per individual tobacco plant under different light quality treatments in Example 2 of the present invention.

[0027] Figure 8 The mass spectrometry diagram of taxadiene in Example 2 of the present invention. Detailed implementation manners

[0028] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention. For those conditions not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments for which the manufacturers are not specified are all conventional products that can be obtained through commercial purchase.

[0029] Example 1 Method for improving the synthesis of exogenous product GFP protein in chassis tobacco

[0030] This example includes the following steps:

[0031] 1. Construction of pEAQ-GFP plasmid and Agrobacterium transformation: Clone the GFP sequence and construct it onto the commercialized pEAQ-HT vector to obtain the pEAQ-GFP plasmid. Heat shock transform JM109 competent cells, culture overnight at 37 °C on a solid medium containing kanamycin antibiotic (50 μg / mL), pick monoclonal colonies, and perform positive detection using PCR and agarose gel electrophoresis. The plasmid with correct sequencing is transformed into Agrobacterium tumefaciens GV3101 by heat shock, and cultured at 28 °C for 48 hours on a medium containing kanamycin (50 μg / mL) and rifampicin (50 μg / mL) antibiotics. Pick monoclonal colonies for positive detection and use them for injecting tobacco leaves. The bacterial solution for injection is cultured overnight in LB and then centrifuged to collect the cells. The cells are resuspended in the resuspension solution to an OD600 of 0.7 and left to stand at 28 °C for 2 hours to obtain an Agrobacterium bacterial solution carrying the plasmid encoding the heterologous target gene.

[0032] 2. Cultivation of Nicotiana benthamiana and synthesis of exogenous products:

[0033] S1. Sow Nicotiana benthamiana seeds in a seedling rockwool plug tray and carry out seedling cultivation under white light LED lamps for 14 days. The light intensity is 180 ± 15 μmol m -2 s -1 , the photoperiod is 16 hours, the day and night temperature is 22 °C / 20 °C. After the cotyledons unfold, transfer the tobacco seedlings together with the rockwool plugs to a rockwool block (7.5 cm × 7.5 cm × 6 cm, Gordan, the Netherlands), and use Hoagland formula nutrient solution (pH = 5.5, EC = 2.0 dS m-1 ), and keep the rock wool blocks moist during cultivation.

[0034] S2. Divide the seedlings of Nicotiana benthamiana into five groups, set five different light quality treatments, and the spectral compositions of the five light qualities are shown in Table 1. Except for UVB, the treatment time of other light qualities is 16 hours. The UVB lamp is Philips TL20W / 01, with a peak wavelength of 311 nm, and the leaf-level UVB intensity is about 1.5 μmol m -2 s -1 , and the treatment time is from 10:00 to 14:00. Treat with light according to the above method for 7 days respectively.

[0035] Table 1 Spectral compositions of light qualities for each group

[0036]

[0037] In Table 1, the wavelength of middle ultraviolet light is 280 - 315 nm, the wavelength of near ultraviolet and blue light is 315 - 500 nm, the wavelength of green light is 500 - 600 nm, the wavelength of red light is 600 - 700 nm, and the wavelength of far red light is 700 - 750 nm.

[0038] S3. Infect the leaves of the plants after the light treatment in S2 with the prepared Agrobacterium liquid. The treatment method is injection. Draw the liquid with a syringe, and press the reverse plate of the syringe with the thumb to inject the liquid from the lower epidermis of the leaf into the leaves of Nicotiana tabacum on the chassis, and perform dark adaptation for 24 hours.

[0039] S4. Transfer the plants after dark adaptation in S3 back to the light environment of each light quality condition and continue to grow. After 3 days, collect the GFP protein leaf samples and detect the expression differences of the target product in the leaves under different light quality conditions.

[0040] Before injecting Agrobacterium in S3, measure the dry weight of the above-ground part and the leaf area of tobacco under different light quality treatments. The above-ground parts of tobacco under different light quality treatments are as Figure 1 shown, the measurement results of the leaf area are as Figure 2 shown, and the measurement results of the dry weight are as Figure 3 shown. The growth differences of Nicotiana benthamiana under different light quality treatment conditions are relatively large. Among them, the leaf area of tobacco under LED-White and LED-Blue treatments is larger, and the dry matter accumulation is also more.

[0041] Use a gel imager and a laser confocal microscope to take leaf fluorescence of the GFP protein leaf samples collected in S4. The results are as Figure 4 shown. The fluorescence brightness of GFP protein in the leaves of tobacco grown under the LED-Red environmental conditions with the ratio of red light and far red light in this example is significantly higher than that of the leaves in other light quality treatment groups.

[0042] The GFP protein content in the chassis tobacco leaves of each group was determined by Western blot: The GFP protein leaf samples collected in S4 were ground into powder after being frozen in liquid nitrogen, and the chassis tobacco cells were lysed by adding RIPA cell lysate containing PMSF. After measuring the soluble protein concentration by the BCA method, the extract was adjusted to the same concentration with the lysate. Protein samples were mixed with protein loading buffer, loaded onto the gel after boiling water bath and centrifugation, and then the protein gel was transferred to a membrane. The PVDF membrane was blocked with skim milk, incubated with the primary antibody against GFP protein and then combined with the secondary antibody, developed and photographed. The immunoblot area was analyzed using ImageJ (v.1.52a), and the relative expression level of the target protein was calculated. The results are as Figure 5 shown. The analysis results showed that the GFP protein content in the tobacco leaves grown under the LED-Red environmental conditions with the red light and far-red light ratio in this example was 1.32 times that under the treatment of ordinary fluorescent lamps (Flu-White), and 1.61 times that under the LED-White conditions.

[0043] Example 2 Method for Improving the Heterologous Protein Synthesis Efficiency of Catalytic Production of Taxadiene

[0044] This example includes the following steps:

[0045] 1. Construction of pEAQ-TS plasmid and Agrobacterium transformation: RNA of Taxus wallichiana was extracted to prepare cDNA, the TS sequence of taxadiene synthase was cloned, and TS was constructed into the linearized pEAQ-HT vector prepared by using the restriction enzymes BshTI and XhoI through homologous recombination to obtain the pEAQ-TS fusion expression plasmid. JM109 competent cells were transformed by heat shock and cultured overnight at 37 °C on a solid medium containing kanamycin (50 μg / mL). Single colonies were picked, and positive detection was carried out by PCR and agarose gel electrophoresis. The plasmid with correct sequencing was transformed into Agrobacterium tumefaciens GV3101 by heat shock and cultured at 28 °C for 48 hours on a medium containing kanamycin (50 μg / mL) and rifampicin (50 μg / mL) antibiotics. Single colonies were picked for positive detection and used for injecting tobacco leaves. The bacterial solution for injection was cultured overnight in LB and then centrifuged to collect the cells. The cells were resuspended in the resuspension solution to OD600 = 0.7 and allowed to stand at 28 °C for 2 hours to obtain the Agrobacterium bacterial solution carrying the plasmid encoding the heterologous target gene.

[0046] 2. Cultivation of Nicotiana benthamiana and synthesis of exogenous products:

[0047] S1. Sow Nicotiana benthamiana seeds in a seedling rockwool plug tray and grow seedlings under white light LED lamps for 14 days, with a light intensity of 180 ± 15 μmol m -2 s -1, with a photoperiod of 16 hours, day / night temperature of 22°C / 20°C. After the cotyledons unfolded, tobacco seedlings together with rockwool plugs were transferred to rockwool blocks (7.5 cm × 7.5 cm × 6 cm, Gordan, the Netherlands), and Hoagland formula nutrient solution (pH = 5.5, EC = 2.0 dS m -1 ) was used, and the rockwool blocks were kept moist during cultivation.

[0048] S2. The seedlings of Nicotiana benthamiana were divided into five groups, and five different light quality treatments were set. The spectral compositions of the five light qualities are shown in Table 1. Except for UVB, the treatment time of other light qualities was 16 hours. The UVB lamp was Philips TL20W / 01, with a peak wavelength of 311 nm, and the leaf-level UVB intensity was about 1.5 μmol m -2 s -1 , and the treatment time was from 10:00 to 14:00. The light treatment was carried out for 7 days according to the above method respectively.

[0049] S3. The leaves of the plants after the light treatment in S2 were infected with the Agrobacterium bacterial liquid prepared in the foregoing of this example. The treatment method was injection. The bacterial liquid was sucked with a syringe, and the liquid was injected from the lower epidermis of the leaf into the bottom tobacco leaf by pressing the reverse plate of the syringe with the thumb, and dark adaptation was carried out for 24 hours.

[0050] S4. The plants after dark adaptation in S3 were transferred back to the light environment under the light quality conditions of each group to continue growing. After 5 days, the taxadiene leaf samples were collected, and the expression differences of the target product in the leaves under different light quality conditions were detected.

[0051] The taxadiene synthase content in the taxadiene leaf samples of each group of chassis tobacco was determined by GC-MS: The taxadiene leaf samples collected in S4 were soaked in methyl tert-butyl ether for 24 hours, then vortexed and centrifuged to aspirate the supernatant. After drying with nitrogen, it was redissolved in MTBE and fixed to a volume of 1 mL for GC-MS injection. The injection volume was 2 μL. The GC-MS conditions were as follows: chromatographic column: DB-5Ul, carrier gas: He 1.2 mL / min, column oven temperature: 100°C, 1 min, 100 - 175°C, 15°C / min, 175 - 220°C, 4°C / min, 220 - 290°C, 20°C / min, injector temperature: 270°C, detector temperature: 250°C. The taxadiene content in the sample was calculated using the corresponding peak area of the taxadiene standard product. As Figure 6 、 Figure 7 and Figure 8As shown, the taxadiene yield per unit leaf weight of tobacco leaves grown under the LED-Red environmental conditions with the red light and far-red light ratio in this embodiment is 84.1% higher than that under the treatment of ordinary fluorescent lamps (Flu-White). Considering the different leaf weights, the taxadiene yield per plant was calculated. The taxadiene yield in tobacco leaves grown under the LED-Red environmental conditions with the red light and far-red light ratio in this embodiment is 46.5% higher than that under the treatment of fluorescent lamps (Flu-White).

[0052] The above are only the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be changed within the scope of the concept described herein through the above teachings or the technology or knowledge in related fields. Any changes and modifications made by those skilled in the art without departing from the spirit and scope of the present invention shall fall within the protection scope of the appended claims of the present invention.

Claims

1. A method for improving the synthesis efficiency of exogenous proteins in Nicotiana benthamiana, characterized in that: The method is to cultivate Nicotiana benthamiana seedlings in a light environment with red light and far-red light, and the ratio of red light to far-red light is 7:3, and the total light intensity is 180±15 µmolm -2 s -1 , the wavelength of the red light is 600-700 nm, and the wavelength of the far-red light is 700-750 nm.

2. The method for improving the synthesis efficiency of exogenous proteins in Nicotiana benthamiana according to claim 1, wherein: It includes the following steps: S1. Sow and raise seedlings of Nicotiana benthamiana; S2. Cultivate the seedlings obtained in S1 in a light environment of red light and far-red light; S3. Infect the leaves of the plants cultivated in S2 with Agrobacterium tumefaciens carrying a plasmid encoding a heterologous target gene, and perform dark adaptation; S4. Transfer the plants after dark adaptation in S3 back to the light environment with a red light ratio of far-red light and continue to grow.

3. The method for improving the synthesis efficiency of exogenous proteins in Nicotiana benthamiana according to claim 2, wherein: The seedling raising time in S1 is 14 days, and the cultivation time in S2 is 7 days.

4. The method for improving the synthesis efficiency of exogenous proteins in Nicotiana benthamiana according to claim 2, wherein: The light intensity of S1 is 180 ± 15 µmol m -2 s -1 , the photoperiod is 16 hours of light, and the temperature is 19 - 23 °C.

5. The method for improving the synthesis efficiency of exogenous proteins in Nicotiana benthamiana according to claim 2, characterized in that: The photoperiod in S2 is 16 hours of light.

6. The method for improving the synthesis efficiency of exogenous proteins in Nicotiana benthamiana according to claim 2, wherein: The expression of the heterologous target gene includes encoding a target protein or a protein catalyzing a target product.

7. The method for improving the synthesis efficiency of exogenous proteins in Nicotiana benthamiana according to claim 2, wherein: The dark adaptation time is 24 hours.

8. Application of the method for improving the synthesis efficiency of exogenous proteins in Nicotiana benthamiana according to any one of claims 1-7 in the expression of exogenous proteins in bioreactors in biosynthesis industries, agriculture and industrial production.

9. Application of the method for improving the synthesis efficiency of exogenous proteins in Nicotiana benthamiana according to any one of claims 1-7 in gene function verification.

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