CN104818293B - A kind of method that foreign protein is quickly expressed in plant - Google Patents

Abstract

The invention belongs to a kind of methods that foreign protein is quickly expressed in plant.This method includes the following steps, 1., the preparation of vegetable material, 2., the structure of viral vectors, 3., the preparation of Agrobacterium re-suspension liquid, 4., using high-pressure spray gun systemic infection plant, 6., the quick expression and detection of GFP 5., infect the culture and observation of rear vegetable material,.A kind of new method of the present invention for easy in plant, quick expression foreign protein, utilizes this method successful expression green fluorescent protein GFP.In a large amount of Optimal Experimentals, applicant has carried out more specific and comprehensive research to the various factors for influencing gene expression, the most suitable infection condition of this method is determined, including bacterium solution re-suspension liquid（MMA OD600）Concentration is suitble to the vegetable material of this method and infects period, temperature condition of vegetable material culture etc..

Description

A method for rapid expression of foreign proteins in plants

technical field

The invention belongs to the field of biotechnology, in particular to a method for rapidly expressing foreign protein in plants.

Background technique

In recent decades, a large number of studies have shown that plant bioreactors can effectively express many foreign proteins, including human serum proteins, growth factors, antibodies, vaccines, etc. At present, medicinal proteins produced by plants abroad have been used in clinical practice, and plant bioreactors are increasingly showing their importance. There are mainly two expression systems in plant bioreactors, namely stable expression system and transient expression system. The plant transient expression system is more and more favored by people because of its fast, safe and effective advantages, and has gradually become one of the domestic research hotspots. Its practical value and development prospects have been generally affirmed.

The plant transient expression system is mediated by plant virus vectors. Agrobacterium suspensions carrying virus vectors and foreign genes are injected into plant leaves by leaf injection method, and the expression of foreign proteins in plants is realized. Commonly used infestation methods such as leaf injection method and vacuum infestation method, leaf injection method needs to be injected leaf by leaf, its disadvantage is that the manual operation is more cumbersome, time-consuming and laborious, and is not suitable for the infestation of a large number of plants, thus affecting its external source. Further promotion and application of protein production.

Using plants as bioreactors to express foreign proteins is a common method. Among them, the plant transient expression system has potential application value and broad development prospects. The main infection methods are leaf injection method and vacuum infection method, but these two methods are not suitable for the infection of a large number of plants and the large-scale production of exogenous proteins.

Contents of the invention

The object of the present invention is to provide a kind of method expressing exogenous protein rapidly in plant, and this method is to be mediated with plant virus vector, utilizes high-pressure spray gun system (model HP-G6) to carry the agrobacterium of virus vector to regenerate The suspension infects the tobacco leaves of the plant, and expresses the foreign protein through a large number of replications of the viral vector carrying the foreign gene in the plant. This method has a simple operation process, strong practicability, and is suitable for the rapid expression and scale of the foreign protein chemical production.

The object of the present invention is achieved in that the method may further comprise the steps:

①. Preparation of plant materials:

The plant material is tobacco, the seeds of tobacco (Nb) are sown in 1/2MS culture (prior art) base, put in the light incubator, and the tobacco seedlings can be grown after cultivating for 7-8 days, and the tobacco seedlings are transplanted in In the flower pot, under the temperature condition of 25±3°C, cultivated for 18-20 days under the cycle condition of 16 hours of light and 8 hours of darkness every day. Standby; in the above steps, the plant material infection period is optimized, that is, the tobacco seedlings whose seedling age is four-leaf stage, five-leaf stage and six-leaf stage are selected respectively, and 20 tobacco seedlings are selected as plant materials in each period.

②. Construction of viral vector:

First, connect the exogenous gene GFP to be expressed to the viral vector p35S-30B, then transform the plasmid p35S-30B-GFP into Agrobacterium EHA105, and culture it overnight at 28°C to obtain the agrobacterium carrying the viral vector and the exogenous gene GFP. Single colony of Bacillus EHA105-p35S-30B-GFP.

③. Preparation of Agrobacterium resuspension:

Pick the single colony EHA105-p35S-30B-GFP cultured in the above step ② and put it into 5ml LB medium, add 5ul of kanamycin at a concentration of 50mg/ml and 5ul of rifampicin at a concentration of 50mg/ml to the 5ml of LB medium , and then put it into the incubator for overnight cultivation for 16 hours. The rotation speed of the incubator is set to 200r/min, and the cultivation temperature is 28°C. After cultivation, the Agrobacterium bacterial liquid can be obtained, and then 1ml of the Agrobacterium bacterial liquid is put into 50ml LB for cultivation In the 50ml LB medium, add 50ul of kanamycin at a concentration of 50mg/ml, 50ul of rifampicin at a concentration of 50mg/ml, and then add 500ul of benzenesulfonic acid Mes (PH5.6) at a concentration of 1mol/L, Acetosyringone AS10ul of 1mol/L, and then put the above-mentioned Agrobacterium bacteria solution into the incubator for overnight cultivation, the speed of the incubator is set to 200r/min, the cultivation temperature is 28°C, overnight cultivation for 16-18h, and the overnight cultivation The bacteria solution was centrifuged at 3000r/min and 4°C for 20min to collect the bacteria, and then the bacteria were resuspended in MMA (10mmol/L Mgcl ₂ , 10mmol/LMes, 100umol/L AS) to obtain the bacteria solution resuspension; In the above steps, the Agrobacterium resuspension was optimized, and the bacterial resuspensions with concentrations (OD ₆₀₀ ) of 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6 were prepared respectively, and the bacterial resuspensions were placed before infection. Stand in the dark room for 2-3 hours before use.

④. Infect plants with high-pressure spray gun system:

Mix the bacterial liquid suspension and corundum prepared in step ③ in a ratio of 3:1, and then put it into the solution tube connected to the high-pressure spray gun (manufactured by Dongguan Anxin Hardware Technology Co., Ltd., model R-21S). Keep it at 0.1Mpa, the nozzle of the high-pressure spray gun is 15-20cm away from the leaf surface, spray the bacterial liquid resuspension vertically on the leaves of the tobacco seedlings prepared in step ①, and pay attention not to spray the spray gun too close to the plant material when spraying, so as not to damage the plants The leaves cause greater damage, and the emery forms tiny wounds on the leaves with the help of the pressure of the high-pressure spray gun, so that the virus vectors and foreign genes carried by the bacterial liquid suspension can easily infect the leaves of the plants to ensure a higher virus Infection efficiency and expression efficiency of exogenous genes.

⑤. Cultivation and observation of plant materials after infection:

Put the plant material infected by the medium and high pressure spray gun in the above step ④ into the light incubator (28°C; 16h daylight light/8h darkness) for cultivation, in a dark room, observe under the irradiation condition of ultraviolet lamp (UV), the expression of GFP The leaf phenotype is green, and the leaves that do not express GFP are red; in the above steps, the cultivation temperature of the plant material after infection was optimized, and different cultivation temperatures were selected in the light incubator: 22°C, 25°C, 28°C, The plant materials were cultured after infection at 31°C and 34°C, the expression of GFP was observed and the expression efficiency of GFP was calculated. The percentage of tobacco plants expressing GFP to the total number of infected plants was calculated.

⑥. Rapid expression and detection of GFP

Extract the total RNA of the plant expressing GFP with Trizol reagent (purchased from TAKARA company), carry out the RT-PCR detection (prior art) of GFP expression on RNA level; Western Blotting Assay (Prior Art).

The present invention has the following advantages and positive effects:

1. The present invention is a new method for expressing exogenous protein easily and rapidly in plants, and the green fluorescent protein GFP is successfully expressed by using this method. In a large number of optimization experiments, the applicant conducted a relatively comprehensive and specific study on various factors affecting gene expression, and determined the optimal infection conditions of the method, including the concentration of the bacterial liquid resuspension (MMA OD ₆₀₀ ), and the suitable method for this method. Plant material and infection period, temperature conditions for plant material cultivation, etc.

2. The present invention uses the vector p35S-30B derived from TMV virus as a medium, connects the foreign gene to the viral vector, and obtains a large amount of target protein through the propagation of the virus in plants. The target plant of the present invention is tobacco, and the target protein is green Fluorescent protein (GFP), the present invention uses the high-pressure spray gun system to infect the plant with the Agrobacterium bacterium liquid carrying the virus vector and the target gene for the first time and expresses the exogenous gene rapidly in the plant, through the rapid amplification of the virus in the plant A large amount of exogenous protein is obtained in a short period of time.

3. The main advantage of the method of the present invention is that the operation process is simple, fast, and easy to popularize and apply. As long as the Agrobacterium resuspension and carborundum are mixed according to the ratio of 3:1, they are sprayed on the plant leaves through a high-pressure spray gun system. The purpose of infecting plants with the Agrobacterium resuspension is achieved.

4. The method of the present invention has low cost and practical value; the operation process is simple and fast, saves time and effort, is convenient and effective, can realize the infection work of a large number of plants, and can be applied to exogenous proteins (such as serum proteins, growth factors, antibodies, vaccines, etc.) Rapid, mass expression and large-scale production of pharmaceutical proteins).

Description of drawings

Fig. 1 is a vector diagram of the p35S-30B-GFP expression vector of the present invention.

Fig. 2 is a bar graph of the effect of the concentration of the Agrobacterium resuspension solution of the present invention on the expression efficiency of the protein GFP.

Fig. 3 is a bar graph of the influence of seedling age of tobacco seedlings of the present invention on the expression efficiency of protein GFP.

Fig. 4 is a bar graph of the effect of the cultivation temperature of tobacco on the expression efficiency of protein GFP after infection of the present invention.

Fig. 5 is a photograph of the phenotype observation of the green fluorescent protein GFP under ultraviolet light of the present invention.

Fig. 6 is a picture of RT-PCR and Western Blot detection of the expression of the green fluorescent protein GFP of the present invention.

Detailed ways

Example one:

Different concentrations of bacterial solutions were used, and the MMA OD ₆₀₀ was 0.6, 0.8, 1.0, 1.2, and 1.4 for infection. Using the seedlings at the four-leaf stage of Nb plants (growing about 18 days after transplanting) as the host material, the efficiency of GFP expression was 65% when the MMA OD ₆₀₀ was 0.6; the efficiency of GFP expression was 75% when the MMA OD ₆₀₀ was 0.8; MMA When OD ₆₀₀ is 1.0, the efficiency of GFP expression is 90%; when MMA OD ₆₀₀ is 1.2, the efficiency of GFP expression is 82%; when MMA OD ₆₀₀ is 1.4, the efficiency of GFP expression is 60%; when MMA OD ₆₀₀ is 1.6, the efficiency of GFP expression is The efficiency is 45%. The expression efficiency is determined by the percentage of the plants with the green fluorescence phenotype in the total number of plants (20 plants for each concentration). The experiment was repeated three times, and the percentages were calculated and averaged (as shown in Figure 2). The results of the study showed that the concentration of the bacterial solution played a key role in the expression efficiency of the exogenous protein, and the optimal concentration of the bacterial solution was 1.0. Too high or too low a concentration would affect the expression efficiency of the foreign protein.

Example two:

Sow the seeds of Nb in 1/2MS medium (searched on the Internet), put them in a light incubator, and cultivate them for about a week. The seedlings were transplanted into flowerpots, and cultivated under the condition of temperature of 25±3°C, under the cycle condition of 16 hours of light and 8 hours of darkness every day, and the seedlings growing for 18-28 days were used as infection materials. With the seedlings at the four-leaf stage of Nb plants as the host material, the efficiency of GFP expression was 90%; with the seedlings at the five-leaf stage as the host material, the efficiency of GFP expression reached 75%; with the seedlings at the six-leaf stage as the host material, GFP The efficiency of expression was 55%. The expression efficiency is determined by the percentage of plants with a green fluorescence phenotype in the total number of plants. The experiment is repeated three times, and the percentages are calculated and averaged (as shown in FIG. 3 ). The research results show that the seedling age of plant materials has an important effect on the expression efficiency of foreign proteins. Under general conditions, the optimum seedling age is the four-leaf stage, and the older the seedling age, the lower the infection efficiency.

Example three:

The expression efficiency of GFP was also influenced by the incubation temperature of the plant material after infection. Put the infected tobacco seedlings in a light incubator and cultivate them for about a week. The culture is carried out under the cycle conditions of 16 hours of light and 8 hours of darkness every day. At 22°C, the efficiency of GFP expression is 65%; at 25°C, the efficiency of GFP expression is 80%; at 28°C Under the condition, the efficiency of GFP expression is 92%; under the temperature condition of 31°C, the efficiency of GFP expression is 75%; under the temperature condition of 34°C, the efficiency of GFP expression is 60%. The expression efficiency is represented by the percentage of the total number of plants with the green fluorescence phenomenon phenotype. The experiment was repeated three times, and the percentages were calculated and averaged (as shown in FIG. 4 ). The results showed that the culture temperature of plant materials after infection had a great influence on the expression efficiency of foreign proteins, and the optimum culture temperature was 28°C.

Example four:

The phenotype of GFP expression was continuously observed for 14 days and photographed and recorded. On the tobacco leaves on the 3rd day after infection, the expression of GFP can be observed under the ultraviolet light (the leaves are green to express GFP, and the normal leaves are red under ultraviolet light), the expression of GFP is strengthened on the 7th day, and the expression of GFP begins to fade on the 10th day (as shown in the figure 5).

Example five:

The total RNA and total soluble protein of the plants expressing GFP were extracted, and the detection of RNA level (semi-quantitative RT-PCR) was carried out. According to observations under UV conditions, the protein GFP mainly accumulated in stems and leaves. We extracted total RNA from tobacco leaves 3, 5, and 7 days after infection as a template, and performed RT-PCR reactions (as shown in FIG. 6A ).

From the total soluble protein extracted from tobacco leaves 7 days after infection, the expression level of GFP protein was detected by Western Blot at the protein level (as shown in FIG. 6B ). Quantitative analysis of GFP protein, that is, using the method established by Bradford to combine Coomassie Brilliant Blue G-250 with protein, and measuring its concentration under an ultraviolet spectrophotometer, the analysis results show that the expression of GFP accounts for about 5.6% of the total soluble protein in plant leaves. %.

Plasmid p35S-30B-GFP (as shown in Figure 1) was provided by the National Key Laboratory of Plant Genome, Institute of Microbiology, Chinese Academy of Sciences.

Claims (1)

1. A kind of 1. method that foreign protein is quickly expressed in plant, it is characterised in that：This method includes the following steps：

   1., the preparation of vegetable material：

   Vegetable material is tobacco, and the seed of tobacco is seeded in 1/2MS culture mediums, is put in illumination box, and culture is 7-8 days long Go out tobacco seedling, by the small transplantation of seedlings of tobacco in flowerpot, under 25 ± 3 DEG C of temperature condition, daily illumination 16h and dark 8h's It is cultivated 18-20 days under cycling condition, the seedling age of tobacco seedling is spare as vegetable material in four leaf stage, five leaf phases, six leaf phases； Infecting period in above-mentioned steps to vegetable material is optimized, i.e., selects seedling age respectively as four leaf stage, five leaf phases and six leaf phases Tobacco seedling, each period selects 20 plants of tobacco seedlings as vegetable material；

   2., the structure of viral vectors：

   The foreign gene GFP that will first express is connected on viral vectors p35S-30B, then plasmid p35S-30B-GFP is converted Agrobacterium EHA105 is incubated overnight under the conditions of 28 DEG C, obtains the Agrobacterium EHA105- for carrying viral vectors and foreign gene GFP The single bacterium colony of p35S-30B-GFP；

   3., the preparation of Agrobacterium re-suspension liquid：

   The single bacterium colony EHA105-p35S-30B-GFP of picking above-mentioned steps 2. middle culture is put into 5ml LB culture mediums, 5ml LB The rifampin 5ul of kanamycins 5ul, 50mg/ml of a concentration of 50mg/ml are added in culture medium, is then placed in incubator progress 16h is incubated overnight, the rotating speed of incubator is set as 200r/min, and cultivation temperature is 28 DEG C, Agrobacterium bacterium solution is obtained after culture, so The Agrobacterium bacterium solution of 1ml is put into 50ml LB culture mediums afterwards, in 50ml LB culture mediums plus the card of a concentration of 50mg/ml that is mould The rifampin 50ul of plain 50ul, 50mg/ml, then add the benzene sulfonic acid Mes500ul, a concentration of of a concentration of 1mol/L PH5.6 The acetosyringone AS10ul of 1mol/L, then above-mentioned Agrobacterium bacterium solution is put into incubator and is incubated overnight, the rotating speed of incubator It is set as 200r/min, cultivation temperature is 28 DEG C, is incubated overnight 16-18h, by the bacterium solution being incubated overnight in 3000r/min, 4 DEG C Under the conditions of centrifugation 20min collect thalline, then thalline is resuspended in 10mmol/LMgcl 2,10mmol/LMes, 100umol/ L AS obtain bacterium solution re-suspension liquid；Agrobacterium re-suspension liquid is optimized in above-mentioned steps, compound concentration OD 600 is respectively Bacterium solution re-suspension liquid before infecting is placed on darkroom and stands 2-3h standby by 0.6,0.8,1.0,1.2,1.4 and 1.6 bacterium solution re-suspension liquid With；

   4., utilize high-pressure spray gun systemic infection plant：

   By step 3. in ready bacterium solution re-suspension liquid and diamond dust according to 3:1 ratio mixing, is then placed in and high-pressure spray gun In connected solution conduit, pressure is maintained at 0.1Mpa, and the nozzle of high-pressure spray gun hangs down bacterium solution re-suspension liquid apart from blade face 15-20cm Straight to be injected in step 1. on the blade of the tobacco seedling of middle preparation, spray gun should not be too near apart from vegetable material when paying attention to spraying, in order to avoid Larger injury is caused to plant leaf blade, the diamond dust forms small wound by the pressure of high-pressure spray gun on blade, The viral vectors of bacterium solution re-suspension liquid carrying and foreign gene is made easily to infect the blade of plant, to ensure that higher virus infection is imitated The expression efficiency of rate and foreign gene；

   5., infect the culture and observation of rear vegetable material：

   The above-mentioned steps vegetable material that 4. mesohigh spray gun infects is put into illumination box to cultivate, in black room, ultraviolet It is observed under lamp UV irradiation conditions, for the leaf morphology of GFP expression for green is presented, the blade presentation for not expressing GFP is red；On It states in step and the cultivation temperature for infecting rear vegetable material is optimized, select different culture temperature respectively in illumination box 22 DEG C, 25 DEG C, 28 DEG C, 31 DEG C and 34 DEG C of degree is cultivated after carrying out the infecting of vegetable materials, and is observed the expression of GFP and is calculated GFP's The tobacco plant of expression efficiency expression GFP accounts for the percentage for always infecting strain number；

   6., the quick expression and detection of GFP

   The total serum IgE of the plant of expression GFP is extracted with Trizol reagents, the RT-PCR detections of GFP expression are carried out on rna level； Plant total soluble protein is extracted using PBS extracting solutions, carries out the Western Blotting detections of GFP expression.