CN111424051B - Method for establishing transient expression system by inducing local tissue expansion of seedlings - Google Patents
Method for establishing transient expression system by inducing local tissue expansion of seedlings Download PDFInfo
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Abstract
The invention provides a method for establishing a transient expression system by inducing local tissue expansion of seedlings, and relates to the technical field of biology. The method comprises the following steps: and (3) recombinant agrobacterium: extracting escherichia coli plasmid, connecting a promoter to the plasmid to obtain a recombinant expression vector, transforming agrobacterium with the recombinant expression vector, selecting positive transformed agrobacterium, and performing amplification culture; activating agrobacterium: culturing positive transformation agrobacterium with a bacterium suspension culture medium to obtain agrobacterium liquid; treating seeds: disinfecting the seeds, inoculating the seeds to a solid culture medium, and culturing until local tissues of seedlings expand; and (3) mediating conversion: and soaking the seedling with local tissue expansion by using agrobacterium liquid, transferring the seedling to a co-culture medium for culture, and inducing agrobacterium to invade the local tissue expansion of the seedling to form an instantaneous expression system. The transient expression system established by the method is beneficial to simply and quickly testing the activity of the seed storage protein promoter, and the cost for purifying the target protein is reduced.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for establishing a transient expression system based on seed germination induction seedling local tissue expansion.
Background
The plant can be used as a bioreactor to produce medicinal protein and other proteins with important values, has the advantages of safety and low price, and the plant bioreactor can greatly improve the economic benefit of agricultural production. Transient expression is an important means for rapidly researching the expression of different promoters in specific genes, compared with the traditional transgenic technology, the transformation period is long, the efficiency is low and the instability is high, and exogenous DNA does not need to be integrated on a host cell chromosome, so that the transformation is easier, faster and higher in transformation efficiency. The plant cell can perform expression and efficient transmission of a plurality of genes within a few days, and particularly the development of synthetic biology ensures that transient expression has greater application prospect.
At present, the conventional plant transient expression system utilizes agrobacterium to inject tobacco leaves for transient expression, so as to test the function of a promoter or express protein, and also utilizes plant protoplast for transient expression, or utilizes plant explants to induce callus for transient expression. The function of the promoter is detected most conveniently and quickly by injecting the agrobacterium into the tobacco leaves for transient expression. However, it is difficult to purify the target protein or drug from tobacco leaves, and there are a lot of inorganic and organic substances in tobacco leaves, and the process of separating and purifying the expressed protein is complicated, and the cost is very high. Although the cost of separating and purifying the protein from the plant callus and the protoplast is not high, the process is very complicated.
Disclosure of Invention
Therefore, there is a need to provide a method for establishing a transient expression system by inducing local tissue expansion of seedlings, which induces local expanded tissue during seed germination, wherein the cell structure of the expanded tissue is loose, which is beneficial to agrobacterium transfection, and the content of inorganic matters and organic matters is very low, which is beneficial to simply and rapidly testing the activity of a seed storage protein promoter, and reducing the cost of purifying a target protein.
A method for inducing local tissue expansion of seedlings to establish a transient expression system comprises the following steps:
and (3) recombinant agrobacterium: extracting escherichia coli plasmid, connecting a promoter to the plasmid to obtain a recombinant expression vector, transforming agrobacterium with the recombinant expression vector, selecting positive transformed agrobacterium, and performing amplification culture;
activating agrobacterium: culturing positive transformation agrobacterium with a bacterium suspension culture medium to obtain agrobacterium liquid;
treating seeds: disinfecting the seeds, inoculating the seeds to a solid culture medium, and culturing until local tissues of seedlings expand;
and (3) mediating conversion: and soaking the seedling with local tissue expansion by using agrobacterium liquid, transferring the seedling to a co-culture medium for culture, and inducing agrobacterium to invade the local tissue expansion of the seedling to form an instantaneous expression system.
According to the method, the solid culture medium is used for inducing the local expansion of the seedling hypocotyl and radicle in a short time, the cell structure of the expanded tissue is loose, and the expanded tissue is easy to be infected by the activated agrobacterium so as to be transformed instantaneously, so that an instantaneous expression system is formed; the hypocotyl and the root of the seedling are delicate and tender, and the content of inorganic and organic components in the seedling is very low, so that the subsequent separation and purification of the needed protein are facilitated, and the extraction cost is reduced; and because the impurity content in the root and hypocotyl of the seedling is low, the function of the promoter can be easily observed after dyeing treatment, and the activity of the seed storage protein promoter can be simply and quickly detected. The transient expression system obtained by the method can be used as a bioreactor to rapidly produce large quantities of genetic engineering drugs, and overcomes the problem that the activity of the genetic engineering drugs is influenced by expressing eukaryotic gene glycosylation by using escherichia coli.
In one embodiment, the Agrobacterium is selected from the EHA105 strain. The EHA105 strain has strong toxicity and good infection effect.
In one embodiment, the seed is canola seed. Rape seeds are rich in oleosin.
In one embodiment, the bacterial suspension culture medium is MS culture medium comprising 0.5 +/-0.1 mg/L6-BA, 0.1 +/-0.05 mg/L NAA, 100 +/-1 mu mol/L acetosyringone and 30 +/-1 g/L sucrose.
In one embodiment, the solid culture medium is MS culture medium containing 0.5 + -0.1 mg/L6-BA, 0.1 + -0.05 mg/L NAA, 30 + -1 g/L sucrose and 7-8g/L agar. The solid culture medium can make seeds generate local tissue expansion in a short time, and is beneficial to agrobacterium infection.
In one embodiment, the co-culture medium is MS culture medium comprising 0.5 + -0.1 mg/L6-BA, 0.1 + -0.05 mg/L NAA, 100 + -1 μmol/L acetosyringone, 30 + -1 g/L sucrose and 8 + -1 g/L agar.
In one embodiment, the step of recombining the agrobacterium is specifically:
extracting a escherichia coli PBI101 plasmid by adopting an alkaline lysis method, carrying out enzyme digestion on an Oleosin promoter and the PBI101 plasmid by using Hind III and BamH I, respectively recovering an Oleosin promoter fragment and a PBI101 vector fragment, and connecting the Oleosin promoter fragment and the PBI101 vector fragment by using T4 ligase to construct a recombinant expression vector;
transforming the recombinant expression vector to agrobacterium by a heat shock method, screening a transformation product by using an LB culture medium containing 50 +/-1 mg/L kanamycin and 20-30mg/L rifampicin, selecting positive transformation agrobacterium, and carrying out expanded culture.
In one embodiment, the step of activating agrobacterium is specifically: and adding the positive transformed agrobacterium into a bacterium suspension culture medium, and performing shaking culture at the rotation speed of 160-200rpm until the concentration OD600 of the agrobacterium liquid is 0.4-0.6.
In one embodiment, the step of treating seeds specifically comprises: with 0.10. + -. 0.05 wt% HgCl2Sterilizing the seeds for 8-10min, washing with sterile water for 3-4 times, inoculating the seeds to a solid culture medium, culturing at 22-25 deg.C for 5-10 days, and allowing the seeds to germinate and grow seedlings with local tissue expansion.
In one embodiment, the step of mediating the transformation is specifically: soaking the seedlings with agrobacterium liquid for 10-30min, and transferring to a co-culture medium for dark culture for 3-5 days to form an instantaneous expression system.
The invention also provides a plant transient expression system prepared by the method. The instantaneous expression system can be used as a bioreactor to rapidly produce a large amount of genetic engineering drugs, and overcomes the problem that the activity of the genetic engineering drugs is influenced by expressing eukaryotic gene glycosylation by using escherichia coli.
Compared with the prior art, the invention has the following beneficial effects:
according to the method, the solid culture medium is used for inducing the local expansion of the hypocotyl and radicle of the seedling in a short time, the expanded tissue has a loose cell structure, and is easy to infect by the activated agrobacterium so as to be transformed instantaneously, so that an instantaneous expression system is formed; the hypocotyl and the root of the seedling are delicate and tender, and the content of inorganic and organic components in the seedling is very low, so that the subsequent separation and purification of the needed protein are facilitated, and the extraction cost is reduced; moreover, the impurity content in the root and hypocotyl of the seedling is low, so that the function of the promoter can be easily observed after dyeing treatment, and the activity of the seed storage protein promoter can be simply and quickly detected. The transient expression system obtained by the method can be used as a bioreactor to rapidly produce large quantities of genetic engineering drugs, and overcomes the problem that the activity of the genetic engineering drugs is influenced by expressing eukaryotic gene glycosylation by using escherichia coli.
Drawings
FIG. 1 is a schematic diagram of the construction of the recombinant expression vector in example 1;
FIG. 2 is a picture of GUS staining of the Oleosin promoter in rape seedlings in example 1;
wherein, a is a blank control group, and b is an example staining group.
Detailed Description
To facilitate an understanding of the invention, a more complete description of the invention will be given below in terms of preferred embodiments. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Example 1
Firstly, establishing a transient expression system
1 test material
Brassica napus (Brassica napus) from the institute of oil crops, Chinese academy of sciences.
2 strains and vectors
Agrobacterium tumefaciens (Agrobacterium tumefaciens) strain is EHA105, and the resistance marker is rifampicin (Rif); the plant expression vector was pBI101 and the resistance marker was kanamycin (kan). Was maintained in the biotechnology focus laboratory of secondary happy academy of agricultural engineering.
3 reagent
Restriction enzymes, T4 DNA ligase, and DNA gel recovery kits were purchased from TaKaRa. The antibiotic is Sigma product, and other biochemical reagents are domestic analytical pure.
4 construction of recombinant expression vectors
The correctly sequenced Escherichia coli cells were cultured in an expanded scale, and plasmids were extracted by the alkaline lysis method according to the molecular cloning Instructions. The promoter and the PBI101 plasmid were digested with Hind III and BamH I, electrophoresed on 1.0% agarose gel, and the promoter fragment and the PBI101 vector fragment were recovered separately with a gel recovery kit. As shown in FIG. 1, the two fragments were ligated by using T4 ligase to construct a recombinant expression vector.
5 selection and transformation of recombinant plasmids
Transforming the constructed recombinant expression vector into agrobacterium by a heat shock method, and coating the transformed product on a medium containing 50mg L-1Kanamycin (Kan) and 25mg L-1Rifampicin (Rif) was cultured in LB medium plate at 28 ℃ for 48 hours. And selecting positive transformation agrobacterium, and carrying out amplification culture for 36h for later use.
6 Agrobacterium mediated transformation of oilseed rape seedlings
With 0.1% HgCl2Sterilizing rape seed for 8-10min, passing through sterile water for 3-4 times, and inoculating to solid culture medium (MS culture medium containing 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose and 7g/L agar). Culturing at 22-25 deg.C for 5-10 days.
The Agrobacterium was cultured with a bacterial suspension medium (MS medium containing 0.5 mg/L6-BA, 0.1mg/L NAA, 100. mu. mol/L acetosyringone and 30g/L sucrose) at 28 ℃ with shaking to a final concentration OD600 of 0.4-0.6 for use.
Randomly dividing the cultured rape seedlings into 2 groups, wherein one group of seedlings is set as a blank control, and the other group of seedlings is soaked in the agrobacterium liquid for 10-30 min. Transferring the seedlings to a co-culture medium (the co-culture medium is an MS culture medium containing 0.5 mg/L6-BA, 0.1mg/L NAA, 100 mu mol/L acetosyringone, 30g/L sucrose and 8g/L agar) and culturing in dark for 3-5 d.
7GUS staining and transient expression
Rape seedlings were transferred to a fixing solution containing 50mM PBS, 1% (v/v) formaldehyde and 0.5% (v/v) Triton-100 for fixation for 45min and washed 3-4 times with distilled water. Then transfer the rape seedlings to staining solution (containing 100mM/L PBS, 0.5% (v/v) Triton-100, 2mM/L X-Gluc, 1mM/L K in staining solution3Fe(CN)6、1mM/L K4Fe(CN)6) Dyeing at 37 ℃ for 24-48 h. Decolorizing with 70% alcohol, observing the coloration of rape seedlings, and taking pictures to record, the result is shown in figure 2.
Second, results and analysis
Oleosin promoter drives GUS gene expression in rape seedlings
Rape Oleosin is a protein family, the Oleosin promoter is a seed-specific promoter, and in order to detect whether the promoter can drive the GUS gene to be transiently expressed, the promoter is connected to a pBI101 vector and subjected to GUS staining detection (FIG. 2). As shown in FIG. 2, when the Oleosin promoter-transfected seedlings of Brassica napus were soaked in the substrate X-Gluc buffer, the hypocotyls and the enlarged areas of radicles were stained blue (dark roots in FIG. 2 b). 55 seedlings are repeatedly co-infected twice, the number of blue-dyed seedlings is 43, and the dyeing rate is 78.2%. The blue-stained seedling tissue cells are transferred into GUS genes, GUS proteins are specifically expressed at the roots of the seedlings, and the blue seedlings are stained.
Comparative example 1
A method for establishing a transient expression system is different from that of example 1 in that in step 6, in the Agrobacterium-mediated transformation of rape seedlings, MS culture medium containing 30g/L sucrose and 7g/L agar is used as the solid culture medium.
When the method is adopted to establish the transient expression system, hypocotyls and radicles of rape seedlings are not expanded, agrobacterium is difficult to infect the seedlings, and the transient expression system is failed to be established.
Comparative example 2
A method for establishing a transient expression system is different from the method in example 1 in that in the step 6 of agrobacterium-mediated transformation of rape seedlings, a solid culture medium is selected from a MS culture medium of 0.5mg/L NAA, 30g/L sucrose and 7g/L agar.
When the method is adopted to establish the transient expression system, because the culture medium does not contain cytokinin, hypocotyls and radicles of rape seedlings are not expanded, the seedlings are difficult to be infected by agrobacterium, and the construction of the transient expression system fails.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (5)
1. A method for establishing a transient expression system by inducing local tissue expansion of seedlings is characterized by comprising the following steps:
and (3) recombinant agrobacterium: extracting escherichia coli plasmids, connecting a promoter to the plasmids to obtain a recombinant expression vector, transforming agrobacterium with the recombinant expression vector, wherein the agrobacterium is an EHA105 strain, selecting positive transformed agrobacterium, and performing amplification culture;
activating agrobacterium: culturing positive transformation agrobacterium with a bacterium suspension culture medium to obtain agrobacterium liquid; the strain suspension culture medium is an MS culture medium comprising 0.5 +/-0.1 mg/L of 6-BA, 0.1 +/-0.05 mg/L of NAA, 100 +/-1 mu mol/L of acetosyringone and 30 +/-1 g/L of cane sugar;
treating seeds: sterilizing seeds, namely rape seeds, inoculating the seeds to a solid culture medium, culturing for 5-10 days at 22-25 ℃ until local tissues of seedlings are expanded, wherein the local tissues are hypocotyls and radicles; the solid culture medium is an MS culture medium which comprises 0.5 plus or minus 0.1mg/L of 6-BA, 0.1 plus or minus 0.05mg/L of NAA, 30 plus or minus 1g/L of sucrose and 7-8g/L of agar;
and (3) mediating conversion: soaking the seedlings with local tissue expansion by using agrobacterium liquid, transferring the seedlings to a co-culture medium for culture, and inducing agrobacterium to invade the local tissue expansion of the seedlings to form an instantaneous expression system; the co-culture medium is an MS culture medium comprising 0.5 +/-0.1 mg/L of 6-BA, 0.1 +/-0.05 mg/L of NAA, 100 +/-1 mu mol/L of acetosyringone, 30 +/-1 g/L of sucrose and 8 +/-1 g/L of agar.
2. The method according to claim 1, wherein the step of recombining the agrobacterium is specifically:
extracting a colon bacillus PBI101 plasmid by adopting an alkaline lysis method, digesting an Oleosin promoter and the PBI101 plasmid by using Hind III and BamH I, respectively recovering an Oleosin promoter fragment and a PBI101 vector fragment, and connecting the Oleosin promoter fragment and the PBI101 vector fragment by using T4 ligase to construct a recombinant expression vector;
transforming the recombinant expression vector to agrobacterium by a heat shock method, screening a transformation product by using an LB culture medium containing 50 +/-1 mg/L kanamycin and 20-30mg/L rifampicin, selecting positive transformation agrobacterium, and carrying out amplification culture.
3. The method according to claim 2, wherein the step of activating agrobacterium is specifically: and adding the positive transformation agrobacterium into a bacterium suspension culture medium, carrying out shaking culture at the rotation speed of 160-200rpm until the concentration OD600 of the agrobacterium liquid is 0.4-0.6.
4. The method according to claim 3, wherein the step of treating the seeds is in particular: with 0.10. + -. 0.05 wt% HgCl2Sterilizing the seeds for 8-10min, washing with sterile water for 3-4 times, inoculating the seeds to a solid culture medium, culturing at 22-25 deg.C for 5-10 days, and allowing the seeds to germinate and grow seedlings with local tissue expansion.
5. The method according to claim 4, characterized in that the step of mediating the transformation is in particular: soaking the seedlings with agrobacterium liquid for 10-30min, and transferring to a co-culture medium for dark culture for 3-5 days to form an instantaneous expression system.
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