Method for improving seed foreign protein expression quantity

Abstract

The invention discloses a method for improving the expression quantity of foreign proteins in seeds, which comprises the following steps: inhibiting the expression of prolamin in plant seeds, and specifically expressing the foreign proteins to realize high expression and accumulation of the foreign proteins. According to the method, the expression quantity of the exogenous recombinant protein in the plant seeds can be greatly improved, the problem that the yield of the recombinant protein produced by utilizing the plant seeds in the existing method is low is solved, the application value and the economic value of a plant bioreactor are improved, and a new choice is provided for large-scale low-cost production of the recombinant protein in the seeds.

Description

(I) Technical field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for increasing the expression amount of exogenous protein in seeds. (II) Background technology

[0002] The production of valuable recombinant proteins in plant cells has the advantages of low production cost, short cycle, easy scalability, and large production capacity flexibility. The research and application of plant bioreactors has always been a hot spot in the bio-industry. Seed endosperm tissue has simple components, no complex secondary metabolites, and is easy to store. Various plant seeds such as rice, corn, and soybeans have been successfully used to produce a variety of recombinant medical, pharmaceutical, or industrial proteins. Although the initial production cost is low, the cost of extracting and purifying plant-derived recombinant proteins is difficult to compress. If the expression level is not high enough, it does not have the value of extraction and purification. The expression level of exogenous proteins determines whether the plant bioreactor has industrial application value. At present, many recombinant proteins have been successfully expressed in seeds, such as cellulase, laccase, trypsin, lactoferrin, lysozyme, phytase, antimicrobial peptides, serum albumin, and many other proteins have been successfully expressed in seeds, but only some highly expressed proteins meet the requirements of industrial application and enter the industrial development stage. Improving the expression level of recombinant proteins is a key issue that urgently needs to be solved in the application of plant bioreactors.

[0003] Plant seeds are widely used in the production of recombinant proteins. After the recombinant proteins accumulate in the seeds, harvesting and storing them in a dry state helps maintain the stability of the proteins. These characteristics make seeds an ideal platform for the production and preservation of recombinant proteins, facilitating subsequent processing and application. Increasing the expression of exogenous proteins in plant seeds is the most direct way to reduce the cost of recombinant protein production. At present, there are many methods and strategies for increasing the expression of exogenous proteins applied to plant bioreactors, such as screening suitable promoters, terminators or regulatory elements that enhance expression, optimizing codons, optimizing protein transport and storage strategies (Jiang Mingxuan et al., Biotechnology Bulletin, 2023, 39(11): 110-122). Inhibiting the expression of proteases in plants and reducing the degradation of exogenous proteins is an effective method to increase the expression and yield of exogenous proteins (Zou Qi, Chinese Journal of Biotechnology, 2023, 43(1): 71-86). The total protein content in seeds is relatively stable, and the room for increasing the expression of exogenous proteins is relatively limited. For example, the total protein content in corn seeds is generally about 10%, of which the alcohol-soluble protein content is greater than 60%. Existing studies have shown that inhibiting the expression of alcohol-soluble protein does not lead to a significant reduction in the total amount of corn grain protein, but rather increases the protein content of other parts through protein rebalancing. Because animals have poor consumption and absorption effects on alcohol-soluble protein, this strategy is generally used to improve the quality of corn protein, but not all other protein expression levels in seeds can be improved (Wu, Front Plant Sci, 2014, 30 (5) 240, PMID: 24910639). Because corn alcohol-soluble protein has the characteristics of high expression and good stability, there are also studies on increasing the expression and stability of exogenous proteins by fusing alcohol-soluble protein with exogenous proteins, but there is no report on increasing the expression of recombinant proteins by inhibiting the expression of corn alcohol-soluble protein. (III) Summary of the invention

[0004] The object of the present invention is to provide a method for increasing the expression amount of exogenous protein in seeds, wherein the method reduces the synthesis of endogenous proteins by inhibiting the expression of alcohol-soluble proteins, thereby achieving high expression of exogenous recombinant proteins in seeds and reducing the production cost of recombinant proteins.

[0005] The technical solution adopted by the present invention is:

[0006] The invention provides a method for increasing the expression amount of foreign protein in seeds. The method comprises inhibiting the expression of alcohol-soluble protein in plant seeds and specifically expressing foreign protein at the same time, thereby achieving high expression and accumulation of foreign protein.

[0007] Furthermore, the prolamin includes 19 kDa and 22 kDa zein.

[0008] Furthermore, the inhibition method provided by the present invention includes but is not limited to RNA interference and gene editing technology. Preferably, the RNA interference sequence is designed based on the highly homologous sequence in the zein gene family, preferably 912-1718 bp shown in SEQ ID NO: 2.

[0009] Furthermore, the expression frame for inhibiting the expression of zein includes a promoter, an RNA interference sequence and a terminator. The promoter includes but is not limited to a seed-specific promoter, preferably a rice glutelin promoter, as shown in 1-911 bp in SEQ ID NO: 2; the RNA interference sequence is preferably shown in 912-1718 bp in SEQ ID NO: 2; the terminator can be a general terminator of a commonly used gene expression frame, preferably shown in 1719-1796 bp in SEQ ID NO: 2.

[0010] Furthermore, the nucleotide sequence of the expression cassette for inhibiting the expression of zein is preferably as shown in SEQ ID NO:2.

[0011] Furthermore, the expression frame for specifically expressing the foreign protein includes a specific promoter, a foreign protein encoding gene, and a terminator; the specific promoter is a promoter such as a rice glutelin promoter, a zein promoter, etc. that can specifically express the foreign protein in seeds, preferably a rice glutelin promoter, and the nucleotide sequence is shown in 1-983 bp in SEQ NO.3. The foreign protein includes but is not limited to industrial, medical or feed proteins that need to be produced on a large scale. The present invention selects recombinant lipase, and the nucleotide sequence is shown in 984-1935 bp in SEQ NO.3. The terminator can be a general terminator of a commonly used gene expression frame, preferably shown in 1936-2424 bp in SEQ ID NO:3.

[0012] Furthermore, the nucleotide sequence of the expression cassette for specifically expressing foreign proteins is shown in SEQ ID NO:3.

[0013] Furthermore, the method includes constructing a selection marker gene expression frame, and the selection marker gene can be a marker gene that can screen out transgenic cells, such as glyphosate-resistant, glufosinate-resistant or hygromycin-resistant, preferably a glyphosate-resistant expression frame, and the nucleotide sequence is shown in SEQ ID NO: 1.

[0014] Furthermore, the method comprises the following steps: (1) constructing a plant transformation vector containing an expression frame for inhibiting the expression of zein and a selection marker gene expression frame, transferring the plant transformation vector into the plant genome, and screening to obtain transgenic plants with low expression of zein; (2) constructing a plant transformation vector containing an expression frame for specifically expressing an exogenous protein and a selection marker gene expression frame, transferring the plant transformation vector into the plant genome, and screening to obtain transgenic plants with high expression of the exogenous protein; (3) hybridizing the transgenic plants screened in step (1) and step (2), and screening to obtain transgenic plants with low expression of zein and high expression of the exogenous protein.

[0015] Furthermore, the basic vector used to construct the plant transformation vector in steps (1) and (2) is a binary vector obtained by removing the hygromycin gene from the basic vector pCambia1300 (NCBI sequence number AF234296) by double digestion with HindIII / XhoI. The method for transferring the plant transformation vector into the plant genome includes the Agrobacterium-mediated method.

[0016] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: the present invention provides a method for specifically expressing exogenous proteins by inhibiting the expression of alcohol-soluble proteins, which can greatly increase the expression amount of exogenous recombinant proteins in plant seeds, solve the problem of low yield of recombinant proteins produced by plant seeds in the existing methods, improve the application value and economic value of plant bioreactors, and provide a new option for large-scale low-cost production of recombinant proteins in seeds. (IV) Description of the drawings

[0017] Figure 1 , map of corn transformation vector LZ that inhibits the expression of alcohol-soluble proteins.

[0018] Figure 2 , recombinant protein transformation vector LP map.

[0019] Figure 3 , SDS protein electrophoresis of low alcohol-soluble protein corn seeds.

[0020] Figure 4 , SDS protein electrophoresis diagram of recombinant protein expression in seeds.

[0021] Figure 5 , SDS protein electrophoresis of the recombinant protein expression level in seeds after polymerization. (V) Specific implementation methods

[0022] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0023] Example 1: Obtaining a plant transformation plasmid vector

[0024] 1. Plant transformation vector LZ

[0025] The artificially synthesized inhibition of alcohol-soluble protein expression frame (rice glutelin promoter, RNA interference sequence designed according to the homologous sequences of 19kDa and 22kDa alcohol-soluble protein family, terminator) has a nucleotide sequence shown in SEQ ID NO.2.

[0026] The artificially synthesized glyphosate-resistant expression frame has a nucleotide sequence shown in SEQ ID NO.1.

[0027] The vector map of corn transformation for inhibiting the expression of prolamin of the present invention is as follows: Figure 1 As shown, the transformation plasmid vector uses pCambia1300 (GenBank: AF234296.1) as the plant transformation vector framework, and the hygromycin selection marker gene in the vector is removed by double restriction digestion with HindⅢ / XhoⅠ. A reading frame containing a complete glyphosate resistance expression frame and a zein expression inhibition frame is added to the multiple cloning site region to obtain the plant transformation vector LZ, i.e., the zein expression inhibition vector LZ. The vector map is shown in Figure 1 The transfection method was used to introduce the vector into Agrobacterium LBA4404 to obtain Agrobacterium containing the transformation vector LZ.

[0028] 2. Plant transformation vector LP

[0029] The artificially synthesized recombinant lipase expression frame has a nucleotide sequence shown in SEQ ID NO.3.

[0030] The vector pCambia1300 (NCBI sequence number AF234296) was double-digested with HindⅢ / XhoⅠ to recover a 6783 bp fragment. The glyphosate-tolerant expression cassette SEQ ID NO.1 and the recombinant lipase expression cassette SEQ ID NO.3 were linked into the vector by recombinant cloning to obtain the plant transformation vector LP. The vector map is shown in Figure 2 The verified plasmid was electroporated into Agrobacterium LBA4404 competent cells, and after restriction enzyme digestion and verification, it was used for corn transformation.

[0031] Example 2: Obtaining transgenic corn with suppressed prolamin expression

[0032] The genetic transformation of corn was carried out by Agrobacterium-mediated transformation, specifically according to the method and medium formula reported by Frame et al. (Plant Physiol, 2002, 129: 13-22), and glyphosate was used as a screening agent. The steps were as follows: Take the non-transgenic inbred line A188 corn ears 8 to 10 days after pollination, and collect the immature embryos with a size of 1.0-1.5 mm. Agrobacterium containing the transformation vector LZ and the immature embryos were co-cultured at 22°C for 3 days. The cultured immature embryos were transferred to a callus induction medium containing a final concentration of 200 mg / L of Timentin antibiotics (GlaxoSmithKline, USA), and cultured in the dark at 28°C for 14 days. Then they were transferred to a screening medium containing 2 mM glyphosate, and cultured in the dark at 28°C and subcultured every 3 weeks. The obviously grown embryonic tissue was transferred to the regeneration medium, cultured in the dark at 28°C for 14 days, transferred to the fresh regeneration medium, cultured in the light at 26°C for 10-14 days, transferred to the rooting medium, and cultured in the light at 26°C until the roots were fully developed. The rooted regenerated seedlings were transplanted into the greenhouse, cross-pollinated with the non-transgenic inbred line A188 as the male parent and the transgenic T0 generation as the female parent, and the LZ carrier T0 generation seeds were harvested for screening and analysis.

[0033] Example 3: Screening of low-alcohol-soluble protein transformants

[0034] The seeds of the T0 generation of LZ vector were harvested, and the transformants LZ1, LZ3 and LZ21 with obvious powdery grains and a powdery grain to hard grain ratio of 1:1 were screened to analyze the expression level of alcohol-soluble protein. The powdery corn seeds were homogenized with an extract containing 70% ethanol and 2% mercaptoethanol, and the supernatant was mixed with SDS sample buffer and denatured by boiling for 10 minutes for SDS protein electrophoresis. The protein electrophoresis method and reagent formula can be found in the Molecular Cloning Experiment Guide (Fourth Edition) . The electrophoresis results showed that compared with the conventional control seeds, the content of 19kDa and 22kDa α-alcohol-soluble proteins in LZ3 and LZ21 was significantly reduced, and the electrophoresis bands were significantly lighter than those in the conventional control. The expression of α-alcohol-soluble protein in LZ1 was also partially inhibited, but the decrease in expression was not as significant as that in LZ3 and LZ21 ( Figure 3 ), therefore LZ3 was selected as an inhibitor of prolamin transformants for polymerization with recombinant lipase maize.

[0035] Example 4: Screening of transformants expressing lipase

[0036] Using the method of Example 2, three seeds were selected from each T0 generation LP transformant harvested, and the seeds were weighed and ground into homogenate. The supernatant was centrifuged and tested with CP4 test strips. The samples that showed positive results were used for protein electrophoresis and enzyme activity test. The supernatant was diluted 100 times and used for enzyme activity test. Using p-nitrophenol butyrate (p-NPB) as the substrate, the enzyme activity was determined and calculated according to the method of Jiang Huifang (Chemistry and Bioengineering 2007.24(8).8(24)72-75), 50ul p-NPB substrate solution, 50ul diluted enzyme solution and 900ul reaction buffer were taken, reacted in a 45°C water bath for 5min, and the absorbance was measured at 405nm. The p-nitrophenol concentration produced was calculated by comparing with the standard curve, and then the enzyme activity was calculated. A number of high lipase activity transformants such as LP-7, LP-15 and LP-72 were screened and obtained (Table 1). The protein extraction supernatant of transformants LP-7, LP-15 and LP-72 with high enzyme activity was mixed with SDS loading buffer, boiled for 10 minutes for denaturation and then used for SDS protein electrophoresis. The commercial enzyme (Novozymes) was used as the positive control. The protein electrophoresis method and reagent formula can be found in the Molecular Cloning Experiment Guide (Fourth Edition) and the electrophoresis results ( Figure 4 ) showed that LP-7, LP-15 and LP-72 had obvious bands at the 35kDa site, and the size of the protein band was consistent with that of the commercial enzyme positive control.

[0037] Table 1 Lipase convertase activity

[0038]

[0039]

[0040] Example 5: Transformant polymerization and enzyme activity analysis

[0041] T1 generation lipase transformants LP-7, LP-15 and LP-72 and low alcohol-soluble protein transformant LZ3 were planted and bag-pollinated. LP-7, LP-15 and LP-72 were used as female parents and LZ3 or conventional non-transgenic A188 was used as male parent for hybridization to obtain LP-7×LZ3, LP-15×LZ3, LP-72×LZ3 and LP-7×A188, LP-15×A188, LP-72×A188 seeds.

[0042] The harvested seeds of different hybrids were weighed and then ground into single grains for homogenization. The samples that showed positive results by CP4 test strips were centrifuged and the supernatant was diluted 100 times for enzyme activity detection. p-Nitrophenol butyrate (p-NPB) was used as the substrate. The enzyme activity was determined and calculated according to the method of Jiang Huifang (Chemistry and Bioengineering 2007.24(8).8(24)72-75). 50ul p-NPB substrate solution, 50ul diluted enzyme solution and 900ul reaction buffer were taken, and the reaction was carried out in a 45℃ water bath for 5min. The absorbance was measured at 405nm. The concentration of p-nitrophenol produced was calculated by comparing with the standard curve, and then the enzyme activity was calculated. The results showed that after the transformant expressing lipase was hybridized with the low alcohol-soluble protein transformant LZ3, the lipase activity in the seeds was greatly increased (Table 2). The supernatant of the transformants LP-7×LZ3, LP-15×LZ3 and LP-72×LZ3 with high enzyme activity was extracted for protein electrophoresis. The commercial enzyme (Novozymes) was used as the positive control and A188 was used as the negative control. The protein electrophoresis method and reagent formula were shown in the Molecular Cloning Experiment Guide (Fourth Edition). The electrophoresis results showed that the recombinant lipase ( Figure 5 ).

[0043] Table 2. Enzyme activity of the hybrid progeny of low-alcohol-soluble corn and lipase transformants

[0044] Transformant name Enzyme activity (unit / g) LP-7×LZ3 525.6±16.81 LP-7×A188 251.2±10.32 LP-15×LZ3 415.3±21.65 LP-15×A188 222.3±12.57 LP-72×LZ3 492.7±21.91 LP-72×A188 225.7±13.57 A188 0.3±0.10 CALB (positive control) 502±12.67

Claims

1. A method for increasing the expression of exogenous protein in seeds, characterized in that: The method comprises inhibiting the expression of alcohol-soluble proteins in plant seeds and specifically expressing foreign proteins at the same time, thereby achieving high expression and accumulation of foreign proteins.

2. The method according to claim 1, characterized in that The prolamins include 19 kDa and 22 kDa zeins.

3. The method according to claim 1, characterized in that The inhibition methods include RNA interference and gene editing technology.

4. The method according to any one of claims 1 to 3, characterized in that: The RNA interference sequence for inhibiting the expression of alcohol-soluble protein in plant seeds is shown as 912-1718 bp in SEQ ID NO:

2.

5. The method according to claim 4, characterized in that The expression cassette for inhibiting the expression of prolamin comprises a promoter, an RNA interference sequence and a terminator, and the nucleotide sequence of the expression cassette is shown in SEQ ID NO:

2.

6. The method according to claim 1, characterized in that The method comprises the following steps: (1) constructing a plant transformation vector containing an expression frame for inhibiting the expression of zein and a screening marker gene expression frame, transferring the plant transformation vector into the plant genome, and screening to obtain a transgenic plant with low expression of zein; (2) constructing a plant transformation vector containing an expression frame for specifically expressing an exogenous protein and a screening marker gene expression frame, transferring the plant transformation vector into the plant genome, and screening to obtain a transgenic plant with high expression of the exogenous protein; and (3) hybridizing the transgenic plants screened in step (1) and step (2), and screening to obtain a transgenic plant with low expression of zein and high expression of the exogenous protein.

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