Recombinant promoter and method for improving expression quantity of exogenous genes in plant seeds

Abstract

The invention discloses a recombinant promoter and a method for improving the expression quantity of an exogenous gene in a plant seed, which are characterized in that a sequence for inhibiting the expression of a seed storage protein is added between the promoter and the exogenous gene, so that the expression of the storage protein in the seed is inhibited while the specific expression of an exogenous recombinant protein in the seed is realized; an environment more suitable for expression and accumulation of foreign proteins is created, the expression quantity of the foreign recombinant proteins in plant seeds is improved, and the cost of producing the recombinant proteins by plants is reduced.

Description

(I) Technical field

[0001] The invention belongs to the field of plant molecular biology and relates to a recombinant promoter and a method capable of increasing the expression amount of foreign proteins in plant seeds. (II) Background technology

[0002] The use of plants to produce recombinant proteins is a new method for large-scale production of recombinant proteins. Currently, a variety of recombinant proteins produced in plants, such as avidin, trypzean, laccase, lactoferrin, and lysozyme, have been commercialized. Plants have the advantages of easy scale-up, large production capacity flexibility, and low hardware requirements, but the expression of recombinant proteins is often not as good as traditional cell culture and microbial fermentation. Therefore, only by increasing the expression of recombinant proteins in plants can the advantages of plant production of recombinant proteins be brought into play to a greater extent.

[0003] Seeds are considered to be an ideal place for plants to produce and store recombinant proteins. Seeds have higher protein production potential, and dry seeds are metabolically inert, so the proteins in seeds can be stably stored for a long time. How to increase the expression of recombinant proteins in seeds is crucial to promoting the commercial application of plant-derived recombinant proteins.

[0004] There are many factors that affect protein expression in seeds, such as transcription level, translation efficiency, recombinant protein modification and localization, etc. In order to increase the expression of recombinant proteins in seeds, it is often necessary to optimize the design of transcription, translation, transport and accumulation at the same time. For example, the optimization of transcription level is mainly achieved by screening high-expression promoters and changing the promoter copy number, cis-acting elements, transcription factor binding spacing and core promoter sequence to obtain artificial promoters that drive high-level transcription of target genes (Zhao Wantong et al., Molecular Plant Breeding, 2019, 17(22): 7377-7358). The translation efficiency is improved by optimizing codons; signal peptides or subcellular localization sequences are designed to improve the transport, localization and accumulation of recombinant proteins. This series of optimizations provides more stable conditions for the expression of recombinant proteins in seeds, but the total amount of protein in seeds is quite stable. The expression of recombinant proteins needs to compete with other storage proteins for resources. Studies have shown that after inhibiting the synthesis of storage proteins in seeds, free amino acids or nitrogen sources will increase and be redistributed for the synthesis of other proteins, thereby achieving a balance in the total amount of protein in seeds. Therefore, combining the reduction of storage protein expression in seeds with the improvement of recombinant protein expression design to create an environment more suitable for recombinant protein expression can increase the expression level of recombinant protein, reduce the production cost of recombinant protein, and improve the competitiveness of plant production of recombinant protein. (III) Summary of the invention

[0005] The present invention aims to provide a recombinant promoter and method for increasing the expression amount of foreign protein in plant seeds. The recombinant promoter can inhibit the expression of seed storage protein genes while specifically expressing foreign protein in seeds, thereby achieving high-level expression and accumulation of foreign protein in seeds.

[0006] The technical solution adopted by the present invention is:

[0007] The invention provides a recombinant promoter for improving the expression level of foreign genes in plant seeds. The recombinant promoter is formed by functionally connecting two parts: a promoter sequence for starting the expression of foreign genes in plants and a nucleic acid sequence for inhibiting the expression of seed storage protein genes.

[0008] The sequence for inhibiting the expression of the seed storage protein gene of the present invention is a recombinant nucleotide sequence designed by a person skilled in the art based on the nucleotide sequence of the storage protein gene and capable of inhibiting the expression of the storage protein gene. The nucleotide sequence for inhibiting the expression of the storage protein gene is connected between the promoter and the exogenous gene.

[0009] Furthermore, the seed storage protein includes corn 19kDa and 22kDa alcohol-soluble proteins, and the nucleotide sequence that inhibits the expression of the seed storage protein gene is shown in SEQ ID NO:2.

[0010] Furthermore, the promoter includes seed-specific promoters and promoters known in the art that can promote the expression of foreign genes in plants, such as rice glutelin promoter, the nucleotide sequence of which is shown in SEQ ID NO: 1. The foreign protein includes various proteins that can be expressed in plant seeds, such as lipase protein.

[0011] The present invention also provides a method for using the recombinant promoter to improve the expression level of foreign genes in seeds. The method comprises transferring the foreign gene expression frame containing the recombinant promoter into the plant genome and screening seeds with significantly improved foreign protein expression.

[0012] Furthermore, the exogenous gene expression cassette is composed of a promoter for initiating exogenous gene expression, a nucleic acid sequence for inhibiting seed storage protein gene expression, an exogenous gene and a terminator functionally connected in sequence from the 5' end to the 3' end. The exogenous gene expression cassette can also be transferred into the plant genome together with a screening marker gene expression cassette, and the screening marker gene expression cassette can be an expression cassette known in the art.

[0013] The method of the present invention is applicable to various situations in which plant seeds are used to express foreign proteins. Sequence combinations that can activate foreign gene expression and inhibit storage protein expression are all within the protection scope of the present invention.

[0014] Compared with the prior art, the beneficial effects of the present invention are mainly embodied in that: the present invention adds a sequence for inhibiting the expression of seed storage protein genes between a promoter and an exogenous gene, and designs a seed-specific promoter for initiating the expression of exogenous genes in plants, and transfers an exogenous recombinant protein expression frame constructed by an artificial synthetic promoter combined with a sequence for inhibiting the expression of seed storage protein genes into the plant genome, thereby achieving the specific expression of exogenous genes in seeds while inhibiting the expression of storage protein genes in seeds, creating an environment more suitable for the expression and accumulation of exogenous proteins, increasing the expression amount of exogenous proteins in plant seeds, and reducing the cost of plant production of recombinant proteins. (IV) Description of the drawings

[0015] Figure 1 , GI-L vector map.

[0016] Figure 2 , GL vector map.

[0017] Figure 3 , seed whole protein electrophoresis. (V) Specific implementation methods

[0018] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0019] The molecular biology and biochemical methods used in the following examples of the present invention are all known techniques. Detailed descriptions are provided in documents such as Current Protocols in Molecular Biology, compiled by Ausubel and published by John Wiley and Sons, and Molecular Cloning: A Laboratory Manual, 3rd ED., compiled by J. Sambrook et al. and published by Cold Spring Harbor Laboratory Press (2001).

[0020] Example 1: Acquisition of promoter and sequence for inhibiting expression of seed storage protein

[0021] The artificially synthesized rice glutelin promoter has a nucleotide sequence as shown in SEQ ID NO: 1. According to the nucleotide sequence of maize α-gluadin (NCBI No. NM_001112530.2, 22 kDa) and α-gluadin (NCBI No. NM_001112275.2, 19 kDa), a 5'-terminal non-coding recombinant nucleotide that inhibits the expression of maize α-gluadin was designed, and the nucleotide sequence is shown in SEQ ID NO: 2.

[0022] Example 2: Construction of plant transformation plasmid vector

[0023] The glyphosate resistance expression frame of the transformation screening marker gene was artificially synthesized, and the nucleotide sequence was shown in SEQ ID NO: 3. The recombinant lipase gene containing a terminator sequence was artificially synthesized after codon optimization based on the lipase protein sequence of Thermomyces lanuginosus (NCBI No.: O59952.1), and the nucleotide sequence was shown in SEQ ID NO: 4. The plant transformation vector pCambia1300 (GenBank: AF234296.1) was used as the vector framework, and the hygromycin selection marker gene in the vector was removed by double restriction digestion with HindⅢ / XhoⅠ. The rice glutelin promoter (SEQ ID NO: 1), 5' non-coding nucleotide (SEQ ID NO: 2), target gene (SEQ ID NO: 4) and glyphosate resistance expression frame (SEQ ID NO: 3) were connected to the transformation vector by homologous recombination to obtain the plant transformation vector GI-L ( Figure 1 ); the plant transformation vector pCambia1300 (GenBank: AF234296.1) was used as the vector framework, the hygromycin selection marker gene in the vector was removed by double restriction digestion with HindⅢ / XhoⅠ, and the rice glutelin promoter (SEQ ID NO: 1), the target gene (SEQ ID NO: 4) and the glyphosate resistance expression frame (SEQ ID NO: 3) were connected to the transformation vector by homologous recombination to obtain the plant transformation vector GL ( Figure 2 ) ; electroporation was used to introduce the transformed Agrobacterium into LBA4404 to obtain Agrobacterium containing the transformation vectors GI-L and GL, respectively.

[0024] Example 3: Obtaining transgenic corn

[0025] 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), using glyphosate as a screening agent, and the steps were as follows: corn ears 8 to 10 days after pollination of the corn inbred line A188 were used for genetic transformation, and immature embryos with a size of 1.0-1.5 mm were collected. Agrobacterium containing the transformation vector and immature embryos were co-cultured at 22°C for 3 days. The immature embryos after co-culture were transferred to a medium containing a final concentration of 200 mg / L of Timentin antibiotics (GlaxoSmithKline, USA) to induce callus tissue, and cultured in the dark at 28°C for 14 days. Then they were transferred to a screening medium containing 2mM glyphosate, cultured in the dark at 28°C, and subcultured every 3 weeks. The resistant tissue obtained by screening was transferred to a regeneration medium, cultured in the dark at 28°C for 14 days, then transferred to a fresh regeneration medium, cultured in the light at 26°C for 10-14 days, then transferred to a rooting medium, and cultured in the light at 26°C until the roots were fully developed. The rooted regenerated seedlings were transplanted into a greenhouse, and cross-pollinated using the non-transgenic inbred line A188 as the male parent and the transgenic T0 generation as the female parent, and 158 GI-L transformants and 182 GL transformants were harvested.

[0026] Example 4: Analysis of recombinant protein expression

[0027] Three seeds were selected from each transformant of the harvested transgenic seeds, and the seeds were weighed and then ground into homogenate. The supernatant was centrifuged and tested with CP4-EPSPS test strips (Shanghai Youlong, catalog number AA0832-BGB). The samples that showed positive results were used for lipase activity testing to screen transformants with high enzyme activity. The inbred line A188 seeds were used as blank controls, and the commercial enzyme TLL (Novozymes, Lipozyme TL100L, cas number: 9001-62-1) was used as a positive control.

[0028] The supernatant of the enzyme extract was diluted 100 times and used 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). 50 μL of p-NPB substrate solution, 50 μL of diluted enzyme solution and 900 μL of reaction buffer were taken, and the reaction was carried out in a water bath at 45°C for 5 minutes. The absorbance was measured at 405 nm. The concentration of p-nitrophenol produced was calculated by comparing with the standard curve, and then the enzyme activity was calculated. The results of enzyme activity analysis showed that the enzyme activity of transgenic corn transformed with GI-L was generally higher than that of transgenic corn transformed with GL vector. The five transformants with the highest enzyme activity obtained by screening are shown in Table 1.

[0029] Table 1 Lipase activity of transgenic corn seeds

[0030] Transformant name Average enzyme activity (unit / g) GI-L15 58.52±3.35 GI-L4 39.61±4.32 GI-L78 25.53±3.22 GI-L36 18.62±3.37 GI-L21 11.34±1.27 G-L8 22.12±2.25 G-L2 9.24±1.12 G-L65 5.13±0.97 G-L19 4.26±0.25 G-L82 4.32±0.34 CK(A188) 0.06±0.01 TLL(Lipozyme TL100L) 231.21±8.33

[0031] Example 5: Analysis of protein composition of transgenic seeds

[0032] The seeds of the transformants GI-L15, GI-L4, G-L8 and G-L2 with the highest enzyme activity were weighed and homogenized with 1-fold protein electrophoresis loading buffer to extract the whole seed protein. The samples that showed positive results by CP4-EPSPS test strips (Shanghai Youlong, catalog number AA0832-BGB) were boiled in boiling water for 10 minutes and then used for protein electrophoresis. The SDS electrophoresis results showed that 19kDa and 22kDa alcohol-soluble proteins were highly expressed in G-L2 and G-L8 transformants that did not contain 5'-end non-coding nucleotides that inhibited alcohol-soluble protein expression. The exogenous recombinant protein bands were significantly weaker than those of GI-L4 and GI-L15. The alcohol-soluble protein bands of 19kDa and 22kDa in GI-L4 and GI-L15 were basically absent, indicating that the use of the promoter containing alcohol-soluble protein expression inhibition provided by the present invention can reduce the expression of alcohol-soluble proteins while increasing the expression of exogenous proteins ( Figure 3 ).

Claims

1. A recombinant promoter for increasing the expression of exogenous proteins in plant seeds, characterized in that: The recombinant promoter is formed by functionally connecting two parts: a promoter sequence for starting the expression of foreign genes in plants and a nucleic acid sequence for inhibiting the expression of seed storage protein genes.

2. The recombinant promoter according to claim 1, characterized in that The nucleotide sequence for inhibiting the expression of the storage protein gene is connected between the promoter and the foreign gene.

3. The recombinant promoter according to claim 1, characterized in that The seed storage proteins include maize 19 kDa and 22 kDa prolamins.

4. The recombinant promoter according to claim 1, characterized in that The nucleotide sequence for inhibiting the expression of the seed storage protein gene is shown in SEQ ID NO:

2.

5. A method for increasing the expression of foreign genes in seeds using the recombinant promoter according to claim 1, characterized in that: The method comprises the following steps: transferring an exogenous gene expression frame containing a recombinant promoter into a plant genome, and screening seeds with significantly improved exogenous protein expression.

6. The method according to claim 5, characterized in that The foreign gene expression frame is composed of a promoter for starting foreign gene expression, a nucleic acid sequence for inhibiting seed storage protein gene expression, a foreign gene and a terminator which are functionally connected in sequence from the 5' end to the 3' end.

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