Codon optimization method for improving protein expression quantity of target gene in plant

Abstract

The invention relates to a codon optimization method for improving the protein expression quantity of a target gene in a plant, and belongs to the technical field of gene engineering. The invention provides a codon optimization method for improving the protein expression quantity of a target gene in a plant, which comprises the step of replacing the third basic group of a codon of a target protein coding sequence with C or G to obtain an optimized target protein coding sequence. The method has effectiveness and universality in plants, and research shows that after codon optimization of the method, the protein expression quantity of target genes in a plant protein transient expression system and a transgenic plant is increased by several times to dozens of times. According to the production application of the functional protein taking the plant as the bioreactor, the yield of the target protein can be increased, and the protein purification cost can be reduced; according to the application of the method in preparation of the transgenic plant, the expression quantity of the target gene can be increased, so that the target character of the transgenic plant is enhanced, and the screening cost of the high-expression transgenic plant is reduced.

Description

Technical Field

[0001] The invention relates to a codon optimization method for improving the protein expression amount of a target gene in a plant, and belongs to the technical field of genetic engineering. Background Art

[0002] In organisms, the DNA sequence of genes determines the sequence of messenger RNA, and the sequence of messenger RNA determines the amino acid sequence in proteins. Codons refer to the coding order and rules of three adjacent nucleotides in a group in messenger RNA or the DNA molecule of its corresponding gene. During protein synthesis, the codon sequence on DNA / RNA is translated by the ribosome into a specific amino acid at a specific position on the target protein. All proteins in organisms are composed of 20 different amino acids; most amino acids are encoded by multiple different codons; codons encoding the same amino acid are called synonymous codons.

[0003] Generally, codons with higher usage frequencies are called optimal codons, and conversely, codons with lower usage frequencies are called rare codons. Different species have different usage frequencies of synonymous codons, resulting in each species having a corresponding optimal codon. For example, in 2022, David Alvarez-Ponce Laboratory in the United States counted the codon usage of more than 15,000 species and annotated the corresponding optimal codons according to the codon usage frequency (see the literature: Subramanian K., Payne B., Feyertag F., Alvarez-Ponce D. (2022) The Codon Statistics Database: A Database of Codon Usage Bias. Mol Biol Evol 39: msac157).

[0004] At present, it is generally believed that rare codons will reduce protein translation efficiency and protein expression. Therefore, in order to increase protein expression, rare codons in the target protein coding sequence are usually replaced with optimal codons. However, in actual operation, according to the existing codon optimization method, the ideal protein expression is often not obtained. It can be seen that although it is known that the same protein encoded by different codons has different expression levels in organisms, there are still many different opinions on what is the optimal codon that can effectively increase the protein expression of the target protein coding sequence in the organism. At this stage, there is still a lack of some truly effective and universal codon optimization methods.

[0005] At the same time, since the number of different codon combinations is astronomical, when the existing codon optimization method cannot achieve the desired protein expression, it is impossible to synthesize and test all possible codon combinations one by one by enumeration, considering the time and economic costs. For example, assuming a protein composed of only 100 amino acids, each amino acid appears 5 times, then the DNA coding sequence corresponding to this protein theoretically has 1 5 ×1 5 ×2 5 ×2 5 ×2 5 ×2 5 ×2 5 ×2 5 ×2 5 ×2 5 ×2 5 ×3 5 ×4 5 ×4 5 ×4 5 ×4 5 ×4 5 ×6 5 ×6 5 ×6 5 =8358844170240 (or 8.3 trillion) possibilities.

[0006] Genetically modified plants refer to plants that use genetic engineering technology to integrate specific recombinant DNA fragments into the plant genome, thereby producing plants that express recombinant RNA and recombinant proteins encoded by the recombinant DNA. The application scenarios of genetically modified plants include changing the agronomic traits of crops, obtaining recombinant pharmaceutical proteins or recombinant nutritional proteins for use in humans or animals, etc. The changes in the traits of genetically modified plants can come from the mutual hybridization between individuals of different species / varieties, or from specific functional genes artificially inserted through genetic engineering (recombinant DNA) technology.

[0007] Generally, the traits of transgenic plants are positively correlated with the expression of their recombinant proteins, and the expression of recombinant proteins is often an important factor limiting the application of excellent genes. At present, it is generally believed that the expression of exogenous proteins is determined by the position effect of the recombinant gene inserted into the genome. Therefore, large quantities of transgenic materials are currently screened to obtain transgenic materials with high recombinant protein expression. This process requires a lot of manpower, material resources, financial resources, time and land costs. Despite this, there are still many genes that cannot obtain transgenic plant materials that meet the requirements of high expression of recombinant proteins. If a codon optimization method with the two advantages of high effectiveness and strong versatility can be found for plants, it will help to increase the expression of recombinant proteins of recombinant genes (target protein coding sequences) in plants, enhance the target traits of transgenic plants, and at the same time, reduce the screening cost of high-expression transgenic plants. Summary of the invention

[0008] To solve the above problems, the present invention provides a codon optimization method for increasing the protein expression amount of a target gene in a plant, the method comprising: replacing the third base of the codon of the target protein coding sequence with C or G to obtain an optimized target protein coding sequence.

[0009] In one embodiment of the present invention, the GC content of the optimized target protein coding sequence is not less than 55%.

[0010] In one embodiment of the present invention, in the optimized target protein coding sequence, the GC content (GC3) of the third base of all codons is not less than 70%.

[0011] In one embodiment of the present invention, in the method, when performing codon replacement, the third base of the codon corresponding to threonine is replaced by C (ie, the codon corresponding to threonine is replaced by ACC).

[0012] In one embodiment of the present invention, in the optimized target protein coding sequence, more than 50% of the segments have undergone replacement of the third base of the codon.

[0013] In one embodiment of the present invention, the target protein coding sequence includes a DNA sequence encoding the target protein, an RNA sequence encoding the target protein, a complementary sequence of the DNA sequence encoding the target protein and / or a complementary sequence of the RNA sequence encoding the target protein.

[0014] In one embodiment of the present invention, the target protein coding sequence includes a single strand and / or a double strand.

[0015] In one embodiment of the present invention, the plant includes dicotyledonous plants and / or monocotyledonous plants.

[0016] In one embodiment of the present invention, the dicotyledonous plant includes Arabidopsis, tobacco, soybean, cotton, lettuce, poplar, alfalfa, rapeseed and / or peanut.

[0017] In one embodiment of the present invention, the monocotyledonous plant comprises corn, rice, sorghum, wheat, sugarcane and / or bamboo.

[0018] In one embodiment of the present invention, the target protein includes a functional protein.

[0019] In one embodiment of the present invention, the functional protein includes a functional protein used to enhance a target trait of a transgenic plant and / or a functional protein produced using the plant as a bioreactor.

[0020] In one embodiment of the present invention, the functional proteins include CRY1a.105 protein, CRY2Ab protein, Bar protein, CP4 protein, SARS-Cov-RBD, HA (Influenza Virus Hemagglutinin), AmFTO, GgFTO and / or OprF.

[0021] The present invention also provides a nucleic acid molecule for expressing a target protein in a plant, wherein the nucleic acid molecule is optimized using the above codon optimization method.

[0022] In one embodiment of the present invention, the plant includes dicotyledonous plants and / or monocotyledonous plants.

[0023] In one embodiment of the present invention, the dicotyledonous plant includes Arabidopsis, tobacco, soybean, cotton, lettuce, poplar, alfalfa, rapeseed and / or peanut.

[0024] In one embodiment of the present invention, the monocotyledonous plant comprises corn, rice, sorghum, wheat, sugarcane and / or bamboo.

[0025] In one embodiment of the present invention, the target protein includes a functional protein.

[0026] In one embodiment of the present invention, the functional protein includes a functional protein used to enhance a target trait of a transgenic plant and / or a functional protein produced using the plant as a bioreactor.

[0027] In one embodiment of the present invention, the functional proteins include CRY1a.105 protein, CRY2Ab protein, Bar protein, CP4 protein, SARS-Cov-RBD, HA (Influenza Virus Hemagglutinin), AmFTO, GgFTO and / or OprF.

[0028] In one embodiment of the present invention, when the target protein is the insect-resistant protein CRY1a.105, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.47;

[0029] When the target protein is the insect-resistant protein CRY2Ab, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.49;

[0030] When the target protein is the herbicide-resistant protein Bar, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.51;

[0031] When the target protein is the herbicide-resistant protein CP4, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.53;

[0032] When the target protein is the novel coronavirus receptor protein ACE2 (Angiotensin-Converting Enzyme 2), the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.55;

[0033] When the target protein is the novel coronavirus spike protein receptor binding domain protein SARS-Cov-RBD, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.57;

[0034] When the target protein is influenza virus hemagglutinin protein HA (InfluenzaVirus Hemagglutinin), the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.59;

[0035] When the target protein is giant panda obesity-related protein AmFTO, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.61;

[0036] When the target protein is Hongyuan chicken obesity-related protein GgFTO, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.63;

[0037] When the target protein is fish immune protein OprF, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.70.

[0038] The present invention also provides a recombinant expression system for expressing a target protein in plants, wherein the recombinant expression system expresses the above nucleic acid molecule.

[0039] In one embodiment of the present invention, the recombinant expression system comprises a recombinant expression vector or a host cell; the recombinant expression vector carries the above-mentioned nucleic acid molecule; and the host cell is transformed with the recombinant expression vector carrying the above-mentioned nucleic acid molecule.

[0040] In one embodiment of the present invention, the recombinant expression vector comprises a plant cell expression vector carrying the above nucleic acid molecule.

[0041] In one embodiment of the present invention, the plant cell expression vector comprises the plant expression vector pCambia1300 and / or a pCambia1300 derivative vector.

[0042] In one embodiment of the present invention, the host cell is Agrobacterium transformed with a recombinant expression vector carrying the above nucleic acid molecule.

[0043] In one embodiment of the present invention, the plant includes dicotyledonous plants and / or monocotyledonous plants.

[0044] In one embodiment of the present invention, the dicotyledonous plant includes Arabidopsis, tobacco, soybean, cotton, lettuce, poplar, alfalfa, rapeseed and / or peanut.

[0045] In one embodiment of the present invention, the monocotyledonous plant comprises corn, rice, sorghum, wheat, sugarcane and / or bamboo.

[0046] In one embodiment of the present invention, the target protein includes a functional protein.

[0047] In one embodiment of the present invention, the functional protein includes a functional protein used to enhance a target trait of a transgenic plant and / or a functional protein produced using the plant as a bioreactor.

[0048] In one embodiment of the present invention, the functional proteins include CRY1a.105 protein, CRY2Ab protein, Bar protein, CP4 protein, SARS-Cov-RBD, HA (Influenza Virus Hemagglutinin), AmFTO, GgFTO and / or OprF.

[0049] The present invention also provides a transgenic plant or a transformed plant cell, wherein the transgenic plant or the transformed plant cell expresses the above nucleic acid molecule.

[0050] In one embodiment of the present invention, the method for preparing the transgenic plant comprises: using transgenic technology to transfer the nucleic acid molecule into a plant body to express the nucleic acid molecule in the plant to obtain the transgenic plant.

[0051] In one embodiment of the present invention, the method for preparing the transformed plant cells comprises: using transgenic technology to transfer the nucleic acid molecules into plant cells to express the nucleic acid molecules in the plant cells, thereby obtaining transformed plant cells.

[0052] In one embodiment of the present invention, the transgenic technology includes microbial mediation, microinjection and / or gene gun bombardment.

[0053] In one embodiment of the present invention, when the transgenic technology is mediated by microorganisms, the method for preparing the transgenic plant comprises: using the above-mentioned recombinant expression system to infect a plant body to express the above-mentioned nucleic acid molecule in the plant to obtain a transgenic plant.

[0054] In one embodiment of the present invention, when the transgenic technology is mediated by microorganisms, the method for preparing the transformed plant cells comprises: using the above-mentioned recombinant expression system to infect plant cells to express the above-mentioned nucleic acid molecules in the plant cells to obtain transformed plant cells.

[0055] In one embodiment of the present invention, the recombinant expression system is a host cell; the host cell is Agrobacterium transformed with a recombinant expression vector carrying the above nucleic acid molecule.

[0056] In one embodiment of the present invention, the recombinant expression system comprises a recombinant expression vector; the recombinant expression vector carries the above-mentioned nucleic acid molecule.

[0057] In one embodiment of the present invention, the recombinant expression vector comprises a plant cell expression vector carrying the above nucleic acid molecule.

[0058] In one embodiment of the present invention, the plant cell expression vector comprises the plant expression vector pCambia1300 and / or a pCambia1300 derivative vector.

[0059] In one embodiment of the present invention, the plant includes dicotyledonous plants and / or monocotyledonous plants.

[0060] In one embodiment of the present invention, the dicotyledonous plant includes Arabidopsis, tobacco, soybean, cotton, lettuce, poplar, alfalfa, rapeseed and / or peanut.

[0061] In one embodiment of the present invention, the monocotyledonous plant comprises corn, rice, sorghum, wheat, sugarcane and / or bamboo.

[0062] In one embodiment of the present invention, the target protein includes a functional protein.

[0063] In one embodiment of the present invention, the functional protein includes a functional protein used to enhance a target trait of a transgenic plant and / or a functional protein produced using the plant as a bioreactor.

[0064] In one embodiment of the present invention, the functional proteins include CRY1a.105 protein, CRY2Ab protein, Bar protein, CP4 protein, SARS-Cov-RBD, HA (Influenza Virus Hemagglutinin), AmFTO, GgFTO and / or OprF.

[0065] The present invention also provides a method for preparing transgenic plants or transformed plant cells, the method comprising: expressing the above nucleic acid molecules in plants to obtain transgenic plants, or expressing the above nucleic acid molecules in plant cells to obtain transformed plant cells.

[0066] In one embodiment of the present invention, the method for preparing the transgenic plant comprises: using transgenic technology to transfer the nucleic acid molecule into a plant body to express the nucleic acid molecule in the plant to obtain the transgenic plant.

[0067] In one embodiment of the present invention, the method for preparing the transformed plant cells comprises: using transgenic technology to transfer the nucleic acid molecules into plant cells to express the nucleic acid molecules in the plant cells, thereby obtaining transformed plant cells.

[0068] In one embodiment of the present invention, the transgenic technology includes microbial mediation, microinjection and / or gene gun bombardment.

[0069] In one embodiment of the present invention, when the transgenic technology is mediated by microorganisms, the method for preparing the transgenic plant comprises: using the above-mentioned recombinant expression system to infect a plant body to express the above-mentioned nucleic acid molecule in the plant to obtain a transgenic plant.

[0070] In one embodiment of the present invention, when the transgenic technology is mediated by microorganisms, the method for preparing the transformed plant cells comprises: using the above-mentioned recombinant expression system to infect plant cells to express the above-mentioned nucleic acid molecules in the plant cells to obtain transformed plant cells.

[0071] In one embodiment of the present invention, the recombinant expression system is a host cell; the host cell is Agrobacterium transformed with a recombinant expression vector carrying the above nucleic acid molecule.

[0072] In one embodiment of the present invention, the recombinant expression system comprises a recombinant expression vector; the recombinant expression vector carries the above-mentioned nucleic acid molecule.

[0073] In one embodiment of the present invention, the recombinant expression vector comprises a plant cell expression vector carrying the above nucleic acid molecule.

[0074] In one embodiment of the present invention, the plant cell expression vector comprises the plant expression vector pCambia1300 and / or a pCambia1300 derivative vector.

[0075] In one embodiment of the present invention, the plant includes dicotyledonous plants and / or monocotyledonous plants.

[0076] In one embodiment of the present invention, the dicotyledonous plant includes Arabidopsis, tobacco, soybean, cotton, lettuce, poplar, alfalfa, rapeseed and / or peanut.

[0077] In one embodiment of the present invention, the monocotyledonous plant comprises corn, rice, sorghum, wheat, sugarcane and / or bamboo.

[0078] In one embodiment of the present invention, the target protein includes a functional protein.

[0079] In one embodiment of the present invention, the functional protein includes a functional protein used to enhance a target trait of a transgenic plant and / or a functional protein produced using the plant as a bioreactor.

[0080] In one embodiment of the present invention, the functional proteins include CRY1a.105 protein, CRY2Ab protein, Bar protein, CP4 protein, SARS-Cov-RBD, HA (Influenza Virus Hemagglutinin), AmFTO, GgFTO and / or OprF.

[0081] The present invention also provides the use of the above codon optimization method or the above nucleic acid molecule or the above recombinant expression system or the above method in preparing transgenic plants or transforming plant cells.

[0082] The technical solution of the present invention has the following advantages:

[0083] The present invention provides a codon optimization method for increasing the protein expression of a target gene in a plant, the method comprising: replacing the third base of a codon of a target protein coding sequence with C or G to obtain an optimized target protein coding sequence. The existing concept is that each species has a corresponding optimal codon combination, therefore, the existing codon optimization methods are all optimized based on the frequency of codon usage of each species, replacing rare codons with high-frequency optimal codons. Contrary to the existing concept, the present invention provides an effective and universal codon optimization method for increasing the expression of a target protein in a plant, the method only requires replacing the third base of a codon of a target protein coding sequence with C or G. Studies have shown that after the codon optimization of the method, the protein expression of the target gene in a plant protein transient expression system and a transgenic plant is increased by several to several dozen times, and the transcription level of the target gene is increased by several to several hundred times, therefore, the method can effectively increase the protein expression of the target gene in a plant. In the production of functional proteins using plants as bioreactors, the method can increase the yield of the target protein, reduce the cost of purifying the target protein, and increase the edible / feeding effect of the plant; in the preparation of transgenic plants, the method can increase the expression amount of the target gene, thereby enhancing the target traits of the transgenic plants and reducing the screening cost of high-expression transgenic plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 : Plasmid map of plant expression vector pHEQ22.

[0085] Figure 2 :Effect of A / G / C / T ending codons on protein expression in tobacco leaves. Figure 2In the figure, A: Effect of A / G / C / T ending codons on the expression level of GFP protein in tobacco leaves; B: Effect of A / G / C / T ending codons on the expression level of RFP protein in tobacco leaves; C: Effect of A / G / C / T ending codons on the expression level of nanoLuc protein in tobacco leaves.

[0086] Figure 3 :Regulation of protein expression in tobacco leaves by degenerate bases C and G.

[0087] Figure 4 : Effects of codon optimization ratios on protein expression in tobacco leaves. Figure 4 In the figure, A: Effect of codon optimization ratio on GFP protein expression in tobacco leaves; B: Effect of codon optimization ratio on nanoLuc protein expression in tobacco leaves; C: Effect of codon optimization ratio on random protein (PR) expression in tobacco leaves.

[0088] Figure 5 :Effects of codon optimization on the expression levels of different functional proteins in tobacco leaves.

[0089] Figure 6 :Effect of A / G / C / T ending codons on protein expression in lettuce leaves.

[0090] Figure 7 : Plasmid map of plant expression vector pQH-GFP.

[0091] Figure 8 :Effect of A / G / C / T ending codons on protein expression in Arabidopsis thaliana.

[0092] Fig. 9 : Infection status of poplar seedlings.

[0093] Fig.10 : Effects of codon optimization on protein expression in cotton.

[0094] Fig.11 : Infection status of soybean immature embryos.

[0095] Fig.12 : Effects of codon optimization on protein expression in rice.

[0096] Fig.13 : Effects of codon optimization on protein expression in maize. DETAILED DESCRIPTION

[0097] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0098] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0099] Experimental Example 1: Effects of A / G / C / T ending codons on protein expression in tobacco leaves

[0100] Experiment 1: Using GFP as the target protein

[0101] The target gene encoding GFP was optimized according to the optimal codons of tobacco and was recorded as GFP-Nb. The corresponding optimized target protein coding sequence is SEQ ID NO: 1.

[0102] Based on GFP-Nb, codon optimization was performed as described in Table 1, and the degenerate base at the third position of the codon was optimized to A, T, G or C, respectively, recorded as GFP-A, GFP-T, GFP-G and GFP-C, and the corresponding optimized target protein coding sequences were SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

[0103] The optimized target protein coding sequences were synthesized and each optimized target protein coding sequence was constructed into the BamHI / StuI position of the plant expression vector pHEQ22 (SEQ ID NO: 66) by seamless cloning to replace the original EGFP fragment of the plant expression vector pHEQ22 to obtain different recombinant pHEQ22 vectors (the plasmid map of the plant expression vector pHEQ22 is shown in Figure 1, the promoter is 35S, and the terminator is 35S-ter in series with NOS-ter); the different recombinant pHEQ22 vectors were transformed into Agrobacterium C58C1 competent cells by electroporation (for electroporation method, see the literature: den Dulk-Ras, A., & Hooykaas, PJ (1995). Electroporation of Agrobacterium tumefaciens. Methods in molecular biology (Clifton, NJ), 55, 63-72.) to obtain different recombinant Agrobacterium; Nicotiana benthamiana was selected as the infection object, and the viable bacteria concentration OD 600 Different recombinant Agrobacterium suspensions with an OD of 0.2 were injected into tobacco leaves from the lower epidermis of the leaves to infect tobacco with different recombinant Agrobacterium; wherein the formula of the recombinant Agrobacterium suspension is: OD 600 =0.2 of recombinant Agrobacterium, 10 mM MgCl2 and 10 mM MES buffer (pH 5.6).

[0104] On the third day after injection, 30 mg of leaves infected with different recombinant Agrobacterium were weighed on a scale, the samples were ground, 800 μL of protein non-denaturing extraction buffer was added for protein extraction, and the expression of fluorescent protein GFP was detected by TECAN spark microplate reader. The specific detection parameters are: excitation light wavelength 485±20 nm, emission light wavelength 535±20 nm, gain value set to 54; among them, the formula of protein non-denaturing extraction buffer is: 150 mM NaCl, 1 mM EDTA, 1% (v / v) Triton X-100, 2% (v / v) glycerol, 1× protease inhibitor cocktail (purchased from Roche, model #04693159001) and 50 mM Tris–HCl buffer (pH 7.5). The test results are as follows Figure 2 As shown in A in the figure, the expression level of synonymous codons after optimization in G or C mode is significantly higher than that of genes optimized in A or T mode. Among them, the expression level of synonymous codons after optimization in G mode is 10 times and 5 times higher than that of A or T mode, respectively, and the expression level of synonymous codons after optimization in C mode is 17 times and 8 times higher than that of A or T mode, respectively.

[0105] Experiment 2: RFP as the target protein

[0106] The target gene encoding RFP was optimized according to the optimal codon of tobacco and recorded as RFP-Nb. The corresponding optimized target protein coding sequence is SEQ ID NO:6.

[0107] Based on RFP-Nb, codon optimization was performed as described in Table 1, and the degenerate base at the third position of the codon was optimized to A, T, G or C, respectively, recorded as RFP-A, RFP-T, RFP-G and RFP-C, and the corresponding optimized target protein coding sequences were SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

[0108] The optimized target protein coding sequences were synthesized, and each optimized target protein coding sequence was constructed into the BamHI / StuI position of the plant expression vector pHEQ22 by seamless cloning to replace the original EGFP fragment of the plant expression vector pHEQ22 to obtain different recombinant pHEQ22 vectors; the different recombinant pHEQ22 vectors were transformed into Agrobacterium C58C1 competent cells by electroporation to obtain different recombinant Agrobacterium; Nicotiana benthamiana was selected as the infection object, and the viable bacterial concentration OD was measured using a 1 mL syringe. 600 Different recombinant Agrobacterium suspensions with an OD of 0.2 were injected into tobacco leaves from the lower epidermis of the leaves to infect tobacco with different recombinant Agrobacterium; wherein the formula of the recombinant Agrobacterium suspension is: OD 600 = 0.2 of recombinant Agrobacterium, 10 mM MgCl2 and 10 mM MES buffer (pH 5.6).

[0109] On the third day after injection, 30 mg of leaves infected with different recombinant Agrobacterium were weighed on a scale, ground, and 800 μL of protein non-denaturing extraction buffer was added for protein extraction. The expression of fluorescent protein GFP was detected by TECAN spark microplate reader. The specific detection parameters were: excitation wavelength 532±20 nm, emission wavelength 588±20 nm, and gain value set to 68. The test results are shown in Figure 2. Figure 2 As shown in B, the expression level of the synonymous codons after optimization in the G or C mode is significantly higher than that of the genes optimized in the A or T mode.

[0110] Experiment 3: nanoLuc as the target protein

[0111] The target gene encoding nanoLuc was optimized according to the optimal codons of tobacco and recorded as nanoLuc-Nb. The corresponding optimized target protein coding sequence is SEQ ID NO:11.

[0112] Based on nanoLuc-Nb, codon optimization was performed as described in Table 1, and the degenerate base at the third position of the codon was optimized to A, T, G or C, respectively, denoted as nanoLuc-A, nanoLuc-T, nanoLuc-G and nanoLuc-C, and the corresponding optimized target protein coding sequences were SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

[0113] The optimized target protein coding sequence was synthesized, and each optimized target protein coding sequence was constructed into the BamHI / StuI position of the plant expression vector pHEQ22 by seamless cloning to replace the original EGFP fragment of the plant expression vector pHEQ22 to obtain different recombinant pHEQ22 vectors; the different recombinant pHEQ22 vectors were transformed into Agrobacterium C58C1 competent cells by electroporation to obtain different recombinant Agrobacterium; Nicotiana benthamiana was selected as the infection object, and a 1 mL syringe was used to inject live bacteria with a concentration of OD 600 Different recombinant Agrobacterium suspensions with an OD of 0.2 were injected into tobacco leaves from the lower epidermis of the leaves to infect tobacco with different recombinant Agrobacterium; wherein the formula of the recombinant Agrobacterium suspension is: OD 600 = 0.2 of recombinant Agrobacterium, 10 mM MgCl2 and 10 mM MES buffer (pH 5.6).

[0114] On the third day after injection, 30 mg of leaves infected with different recombinant Agrobacterium were weighed on a scale, the samples were ground, and 800 μL of protein non-denaturing extraction buffer was added for protein extraction to obtain a protein extract; 10 μL of protein extract was added to 990 μL of protein non-denaturing extraction buffer and diluted 100 times, mixed to obtain a first dilution; 9 μL of the first dilution was added to 351 μL of protein non-denaturing extraction buffer and diluted 40 times, mixed to obtain a second dilution; 2 μL of 5 mM Furimazine substrate (purchased from Jinzhida (Shanghai) Biotechnology Co., Ltd.) was added to 360 μL of the second dilution, vortexed to mix, and the timing started from the addition of the substrate. After 3 minutes, the expression level of the chemiluminescent protein nanoLuc was detected by the TECAN spark microplate reader. The specific detection parameters are: detecting the number of photons emitted between wavelengths of 360 and 700 nm per second (Integration time). The detection results are as follows: Figure 2 As shown in C, the expression level of the synonymous codons after optimization in the G or C mode is significantly higher than that of the genes optimized in the A or T mode.

[0115] comprehensive Figure 2The results show that the expression level of the gene codon optimized to C is higher than that of the gene codon optimized to G. This may be because there are 15 amino acids with synonymous codons ending in C, while there are only 11 amino acids with synonymous codons ending in G, that is, the number of codons corresponding to the amino acid that needs to be optimized when the third degenerate base is optimized to C is greater than that of the amino acid that needs to be optimized to G. These results show that the optimal codons of tobacco tend to use codons ending in G or C.

[0116] Table 1 Optimization methods for codons ending with A / T / G / C

[0117]

[0118] Experimental Example 2: Effects of degenerate bases G or C on protein expression in tobacco leaves

[0119] The results of Experimental Example 1 showed that the protein expression level of G / C ending codons was higher than that of A / T ending codons. However, it was found from Table 1 that the third degenerate bases of alanine A, glycine G, proline P, threonine T, valine V, arginine R, leucine L and serine S contained both G and C.

[0120] In order to identify whether the third degenerate bases G and C of the above amino acids have different regulatory effects on protein expression, the codons corresponding to alanine A, glycine G, and proline P were optimized to GCC, GGC, and CCC on the basis of GFP-G, denoted as GFP-G / C--AGP, and the corresponding optimized target protein coding sequence is SEQ ID NO: 16;

[0121] The codons corresponding to threonine T and valine V were optimized to ACC and GUC, denoted as GFP-G / C--TV, and the corresponding optimized target protein coding sequence is SEQ ID NO: 18;

[0122] The codons corresponding to leucine L and serine S were optimized to CUC and AGC, denoted as GFP-G / C--LS, and the corresponding optimized target protein coding sequence is SEQ ID NO: 20;

[0123] The codons corresponding to threonine T and serine S were optimized to ACC and AGC, denoted as GFP-G / C--TS, and the corresponding optimized target protein coding sequence is SEQ ID NO: 22;

[0124] The codons corresponding to valine V and leucine L were optimized to GUC and CUC, denoted as GFP-G / C--VL, and the corresponding optimized target protein coding sequence is SEQ ID NO: 24;

[0125] The codons corresponding to alanine A and glycine G were optimized to GCC and GGC, denoted as GFP-G / C--AG, and the corresponding optimized target protein coding sequence is SEQ ID NO: 26;

[0126] The codons corresponding to proline P and arginine R were optimized to CCC and CGC, denoted as GFP-G / C--PR, and the corresponding optimized target protein coding sequence was SEQ ID NO:28.

[0127] At the same time, based on GFP-C, the codons corresponding to alanine A, glycine G, and proline P were optimized to GCG, GGG, and CCG, denoted as GFP-C / G--AGP, and the corresponding optimized target protein coding sequence is SEQ ID NO: 17;

[0128] The codons corresponding to threonine T and valine V were optimized to ACG and GUG, denoted as GFP-C / G--TV, and the corresponding optimized target protein coding sequence is SEQ ID NO: 19;

[0129] The codons corresponding to leucine L and serine S were optimized to CUG and UCG, denoted as GFP-C / G--LS, and the corresponding optimized target protein coding sequence is SEQ ID NO: 21;

[0130] The codons corresponding to threonine T and serine S were optimized to ACG and UCG, denoted as GFP-C / G--TS, and the corresponding optimized target protein coding sequence is SEQ ID NO: 23;

[0131] The codons corresponding to valine V and leucine L were optimized to GUG and CUG, denoted as GFP-C / G--VL, and the corresponding optimized target protein coding sequence is SEQ ID NO: 25;

[0132] The codons corresponding to alanine A and glycine G were optimized to GCG and GGG, denoted as GFP-C / G--AG, and the corresponding optimized target protein coding sequence is SEQ ID NO: 27;

[0133] The codons corresponding to proline P and arginine R were optimized to CCG and AGG, denoted as GFP-C / G--PR, and the corresponding optimized target protein coding sequence was SEQ ID NO:29.

[0134] After synthesizing the optimized target protein coding sequence, the expression level of fluorescent protein GFP was detected according to the method of Experiment 1 in Experiment 1. The detection results are as follows Figure 3As shown in the figure, the relative expression levels of GFP-G, GFP-C, GFP-G / C--AGP, GFP-C / G--AGP, GFP-G / C--AG, GFP-C / G--AG, GFP-G / C--PR and GFP-C / G--PR proteins were compared, and the results showed that the codons ending with G or C detected by alanine A, glycine G, proline P and arginine R had similar effects, and were all optimal codons; the relative expression levels of GFP-G, GFP-C, GFP-G / C--TV, GFP-C / G--TV, GFP-G / C--TS, GFP-C / G--TS, GFP-G / C--VL and GFP-C / G--VL proteins were compared, and the results showed that the codons ending with G or C detected by valine V, serine S and leucine L had similar effects, and were all optimal codons, but the protein expression level increased after the threonine T codon ACG was changed to ACC, and conversely, the protein expression level decreased after ACC was changed to ACG, indicating that ACC is the optimal codon for threonine T.

[0135] Experimental Example 3: Effect of codon optimization ratio on protein expression in tobacco leaves

[0136] Experiment 1: Using GFP as the target protein

[0137] In order to study the relationship between codon optimization ratio and protein expression, GFP-Nb was gradient optimized and recorded as GFP-1, GFP-2, GFP-3, GFP-4, and GFP-5, and the corresponding optimized target protein coding sequences were SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34. The GC% of GFP-Nb, GFP-1, GFP-2, GFP-3, GFP-4, and GFP-5 were 32%, 40%, 45%, 50%, 55%, and 62%, respectively, and the GC3% were 11%, 35%, 50%, 65%, 77%, and 100%, respectively.

[0138] After synthesizing the optimized target protein coding sequence, the expression level of fluorescent protein GFP was detected according to the method of Experiment 1 in Experiment 1. The detection results are as follows Figure 4 As shown in A, as the optimization ratio increases, the protein expression level gradually increases; when the GC3 is greater than 50% after gene codon optimization, the protein expression levels are 4 times, 5 times and 9 times that of GFP-Nb, respectively.

[0139] Experiment 2: nanoLuc as the target protein

[0140] In order to further verify the results of Experiment 1, nanoLuc-Nb was subjected to similar gradient optimization and recorded as nanoLuc-1, nanoLuc-2, nanoLuc-3, nanoLuc-4, and nanoLuc-5, and the corresponding optimized target protein coding sequences were SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39. The GC% of nanoLuc-Nb, nanoLuc-1, nanoLuc-2, nanoLuc-3, nanoLuc-4, and nanoLuc-5 were 32%, 40%, 45%, 50%, 55%, and 64%, respectively, and the GC3 were 8%, 30%, 43%, 56%, 72%, and 100%, respectively.

[0141] After synthesizing the optimized target protein coding sequence, the expression level of the chemiluminescent protein nanoLuc was detected by referring to the method of Experiment 3 in Experimental Example 1. The detection results are as follows Figure 4 As shown in B, consistent with GFP, the expression level of nanoLuc protein gradually increased with the increase of optimization ratio; when GC3 was greater than 50% after gene codon optimization, the protein expression levels were 3, 4 and 5 times that of nanoLuc-Nb, respectively.

[0142] Experiment 3: Random sequence protein as target protein

[0143] In order to further verify whether the codon optimization method proposed in the present invention is applicable to all genes, three random protein sequences consisting of 200 amino acids were designed by computer. NCBI blast found that they had no correlation with existing proteins. The random protein sequences were named PR1 (Protein Random 1), PR2 and PR3.

[0144] PR1, PR2 and PR3 were reverse translated into protein coding sequences according to the optimal codons of tobacco, recorded as PR1-Nb, PR2-Nb and PR3-Nb, and the corresponding protein coding sequences were SEQ ID NO:40, SEQ ID NO:42, and SEQ ID NO:44, respectively.

[0145] PR1-Nb, PR2-Nb and PR3-Nb were further optimized, and their GC3 were optimized to 100%, recorded as PR1-GC, PR2-GC and PR3-GC, and the corresponding optimized target protein coding sequences were SEQ ID NO:41, SEQ ID NO:43, and SEQ ID NO:45, respectively.

[0146] After synthesizing the protein coding sequence and the optimized target protein coding sequence, each protein coding sequence and each optimized target protein coding sequence were constructed into the SalI / BamHI position of the plant expression vector pHEQ22 by seamless cloning, and the expression amount of the target protein was detected by referring to the method of Experiment 1 in Experimental Example 1. The detection results are as follows Figure 4 As shown in C, after the PR codon was completely optimized to a G / C ending codon, the protein expression levels were 8, 7, and 9 times higher than before optimization, respectively.

[0147] comprehensive Figure 4 The results show that the optimal codon of dicotyledonous tobacco ends with G / C instead of A / T, and the higher the codon optimization ratio, the higher the protein expression. Importantly, the codon optimization method proposed in the present invention is not only applicable to reporter genes, but may be applicable to all genes.

[0148] Experimental Example 4: Effects of codon optimization on the expression of different functional proteins in tobacco leaves

[0149] In order to further verify that the codon optimization method proposed in the present invention is applicable to common transgenic plant functional genes, commonly used transgenic proteins in plants were selected: insect-resistant protein CRY1a.105, insect-resistant protein CRY2Ab, herbicide-resistant protein Bar, herbicide-resistant protein CP4, new coronavirus receptor protein ACE2 (Angiotensin-Converting Enzyme 2), new coronavirus spike protein receptor binding domain protein SARS-Cov-RBD, influenza virus hemagglutinin protein HA (Influenza Virus Hemagglutinin), giant panda obesity-related protein AmFTO, Hongyuan chicken obesity-related protein GgFTO and fish immune protein OprF, and the corresponding protein coding sequences are SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:69, respectively.

[0150] The above protein coding sequences were codon optimized, and their GC3 was optimized to 100%, recorded as CRY1a.105-GC, CRY2Ab-GC, Bar-GC, CP4-GC, ACE2-GC, SARS-Cov-RBD-GC, HA-GC, AmFTO-GC, GgFTO-GC, OprF-GC, and the corresponding optimized target protein coding sequences were SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, and SEQID NO:70, respectively.

[0151] After synthesizing the optimized target protein coding sequence, each protein coding sequence and each optimized target protein coding sequence were constructed into the SalI / BamHI position of the plant expression vector pHEQ22 by seamless cloning and fused with the eGFP contained in the vector, and the expression amount of the target protein was detected by referring to the method of Experiment 1 in Experimental Example 1. The detection results are as follows Figure 5 As shown, the expression of these functional gene proteins is at least increased by 1 times, which will help to further improve the expression level of transgenic plant resistance genes and improve their performance such as herbicide resistance and pest resistance. These results further show that the codon optimization method proposed in the present invention is suitable for common transgenic plant functional genes. Among them, the effect of increasing the expression of functional genes is not as obvious as that of reporter genes. It may be that the functional genes are fused with GFP and each pair of genes contains the same GFP sequence, thereby reducing the effect of codon optimization on the expression of functional gene proteins.

[0152] Experimental Example 5: Effect of A / G / C / T ending codons on protein expression in lettuce

[0153] In order to study whether the optimal codon of the dicotyledonous plant lettuce also ends with G / C, the Agrobacterium containing GFP-Nb and GFP-5 obtained in Experiment 1 of Experimental Example 3 was used to select the young leaves of lettuce (glass lettuce) grown for 3 weeks as the infection objects, and the viable bacteria concentration OD 600 The recombinant Agrobacterium suspension with an OD of 0.2 was injected into the lettuce leaves from the lower epidermis of the leaves to infect the lettuce with the recombinant Agrobacterium; wherein the formula of the recombinant Agrobacterium suspension was: OD 600 = 0.2 of recombinant Agrobacterium, 10 mM MgCl2 and 10 mM MES buffer (pH 5.6).

[0154] On the third day after injection, the expression of GFP was observed using a handheld fluorescent microscope. Figure 6As shown, almost no expression was observed for GFP-Nb with A / T ending optimized, while obvious fluorescence signal was observed for GFP-5 with GC3 optimized to 100%, indicating that the most suitable codon in lettuce also ends with G / C, just like tobacco.

[0155] Experimental Example 6: Effects of A / G / C / T ending codons on protein expression in Arabidopsis

[0156] In order to study whether the optimal codon of the dicotyledonous plant Arabidopsis thaliana also ends with G / C, the GFP-A, GFP-T, GFP-G, and GFP-C obtained in Experiment 1 of Experimental Example 1 were used to construct the optimized target protein coding sequences into the BamHI / StuI position of the plant expression vector pQH-GFP (SEQ ID NO: 67) by seamless cloning to replace the original GFP fragment of the plant expression vector pQH-GFP, and different recombinant pQH-GFP vectors were obtained (the plasmid map of the plant expression vector pQH-GFP is shown in Figure 7 , the promoter is 35S, and the terminator is 35S-ter in series with NOS-ter); different recombinant pQH-GFP vectors were transformed into Agrobacterium C58C1 competent cells by electric shock method to obtain different recombinant Agrobacterium; Arabidopsis thaliana (Columbia) was selected as the infection object, and different recombinant Agrobacterium were used to infect Arabidopsis thaliana using the floral dip method (see reference: Clough SJ, Bent A.F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743.).

[0157] The T1 generation seeds were harvested, and the number of T1 generation positive strains (hygromycin resistance) with GFP fluorescence was observed by handheld fluorescence observation at the cotyledon stage. The test results are shown in Table 2. Among the 41 GFP-A and 96 GFP-T independently transformed Arabidopsis T1 generation plants, no single plant with GFP fluorescence signal was found, but among the GFP-G and GFP-C Arabidopsis T1 generation plants, about 30% of the single plants had obvious fluorescence signals. These results show that G or C ending codons are the optimal codons for Arabidopsis. After the codons are optimized to G or C endings, not only can the protein expression amount be significantly increased, but also the screening efficiency of high-expression transgenic plants can be improved.

[0158] In order to further analyze the expression levels of GFP-A, GFP-T, GFP-G, and GFP-C transgenic Arabidopsis, 8 strains of Arabidopsis infected with each recombinant Agrobacterium were randomly selected on the third day after injection and qPCR analysis was performed using a fluorescent PCR kit (Takara, RR820A). The qPCR results are shown in Figure 8 As shown, the transcription levels of GFP-A and GFP-T in all tested strains were very low, while the transcription levels of GFP-G and GFP-C were several times or even hundreds of times higher than that of GFP-A or GFP-T.

[0159] Comprehensive Table 2 and Figure 8 The results show that the optimal codon of Arabidopsis thaliana also ends with G or C. After the codon is optimized to end with G or C, the transcription level of the target gene increases several times or even hundreds of times, making it easier to screen high-expressing transgenic materials.

[0160] Table 2 Fluorescence rate of Arabidopsis thaliana T1 transgenic materials

[0161] Total number of plants Number of fluorescent plants Fluorescence rate GFP-A 41 0 0.00％ GFP-T 96 0 0.00％ GFP-G 17 5 29.41％ GFP-C 40 12 30.00％

[0162] Experimental Example 7: Effects of A / G / C / T ending codons on protein expression in poplar

[0163] In order to study whether the optimal codon of poplar also ends with G / C, ZsGreen with higher fluorescence intensity was selected as the target protein, and the protein coding sequence of the target protein was codon optimized. The degenerate base corresponding to the third position of the codon was optimized to G / C or A / T, respectively, recorded as ZsGreen-G / C and ZsGreen-A / T, and the corresponding optimized target protein coding sequences are SEQ ID NO:64 and SEQ ID NO:65.

[0164] After synthesizing the optimized target protein coding sequence, each optimized target protein coding sequence was constructed into the BamHI / StuI position of the plant expression vector pQH-GFP by seamless cloning to replace the original GFP fragment of the plant expression vector pQH-GFP, and different recombinant pQH-GFP vectors were obtained; the different recombinant pQH-GFP vectors were transformed into Agrobacterium C58C1 competent cells by electric shock to obtain different recombinant Agrobacterium; poplar seedlings (Populus argentea) were selected as the infection objects, and the young leaves of poplar seedlings were infected with different recombinant Agrobacterium using the leaf disc method (see literature: Bruegmann T., Polak O., Deecke K., Nietsch J., Fladung M. (2019) Poplar Transformation. Methods Mol Biol 1864: 165-177.).

[0165] One month after infection, the fluorescence of T1 seedlings was observed using a handheld fluorescent lamp. Fig. 9 As shown, no fluorescence was observed in seedlings infected with ZsGreen-A / T, while obvious fluorescence appeared in seedlings infected with ZsGreen-G / C, indicating that the optimal codon for the dicotyledonous plant poplar also ends with G or C, and the protein expression level was significantly improved after optimization.

[0166] Experimental Example 8: Effect of codon optimization on protein expression in cotton

[0167] Using the Agrobacterium containing GFP-Nb and GFP-5 obtained in Experiment 1 of Experimental Example 3, 2-week-old cotton seedlings (Zhongmian Institute 29) were selected as the infection objects, and the viable bacterial concentration OD 600 The recombinant Agrobacterium suspension with an OD of 0.2 was injected into the young leaves of cotton seedlings from the lower epidermis of the leaves to infect the young leaves of cotton; wherein the formula of the recombinant Agrobacterium suspension was: OD 600 =0.2 of recombinant Agrobacterium, 10 mM MgCl2 and 10 mM MES buffer (pH 5.6).

[0168] On the third day after injection, the expression of GFP was observed using a handheld fluorescent microscope. Fig.10 As shown, regardless of whether the transcriptional repressor protein p19 is co-expressed (the transcriptional repressor protein p19 can inhibit plant post-transcriptional silencing and promote the expression of exogenous genes, see the literature: Voinnet O., Rivas S., Mestre P., Baulcombe D. (2003) An enhanced transient expression system in plants based on suppression of genesilencing by the p19 protein of tomato bushy stunt virus. Plant J 33: 949-956.), after the codons are optimized in the G / C mode, the protein expression level is significantly higher than that in the A / T ending codon optimization mode, indicating that in the dicotyledonous plant cotton, the optimal codon also ends with G or C, and the protein expression level is significantly improved after optimization.

[0169] Experimental Example 9: Effect of codon optimization on protein expression in soybean

[0170] The hygromycin resistance gene in pQH-GFP was replaced with the glufosinate-resistant bar gene by enzyme digestion and seamless cloning, and the vector was named pQB-GFP (SEQ ID NO: 68).

[0171] The ZsGreen-G / C and ZsGreen-A / T obtained in Experimental Example 7 were seamlessly cloned into the BamHI / StuI position of the vector pQB-GFP to replace the original GFP fragment of the plant expression vector pQB-GFP, thereby obtaining different recombinant pQB-GFP vectors; the different recombinant pQB-GFP vectors were transformed into Agrobacterium C58C1 competent cells by electroporation to obtain different recombinant Agrobacterium; soybean immature embryos (Tianlong No. 1) were selected as the infection objects, and different recombinant Agrobacterium were used to infect soybean immature embryos through cotyledon nodes (for the infection method, see the literature: Ko TS, Korban SS, Somers DA (2006) Soybean (Glycinemax) transformation using immature cotyledon explants. Methods Mol Biol 343: 397-405.).

[0172] After infection, the soybean seedlings were cultured in the dark for 3 days and in recovery culture for 7 days (for dark culture and recovery culture, see the literature: Ko TS, Korban SS, Somers DA (2006) Soybean (Glycine max) transformation using immature cotyledon explants. Methods Mol Biol 343: 397-405.), and the infection was observed by handheld fluorescence. Fig.11 As shown, the protein expression level of ZsGreen-G / C was significantly higher than that of ZsGreen-A / T, indicating that the optimal codon of the dicotyledonous plant soybean also ends with G or C, and the protein expression level was significantly improved after optimization.

[0173] Experimental Example 10: Effect of codon optimization on protein expression in rice

[0174] Using the Agrobacterium containing ZsGreen-G / C and ZsGreen-A / T obtained in Experimental Example 9, rice seedlings (Japanese early-maturing japonica rice Kitaake) were selected as the infection objects, and different recombinant Agrobacterium were used to infect the young leaves of rice seedlings (for the infection method, see the document: Nishimura A. (2020) Agrobacterium Transformation in the Rice Genome. Methods Mol Biol 2072: 207-216.).

[0175] After infection, the rice seedlings were subjected to dark culture, dedifferentiation, differentiation, and rooting culture (for dark culture, dedifferentiation, differentiation, and rooting culture, see the literature: Nishimura A. (2020) Agrobacterium Transformation in the Rice Genome. Methods Mol Biol 2072: 207-216.) to obtain rice T1 transgenic seedlings. Five independent rice T1 transgenic seedlings were randomly selected, and protein expression was detected by Western Blot (for Western Blot, see the literature: Hnasko TS, Hnasko RM (2015) The Western Blot. Methods Mol Biol 1318: 87-96.). The results are as follows Fig.12 As shown, after the codon of the rice transgenic material was optimized to end with G / C, the protein expression level was significantly increased, indicating that the optimal codon of the monocotyledonous plant rice also ends with G or C, and the protein expression level was significantly increased after optimization.

[0176] Experimental Example 11: Effect of codon optimization on protein expression in corn

[0177] Using the Agrobacterium containing ZsGreen-G / C and ZsGreen-A / T obtained in Experimental Example 9, immature maize embryos (maize inbred line B104) about 10 days after maize pollination were selected as the infection objects, and different recombinant Agrobacterium were used to infect the immature maize embryos (for the method of obtaining and infecting immature maize embryos, see the literature: Ishida Y., Hiei Y., Komari T. (2007) Agrobacterium-mediated transformation of maize. Nat Protoc 2: 1614-1621.).

[0178] After infection, the maize immature embryos were cultured in the dark for 5 days and then in recovery culture for 5 days (for dark culture and recovery culture, see the reference: Ishida Y., Hiei Y., Komari T. (2007) Agrobacterium-mediated transformation of maize. Nat Protoc 2: 1614-1621.), and the expression of exogenous gene proteins in maize immature embryos was observed by handheld fluorescence. Fig.13 As shown, fluorescent protein expression was only observed in immature maize embryos infected with ZsGreen-G / C, and no fluorescent protein expression was observed in more than 100 immature maize embryos infected with ZsGreen-A / T. These results indicate that the optimal codon for monocot maize also ends with G or C, and the protein expression level was significantly improved after optimization.

[0179] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A codon optimization method for increasing the protein expression of a target gene in a plant, characterized in that: The method comprises: replacing the third base of the codon of the target protein coding sequence with C or G to obtain an optimized target protein coding sequence.

2. The codon optimization method according to claim 1, characterized in that The GC content of the optimized target protein coding sequence is no less than 55%.

3. The codon optimization method according to claim 1 or 2, characterized in that In the optimized target protein coding sequence, the GC content of the third base of all codons is not less than 70%.

4. The codon optimization method according to any one of claims 1 to 3, characterized in that: In the method, when codon replacement is performed, the third base of the codon corresponding to threonine is replaced with C.

5. The codon optimization method according to any one of claims 1 to 4, characterized in that: In the optimized target protein coding sequence, more than 50% of the segments have undergone replacement of the third base of the codon.

6. A nucleic acid molecule for expressing a target protein in a plant, characterized in that: The nucleic acid molecule is optimized using the codon optimization method according to any one of claims 1 to 5.

7. The nucleic acid molecule according to claim 6, wherein The target protein includes a functional protein; the functional protein includes a functional protein used to enhance the target trait of a transgenic plant and / or a functional protein produced using the plant as a bioreactor; The functional proteins include CRY1a.105 protein, CRY2Ab protein, Bar protein, CP4 protein, novel coronavirus receptor protein ACE2, novel coronavirus spike protein receptor binding domain protein SARS-Cov-RBD, influenza virus hemagglutinin protein HA, giant panda obesity-related protein AmFTO, Hongyuan chicken obesity-related protein GgFTO and / or fish immune protein OprF; When the target protein is the insect-resistant protein CRY1a.105, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.47; When the target protein is the insect-resistant protein CRY2Ab, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.49; When the target protein is the herbicide-resistant protein Bar, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.51; When the target protein is the herbicide-resistant protein CP4, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.53; When the target protein is the novel coronavirus receptor protein ACE2, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.55; When the target protein is the novel coronavirus spike protein receptor binding domain protein SARS-Cov-RBD, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.57; When the target protein is influenza virus hemagglutinin protein HA, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.59; When the target protein is giant panda obesity-related protein AmFTO, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.61; When the target protein is Hongyuan chicken obesity-related protein GgFTO, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.63; When the target protein is fish immune protein OprF, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.

70.

8. A recombinant expression system for expressing a target protein in a plant, characterized in that: The recombinant expression system expresses the nucleic acid molecule according to claim 6 or 7.

9. A transgenic plant or transformed plant cell, characterized in that: The transgenic plant or transformed plant cell expresses the nucleic acid molecule according to claim 6 or 7.

10. Use of the codon optimization method according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6 or 7, or the recombinant expression system according to claim 8 in preparing transgenic plants or transforming plant cells.

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