Method for improving expression abundance of target gene protein by optimizing peanut nucleotide codon sequence

Abstract

The invention provides a method for improving the protein expression abundance of a target gene by optimizing a peanut nucleotide codon sequence, and relates to the technical field of biology. The method for improving the expression abundance of the target gene protein mainly comprises the following steps: obtaining the occurrence frequency of peanut gene codons and the abundance of tRNA corresponding to each codon combination in peanut cells through peanut genome sequencing analysis, and optimizing a codon sequence corresponding to low-abundance tRNA into a codon corresponding to high-abundance tRNA. According to the invention, the defects in the prior art are overcome, and a part of triad codes in an original target gene sequence are replaced with the same amino acid with higher tRNA abundance, so that the target gene can be obtained by optimizing codons of an original nucleotide sequence of a gene for coding protein on the premise of not changing an expression amino acid sequence; therefore, the target gene protein expression abundance in the cells is improved by utilizing the difference of different tRNA abundance in the cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for optimizing peanut nucleotide codon sequences to increase the expression abundance of target gene proteins. Background Art

[0002] Codon preference refers to the fact that different organisms have different frequencies of using degenerate codons during the translation process, and a set of commonly used codons that are adapted to them have been formed during the evolution process. During the translation of proteins, tRNA transports amino acids to the ribosome, and the triplet codon in mRNA determines the amino acid sequence. Due to the degeneracy of codons, the same amino acid can be encoded by multiple codons. Although there are as many as 61 types of tRNA, organisms do not have all types, and the abundance of various tRNAs in different organisms and different cells also varies significantly. If some tRNAs required for gene translation are low in abundance or non-existent in the cell, it will greatly affect the efficiency of the gene in the mRNA translation stage, and then affect the abundance of protein expression of the gene. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention provides a method for optimizing peanut nucleotide codon sequences to increase the abundance of target gene protein expression.

[0004] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0005] A method for optimizing peanut nucleotide codon sequences to increase target gene protein expression abundance, the method comprising the following optimization methods:

[0006] (1) The frequency of occurrence of peanut gene codons and the abundance of tRNA corresponding to each codon combination in peanut cells are obtained through peanut genome sequencing analysis, and the codon sequence corresponding to the low-abundance tRNA is optimized to the codon corresponding to the high-abundance tRNA;

[0007] (2) Based on the nucleotide sequence composition and tRNA abundance of peanuts, the original nucleotide sequence of the protein-encoding gene is optimized without changing the expressed amino acid sequence, thereby adapting to the differences in tRNA abundance in peanut cells, thereby increasing the protein expression abundance of the target gene in peanut cells;

[0008] (3) Based on the degeneracy of codons, without changing the results of gene expression, by changing the arrangement of individual nucleotides in the DNA or RNA sequence, the codon combination at some sites on the mRNA chain to be translated is changed, and the triplet codons at some positions with low tRNA abundance and relatively more difficult to participate in translation are replaced with triplet codons with high tRNA abundance and higher translation efficiency.

[0009] Preferably, the expression product corresponding to the nucleic acid is an exogenous protein or a gene expression product of peanut itself.

[0010] Preferably, the codon sequence replacement operation rule follows that the codon combination with lower tRNA abundance is replaced with the codon combination with the highest tRNA abundance in the same amino acid, and the replacement priority is as follows:

[0011] (1) The priority order for leucine replacement is: TTG—CTT—CTC—CTG—TTA—CTA;

[0012] (2) The priority order for phenylalanine substitution is: TTC-TTT;

[0013] (3) The priority order for isoleucine substitution is: ATT—ATC—ATA;

[0014] (4) The priority order for valine replacement is: GTT—GTG—GTC—GTA;

[0015] (5) The priority order for tyrosine substitution is: TAT-TAC;

[0016] (6) The priority order for histidine replacement is: CAT-CAC;

[0017] (7) The priority order for glutamine replacement is: CAA—CAG;

[0018] (8) The priority order for asparagine substitution is: AAT-AAC;

[0019] (9) The priority order for lysine replacement is: AAG-AAA;

[0020] (10) The priority order for aspartate substitution is: GAT—GAC;

[0021] (11) The priority order for glutamate replacement is: GAG—GAA;

[0022] (12) The priority order for serine substitution is: TCA—TCT—TCC—AGC—AGT—TCG;

[0023] (13) The priority order for proline substitution is: CCA—CCT—CCC—CCG;

[0024] (14) The priority order for threonine substitution is: ACT—ACA—ACC—ACG;

[0025] (15) The priority order for alanine substitution is: GCT—GCA—GCC—GCG;

[0026] (16) The priority ranking for cysteine ​​substitution is: TGC—TGT;

[0027] (17) The priority order for glycine substitution is: GGA—GGT—GGC—GGG;

[0028] (18) The priority order for arginine substitution is: AGA—AGG—CGT—CGC—CGA—CGG;

[0029] (19) The priority order for stop codon replacement is: TGA—TAA—TAG.

[0030] Preferably, when the amino acid is replaced, methionine and tryptophan do not have two or more codons and do not involve any codon replacement operation.

[0031] Preferably, in the optimization method, the replacement amount of triplet codes in the nucleotide sequence is ≥25%, and the change amount of the amino acid sequence is ≤5%.

[0032] And the above method is applied to breeding new peanut varieties.

[0033] Preferably, the application method comprises the following steps:

[0034] (1) causing the peanut cell genome to produce an optimized mutation as described in any one of claims 1 to 5;

[0035] (2) making the peanut plant contain cells, tissues, organs, or materials obtained by the optimization method according to any one of claims 1 to 5;

[0036] (3) enabling peanut plants to express genes optimized by the optimization method described in any one of claims 1 to 5.

[0037] The present invention provides a method for optimizing peanut nucleotide codon sequences to increase the abundance of target gene protein expression, which has the following advantages over the prior art:

[0038] The present invention replaces some triplet codons in the original target gene sequence with triplet codons corresponding to the same amino acid but with higher tRNA abundance, thereby optimizing the codons of the original nucleotide sequence of the gene encoding the protein without changing the expressed amino acid sequence, thereby utilizing the difference in the abundance of different tRNAs in the cell to increase the abundance of the target gene protein in the cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a codon replacement comparison diagram of the GFP-PNT optimized gene compared to the GFP gene in Example 1 of the present invention;

[0040] Figure 2This is a schematic diagram of the complete structure of the transformation vector used for gene editing in Example 1 of the present invention;

[0041] Figure 3 This is a schematic diagram of the fluorescence reaction of Yuhua 9326 (GFP-PNT optimized gene variety) in Example 2 of the present invention;

[0042] Figure 4 This is a schematic diagram of the fluorescence reaction of Yuhua 9326 (introduced non-optimized GFP variety) in Example 2 of the present invention;

[0043] Figure 5 This is a schematic diagram of the fluorescence reaction of Yuhua 9326 (a variety without GFP introduction) of the present invention. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] Embodiment 1:

[0046] In order to verify the effectiveness of the method for increasing the expression abundance of the target gene protein involved in the present invention, GFP fluorescent protein was introduced into the Yuhua 9326 variety of peanuts. It was noted that the intensity of green fluorescence of the Yuhua 9326 variety of peanut tissues that developed into complete plants after being treated by this method was lower than expected after blue-violet light excitation. According to molecular biological analysis, the transcription amount of the GFP gene was basically in line with expectations, but the corresponding mRNA translation speed was limited by the low abundance of tRNA corresponding to some medium and high frequency codons, and the translation speed was always low, which was not enough to fully express and improve the manifestation of fluorescence traits.

[0047] The effectiveness of the method for increasing the expression abundance of the target gene protein of the present invention can be verified by taking the fluorescence intensity of the peanut material before and after the GFP gene sequence is optimized as an indicator.

[0048] After sequencing, the complete sequence of the GFP gene is as follows:

[0049] ATG GTG AGC AAG GGC GAG GAG CTG TTC ACC GGG GTGGTG CCC ATC CTG GTCGAG CTG GAC GGC GAC GTAAAC GGCCAC AAG TTC AGC GTG TCC GGC GAG GGC GAG GGCGATGCC ACC TAC GGC AAG CTG ACC CTG AAG TTC ATC TGC ACCACC GGC AAG CTG CCC GTGCCC TGG CCC ACC CTC GTG ACCACC CTG ACC TAC GGC GTG CAG TGC TTC AGC CGC TACCCCGAC CAC ATG AAG CAG CAC GAC TTC TTC AAG TCC GCC ATGCCC GAA GGC TAC GTC CAGGAG CGC ACC ATC TTC TTC AAGGAC GAC GGC AAC TAC AAG ACC CGC GCC GAG GTG AAGTTCGAG GGC GAC ACC CTG GTGAAC CGCATC GAG CTGAAGGGC ATC GAC TTC AAG GAG GAC GGCAAC ATC CTG GGG CACAAG CTG GAG TAC AAC TAC AAC AGC CAC AAC GTC TAT ATCATG GCCGAC AAG CAGAAGAAC GGC ATCAAG GTG AAC TTCAAG ATC CGC CAC AAC ATC GAG GAC GGCAGC GTG CAG CTCGCC GAC CAC TAC CAG CAG AAC ACC CCCATC GGC GAC GGCCCC GTG CTGCTG CCC GAC AAC CAC TAC CTG AGC ACC CAGTCC GCC CTGAGC AAA GAC CCC AAC GAGAAGCGC GAT CACATG GTC CTG CTG GAG TTC GTG ACC GCC GCC GGG ATC ACTCTC GGC ATG GACGAG CTG TACAAG TAA

[0050] The gene optimization expression operation of the present method is based on gene editing at the molecular biology level. Specifically, based on the degeneracy of codons, that is, the phenomenon that the same amino acid has two or more codons, the codon combination of some sites on the mRNA chain to be translated can be changed by changing the arrangement of individual nucleotides in the DNA or RNA sequence without changing the gene expression result, such as protein, and the triplet codons at some positions corresponding to low tRNA abundance and relatively more difficult to participate in translation are replaced with triplet codons corresponding to high tRNA abundance and higher translation efficiency, which helps to improve the efficiency of protein translation in the gene expression process.

[0051] In this practice, the operation rule follows that the codon combination with lower tRNA abundance is replaced with the codon combination with the highest tRNA abundance for the same amino acid. The replacement priority is shown in Table 1 below. For the same amino acid, the codon combination priority decreases from top to bottom.

[0052] Table 1

[0053]

[0054]

[0055] Based on this method, all replacement contents of the following codon combinations include:

[0056] (1) Replace all CTT CTC CTG TTA CTA codon combinations with TTG

[0057] (2) Replace all TTT codon combinations with TTC

[0058] (3) Replace all ATCATA codon combinations with ATT

[0059] (4) Replace all GTA GTC GTG codon combinations with GTT

[0060] (5) Replace all TAC codon combinations with TAT

[0061] (6) Replace all CAC codon combinations with CAT

[0062] (7) Replace all CAG codon combinations with CAA

[0063] (8) Replace all AAC codon combinations with AAT

[0064] (9) Replace all AAA codon combinations with AAG

[0065] (10) Replace all GAC codon combinations with GAT

[0066] (11) Replace all GAA codons with GAG

[0067] (12) Replace all TCT TCC AGC AGT TCG codon combinations with TCA

[0068] (13) Replace all CCT CCG CCC codon combinations with CCA

[0069] (14) Replace all ACC ACGACA codon combinations with ACT

[0070] (15) Replace all GCC GCA GCG codon combinations with GCT

[0071] (16) Replace all TGT codon combinations with TGC

[0072] (17) Replace all GGC GGG GGT codon combinations with GGA

[0073] (18) Replace all AGG CGT CGC CGA CGG codon combinations with AGA

[0074] (19) Replace all TAATAG codon combinations with TGA

[0075] The complete sequence after optimized expression by this method is the GFP-PNT optimized gene:

[0076] ATG GTT TCA AAG GGA GAG GAG TTG TTC ACT GGA GTTGTT CCC ATT TTG GTTGAG TTG GAT GGA GAT GTT AAT GGACAC AAG TCA GTT TCA GGA GAG GAG GGAGAT GCTAG TAT GGA AAG TTG CAG ACT ACT TTG GTTCCC TGG CCC ACT TTG GTT ACT ACTTTG ACT TAT GGA GTT CAA TGC TTC TCA AGA TATCCC GATCAC ATG AAG CAA CAC GAT TTC AAG TCA GCT ATG CCCGAG GGA TAT GTT CAAGAG ACTAG GATAG GATGA TTC ACT TTC GTT AAG TTCGAGGGA GAT ACT TTG GTT AAT AGA ATT GAG TTG AAG GGA ATTGAT TTC AAG GAG GATGGAAAT ATT TTG GGA CAC AAG TTGGAG TATAAT TATAAT TCA CAC AAT GTT TATATTATG ATT ATT ATT ATT ATTAG GATAG GTTAG CAA AGACAC AAT ATT GAG GAT GGATCA GTT CAA TTG GCT GAT CACTAT CAA CAA ACT CCC ATT GGA GAT GGA CCC GTTTTGTTG CCC GAT AAT CAC TAT TTG TCA ACT CAA GCT TTGTCAAAG GAT CCCATGAT GATGA TGAACTTAG ACT GCT GCT GGA ATT ACT TTG GGAATGGAT GAG TTG TATAAG TGA

[0077] You can also have a sweet tooth. Figure 1 What is wrong:

[0078] When constructing a vector for gene editing, the pCAMBIA2300 plasmid sequence was used as a vector, and the vector was constructed by enzyme digestion and ligation. Figure 2 The DNA double strands shown are used to transform the target plant to complete genetic transformation.

[0079] Embodiment 2:

[0080] The concentration of the expression product of the plants treated by the method described in Example 1 was measured to determine the effectiveness of the gene sequence optimization expression method based on peanut codon preference.

[0081] The callus tissues of Yuhua 9326 peanut were aseptically cultured in MB medium to obtain 50 peanut callus tissue materials of the original Yuhua 9326 variety and the transgenic variety Yuhua 9326 (GFP gene optimized variety) in which the GFP-PNT optimized gene was inserted using the above-mentioned pCAMBIA2300 plasmid sequence.

[0082] The callus of Yuhua 9326 peanut was subsequently induced to differentiate, and 20 complete plants of the original Yuhua 9326 peanut variety and the transgenic Yuhua 9326 (GFP gene optimized variety) peanut variety in which the GFP-PNT optimized gene was inserted using the above-mentioned pCAMBIA2300 plasmid sequence were obtained after tissue culture. The plants were planted under normal greenhouse conditions for 120 days until the plants matured and bore fruit. The seeds of the two plants were collected and the cotyledons and embryos were separated separately. The two peanut seeds were observed for the presence and intensity of fluorescence under ultraviolet light to investigate the enhanced expression of the GFP fluorescent protein gene.

[0083] The effectiveness of the method for increasing the expression abundance of the target gene protein involved in the present invention can be verified by taking the ultraviolet fluorescence intensity of the two types of Yuhua 9326 peanut callus materials before and after GFP gene sequence optimization as an indicator.

[0084] Specific results such as Figure 3-5 As shown, Yuhua 9326 (GFP-PNT optimized gene variety) can be observed with strong fluorescence, Yuhua 9326 (introduced non-optimized GFP variety) can be observed with weak fluorescence, and Yuhua 9326 (unintroduced GFP variety) has no obvious fluorescence;

[0085] Based on the above experiments, it can be concluded that the gene sequence optimization expression method based on peanut codon preference can improve the intensity of fluorescence emitted by Yuhua 9326 variety peanut callus material by optimizing the expression abundance of GFP fluorescent protein in Yuhua 9326 variety peanut callus material.

[0086] Based on the same principle, the gene sequence optimization expression method based on peanut codon preference is sufficiently effective.

[0087] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for optimizing peanut nucleotide codon sequences to increase target gene protein expression abundance, characterized in that: The method includes the following optimization modes: (1) The frequency of occurrence of peanut gene codons and the abundance of tRNA corresponding to each codon combination in peanut cells are obtained through peanut genome sequencing analysis, and the codon sequence corresponding to the low-abundance tRNA is optimized to the codon corresponding to the high-abundance tRNA; (2) Based on the nucleotide sequence composition and tRNA abundance of peanuts, the original nucleotide sequence of the protein-encoding gene is optimized without changing the expressed amino acid sequence, thereby adapting to the differences in tRNA abundance in peanut cells, thereby increasing the protein expression abundance of the target gene in peanut cells; (3) Based on the degeneracy of codons, without changing the results of gene expression, by changing the arrangement of individual nucleotides in the DNA or RNA sequence, the codon combination at some sites on the mRNA chain to be translated is changed, and the triplet codons at some positions with low tRNA abundance and relatively more difficult to participate in translation are replaced with triplet codons with high tRNA abundance and higher translation efficiency.

2. The method according to claim 1, characterized in that: The expression product corresponding to the nucleic acid is an exogenous protein or a gene expression product of peanut itself.

3. The method according to claim 1, characterized in that: The codon sequence replacement operation rule follows that the codon combination with lower tRNA abundance is replaced with the codon combination with the highest tRNA abundance in the same amino acid, and the replacement priority is as follows: (1) The priority order for leucine replacement is: TTG—CTT—CTC—CTG—TTA—CTA; (2) The priority order for phenylalanine substitution is: TTC-TTT; (3) The priority order for isoleucine substitution is: ATT—ATC—ATA; (4) The priority order for valine replacement is: GTT—GTG—GTC—GTA; (5) The priority order for tyrosine substitution is: TAT-TAC; (6) The priority order for histidine replacement is: CAT-CAC; (7) The priority order for glutamine replacement is: CAA—CAG; (8) The priority order for asparagine substitution is: AAT-AAC; (9) The priority order for lysine replacement is: AAG-AAA; (10) The priority order for aspartate substitution is: GAT—GAC; (11) The priority order for glutamate replacement is: GAG—GAA; (12) The priority order for serine substitution is: TCA—TCT—TCC—AGC—AGT—TCG; (13) The priority order for proline substitution is: CCA—CCT—CCC—CCG; (14) The priority order for threonine substitution is: ACT—ACA—ACC—ACG; (15) The priority order for alanine substitution is: GCT—GCA—GCC—GCG; (16) The priority ranking for cysteine ​​substitution is: TGC—TGT; (17) The priority order for glycine substitution is: GGA—GGT—GGC—GGG; (18) The priority order for arginine substitution is: AGA—AGG—CGT—CGC—CGA—CGG; (19) The priority order for stop codon replacement is: TGA—TAA—TAG.

4. The method according to claim 3, characterized in that: During the amino acid substitution, methionine and tryptophan do not have two or more codons, and no codon substitution operation is involved.

5. The method according to claim 1, characterized in that: In the optimization method, the replacement amount of triplet codes in the nucleotide sequence is ≥25%, and the change amount of the amino acid sequence is ≤5%.

6. Use of any method according to claims 1 to 5 in breeding new peanut varieties.

7. The use according to claim 6, characterized in that: The application method comprises the following steps: (1) causing the peanut cell genome to produce an optimized mutation as described in any one of claims 1 to 5; (2) making the peanut plant contain cells, tissues, organs, or materials obtained by the optimization method according to any one of claims 1 to 5; (3) enabling peanut plants to express genes optimized by the optimization method described in any one of claims 1 to 5.

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