CCD4 protein mutant P301L and application thereof

Abstract

The invention belongs to the technical field of tobacco gene engineering, and particularly relates to a CCD4 protein mutant P301L and application thereof. Compared with a wild type CCD4 protein, the mutant has the following mutation: p. P301L, and the NCBI (National Center of Biotechnology Information) accession number of the wild type CCD4 protein is AKO22632.1. According to the invention, the substrate beta-carotene is docked to a CCD4 enzyme activity pocket, a saturated mutant library is constructed, screening is carried out through a bacterial color complementation experiment, and after specific site amino acid mutation, degradation of the substrate beta-carotene is accelerated, so that a certain technical foundation is laid for further cultivation of new varieties of tobacco crops.

Description

Technical Field

[0001] This disclosure belongs to the field of tobacco genetic engineering technology, specifically relating to a CCD4 protein mutant P301L and its applications. Background Technology

[0002] Carotenoids are important plastid pigments with crucial physiological functions, closely related to plant growth, development, and photosynthesis, and influencing crop quality traits. Carotenoid cleavage dioxygenases (CCDs) are a highly isomerized family of polyene chain oxidases, which... 2+ With the assistance of these enzymes, a catalytic reaction occurs by binding four histidine residues. CCDs, as key enzymes, participate in the production of plant hormones and play an important role in the formation of color and aroma in plants (leaves, flowers, and fruits). CCD1 and CCD4, through cleavage, form volatile apocarotenoids, producing aroma compounds.

[0003] CCD4, a key enzyme involved in the catalytic cleavage of carotenoids, produces various flavor and aromatic compounds, such as β-ionone, which impart fragrance to flowers, fruits, and leaves, playing a crucial role in pollen and seed dispersal. While existing research on carotenoid cleavage pathways is extensive, targeted breeding and cultivation using genetic engineering techniques are essential for maximizing plant utilization. Tobacco, as a model crop for genetic engineering research, directly impacts quality traits due to the efficiency of carotenoid degradation. Therefore, targeted modification of tobacco terpene metabolism genes through directed evolution is of significant practical importance for accelerating tobacco breeding and can also lay a technological foundation for the genetic breeding of other crops. Summary of the Invention

[0004] To address the shortcomings of existing technologies and practical needs, this disclosure provides a carotenoid cleavage dioxygenase (CCD4) mutant and its applications. The aim is to provide several CCD4 proteins with better enzyme activity and the tobacco CCD4 gene, thereby providing a new technical approach for the breeding of new tobacco varieties, and also laying a certain technical foundation for the breeding and variety improvement of other crops.

[0005] To achieve this objective, the present disclosure adopts the following technical solution: According to one aspect of this disclosure, a CCD4 protein mutant P301L is provided, which, compared with the wild-type CCD4 protein, has the following mutation: p. P301L, the wild-type CCD4 protein having NCBI accession number AKO22632.1.

[0006] In some embodiments, the amino acid sequence of the CCD4 protein mutant P301L is shown in SEQ ID No. 3.

[0007] According to another aspect of this disclosure, a CCD4 gene mutant is provided, the nucleotide sequence of which is a DNA sequence encoding the CCD4 protein mutant P301L described in this disclosure.

[0008] In some embodiments, the CCD4 gene mutant has the following mutation compared to the wild-type CCD4 gene: g.902C>T.

[0009] In some embodiments, the NCBI accession number for the nucleotide sequence of the wild-type CCD4 gene is KM605433.1.

[0010] In some embodiments, the nucleotide sequence of the CCD4 gene mutant is shown in SEQ ID No. 4.

[0011] According to another aspect of this disclosure, a biomaterial is provided, the biomaterial comprising: (A) A vector comprising the CCD4 gene mutant described in this disclosure; (B) A cell comprising the carrier described in (A).

[0012] In some embodiments, the biomaterial is plant biomaterial, and in other embodiments it is herbaceous plant.

[0013] In some embodiments, the plant is selected from one or more of tobacco, wheat, corn, and sorghum.

[0014] According to another aspect of this disclosure, a method for constructing a transgenic plant expressing the CCD4 protein mutant P301L described in this disclosure is provided.

[0015] In some embodiments, the construction method includes introducing the CCD4 gene mutant described in this disclosure.

[0016] In some embodiments, the plant is a herbaceous plant.

[0017] In some embodiments, the herbaceous plant is selected from one or more of tobacco, wheat, corn, and sorghum.

[0018] According to another aspect of this disclosure, the application of the CCD4 protein mutant P301L, the CCD4 gene mutant, and the biomaterial described herein in pigment degradation is provided.

[0019] In some embodiments, the pigment includes carotenoids.

[0020] In some embodiments, the carotenoids include β-carotene.

[0021] According to another aspect of this disclosure, the application of the CCD4 protein mutant P301L, the CCD4 gene mutant, and the biomaterial described herein in the preparation of β-ionone is provided.

[0022] Compared with the prior art, this disclosure has at least the following beneficial effects: This disclosure involves hooking the substrate β-carotene into a CCD4 enzyme activity pocket to construct a saturated mutant library. Screening is performed using a bacterial color complementation experiment. After specific site amino acid mutations, the degradation of the substrate β-carotene is accelerated, laying a certain technical foundation for further breeding of new tobacco crop varieties. Attached Figure Description

[0023] Figure 1 The results of the bacterial color complementary experiment are shown in the figure; among them, Figure 1 Figure a shows the bacterial color experiment results of the P301L mutant; Figure 1 b shows the results of the relative β-carotene content determination; Figure 2 The appearance of the P301L mutant plant (EMS mutant) and the wild-type plant (Yunyan 87) are shown. Figure 3 The results of β-ionone content determination in P301L mutant plants (EMS mutant) and wild-type plants (Yunyan 87) are shown in the figure. Detailed Implementation

[0024] The present disclosure will be further illustrated below by means of embodiments. It should be understood that the embodiments of the present disclosure are only for illustration and not for limiting the present disclosure. Simple improvements to the present disclosure under the premise of the present disclosure are within the scope of protection claimed by the present disclosure.

[0025] Example 1: Modification of the codon Following standard methods, the wild-type CCD4 gene was obtained from the flue-cured tobacco variety Yunyan 87 through molecular cloning and sequencing. Its sequence was completely identical to the one published in the NCBI database (NCBI accession number KM605433.1). To avoid protein expression differences caused by codon bias, the NtCCD4 codon was modified to better facilitate expression in *E. coli*. Codon optimization and vector construction were commissioned to Beijing Qingke Biotechnology Co., Ltd. The protein expression vector was pMAL-c5x, with the BamHI homologous cloning site, resulting in the recombinant vector pMAL-CCD4. The specific steps are as follows: 1. Modification of wild-type CCD4: The wild-type CCD4 protein, with NCBI accession number AKO22632.1, is 601 amino acids in length. To avoid codon bias-induced differences in protein expression, the codons of the tobacco wild-type CCD4 protein were modified to better facilitate expression in *E. coli*. Specifically, the signal peptide formed by removing the first 28 amino acids from the N-terminus of the wild-type CCD4 protein was removed, resulting in a peptide of 573 amino acids, as shown in SEQ ID No. 1 (CCD4-573). SSSPILKVSSVRIEERPQTTTTTTRTKPQEKPTPSPYTPPKDTPKRQLPTKSITTKKPVEPSFPSVIFNAFDDFVNTFIDPPLKPCVDPKYILSNNFAPVDELPPTECEVVVGSLPPCLDGAYIRNGPNPQYLPRGPYHLFDG DGMLHSIKISQSKATLCSRYVKTYKYTIEREAGSPVFPNVFSGFNGLTASAARGAITAARAIAGQFNPTNGIGLANTSLALFGGKLFALGESDLPYAVKLAPDGDIITLGRYDFDGKLFMSMTAHPKIDPDTNEAFAFRYGPM PPFLTYFRIEPNGTKTPDVPIFSMTRPSFLHDFAITNKFAIFSDIQIGMNPLEFITGGSPVSSDSGKIPRLGVIPRYAKNESEMKWFDVPGINIVHAINAWDEDDGDTIVMVAPNILSVEHTLERMDMIHASVEKVKIDLKSG MVSRQPLSTRNLDFGVINPAYVGKKNKYVYAAIGDPMPKIAGIAKLDVSVAEADRRDCIVACRLFGEGCFGGEPYFVANNSAADEDDGYVVSFVHNEKTGESRFLVMDAKSPNLDIVAAVKLPRRVPYGFHGLFVRETDLRKLM The wild-type CCD4 gene, with NCBI accession number KM605433.1, is 1806 nucleotides in length. Correspondingly, removing the first 84 nucleotides from the 5' end and the stop codon TAG from the wild-type CCD4 gene results in a 1719-nucleotide sequence, as shown in SEQ ID No. 2 (CCD4-1719). 2. Based on the modification of wild-type CCD4, the Pro amino acid (P) at position 273 of SEQ ID No. 1 (corresponding to position 301 of the AKO22632.1 sequence) was mutated to the amino acid Leu (L), as shown in SEQ ID No. 3 (CCD4-P301L): SSSPILKVSSVRIEERPQTTTTTTRTKPQEKPTPSPYTPPKDTPKRQLPTKSITTKKPVEPSFPSVIFNAFDDFVNTFIDPPLKPCVDPKYILSNNFAPVDELPPTECEVVVGSLPPCLDGAYIRNGPNPQYLPRGPYHLFDG DGMLHSIKISQSKATLCSRYVKTYKYTIEREAGSPVFPNVFSGFNGLTASAARGAITAARAIAGQFNPTNGIGLANTSLALFGGKLFALGESDLPYAVKLAPDGDIITLGRYDFDGKLFMSMTAHPKIDLDTNEAFAFRYGPM PPFLTYFRIEPNGTKTPDVPIFSMTRPSFLHDFAITNKFAIFSDIQIGMNPLEFITGGSPVSSDSGKIPRLGVIPRYAKNESEMKWFDVPGINIVHAINAWDEDDGDTIVMVAPNILSVEHTLERMDMIHASVEKVKIDLKSG MVSRQPLSTRNLDFGVINPAYVGKKNKYVYAAIGDPMPKIAGIAKLDVSVAEADRRDCIVACRLFGEGCFGGEPYFVANNSAADEDDGYVVSFVHNEKTGESRFLVMDAKSPNLDIVAAVKLPRRVPYGFHGLFVRETDLRKLM Its corresponding nucleotide bases are shown in SEQ ID No. 4 (CCD4-g.902C>T): Example 2 Construction of saturated mutants A single-point saturation mutant is constructed based on the mutation site at position 301, which involves mutating one amino acid into the remaining 19 amino acids. The specific process is as follows: 1) Based on the above sites, the degenerate primer sequences are designed as follows: P273-F: 5'-CATCCCAAAATTGACNNKGATACTAACGAGGCT-3' (SEQ ID No. 5), general-R: 5'-CATTGTGCACAACGACACAACGTAG-3' (SEQ ID No. 6); NNK is a special codon representation in degenerate primers, where N represents any base (A, C, G, T) and K represents either a G or T base. In the sequence 5'-TTTGCTTTCCGTTACNNKCCAATGCCTCCTTTT-3', NNK represents a degenerate site containing 32 possible codon combinations, specifically N=A / C / G / T, N=A / C / G / T, and K=G / T. The combinations are as follows: The first N: can be A, C, G, or T (4 possibilities) The second N can be A, C, G, or T (4 possibilities) K: can be G or T (2 possibilities); 2) Using pMAL-CCD4 constructed in Example 1 as a template, and then using P273-F as the forward primer and general-R as the reverse primer, full plasmid PCR was performed. 3) The PCR amplification product was digested with DpnI, and then the digested product was co-transformed with the pAC-β double plasmid into E. coli BL21(DE3) competent cells. After transformation, the bacterial culture was plated on plates containing 100 μg / L ampicillin and 34 μg / L chloramphenicol to obtain saturation mutagenesis. The empty vector pMAL-c5x was co-transformed with the pAC-β plasmid as a negative control strain.

[0026] Example 3: Bacterial Color Complement Experiment 1) Select positive single clones. Pick one single clone from each of the plates containing double antibiotics (Amp+, 100 μg / mL) / (Amp+, 68 μg / mL) and put it into a centrifuge tube containing liquid culture medium. Incubate at 37℃ and 200 rpm for 8-10 h. 2) Under aseptic conditions, transfer 100 μL of bacterial culture to 10 mL of TB medium for expansion culture. Add 10 μL of ampicillin antibiotic (Amp+, 100 μg / mL) and incubate at 37°C with a shaker at 200 rpm for approximately 3 hours until OD (dose retardation). 600 When the temperature reaches 0.4-0.6, add 10 μL of IPTG (0.1 mmol / L) induction solution and incubate overnight at 37°C. 3) Centrifuge the induced bacterial culture at 4000 rpm / min and 4℃ for 8 min, discard the supernatant and collect the bacterial cells; 4) Wash the collected bacterial cells once with phosphate buffer, add 200 μL of sterile water to resuspend, and then add the bacterial cells sequentially to a clear 96-well cell culture plate. Take a picture and save it.

[0027] 5) The absorbance at 440 nm was measured using a spectrophotometer and calculated.

[0028] Using the high-throughput screening method described above, the absorbance (OD) was finally determined. 440 The mutants with significantly reduced values ​​were further subjected to nucleic acid sequencing, and the corresponding nucleotide bases of the sequencing results are shown in SEQ ID No. 4.

[0029] Test results as follows Figure 1 As shown, from Figure 1 As can be seen, the vector pAC-β contains β-carotene synthase. Co-transformation of pAC-β and the empty vector pMAL in E. coli can synthesize β-carotene, turning the E. coli from white to yellow (pMAL). When the 301st site of CCD4 is saturated and the Pro amino acid is mutated to 19 other different amino acids, and co-transformed with pAC-β double plasmid into E. coli BL21(DE3), the color of the bacterial culture changed significantly after IPTG induction. Using the pMAL-c5x empty vector as a negative control, it can be seen that the P301L mutant is significantly lighter in color than the wild type (WT) and other mutants.

[0030] Figure 1 Figure b shows the results of relative β-carotene content determination. pAC-β contains carotenoid synthase, which can synthesize β-carotene in E. coli, causing the E. coli to change from white to yellow. Therefore, compared with the control, the color of the bacteria co-transformed by the recombinant plasmid was significantly lighter, indicating that CCD4 has catalytic activity, and the substrate β-carotene can be oxidized and degraded. The reduction in substrate amount leads to the lighter color. β-carotene in OD 440 The CCD4 exhibits a strong absorbance value and can degrade β-carotene. Therefore, the stronger the CCD4 enzyme activity, the higher the efficiency of β-carotene degradation, ultimately leading to a higher OD value. 440The absorbance value decreased. Using empty pMAL-c5x as a negative control, it can be seen that the mutant P301L (indicated by the arrow) showed a lower absorbance value compared to wild-type CCD4 (WT). 440 The absorbance at position 301 was significantly reduced. The saturation mutation replaced the Pro amino acid at position 301 with the remaining 19 amino acids. It can be seen that when the Pro amino acid at position 301 (P) was mutated to the amino acid Leu (L) (as shown by the arrow), the relative β-carotene content was significantly reduced compared with the wild type (WT) and other mutations, indicating that its carotenoid cleavage dioxygenase CCD4 had a significant change.

[0031] Example 4: Determination of β-ionone content in P301L EMS mutant Mutants of the CCD4 gene were screened in the Yunyan 87 EMS mutant library constructed by the research group. The planting and specific screening steps of the mutants were carried out in accordance with the paper "Gao Yulong; Zhao Lu; Wang Bingwu; Kong Guanghui; Wang Yahui; Liu Jianjin; Duan Jie; Wu Xingfu; Li Qing; Zhe Kaiming. New high-nicotine flue-cured tobacco material obtained by mutant NtJAZ1 gene [J]. Crop Journal, 2025(01):83-88.DOI:10.16035 / j.issn.1001-7283.2025.01.010." The screening primers used were: CCD4_F: TCATTGATCCTCCTTTGAAACCTTGT (SEQ ID No.7), CCD4_R: ATGATGACTTGTCTGTAATTATCCATTAATTATG (SEQ ID No. 8).

[0032] Obtain a mutant ( Figure 2 After Sanger sequencing, it was confirmed that the nucleotide at position 902 was mutated from C to T, and the corresponding amino acid residue at position 301 was mutated from proline (P) to leucine (L).

[0033] Homozygous mutant plants and wild-type Yunyan 87 were grown in a greenhouse. Leaves were collected during the flowering period, blanched, and dried as samples. Approximately 20.0 g of each sample, accurate to 0.1 mg, was weighed and placed in a 500 mL flat-bottom flask. 350 mL of distilled water and 1.0 mL of n-heptadecane (3.2) were accurately added. A simultaneous distillation extractor with a heating mantle attached to one side of the flask was used for distillation at 200 °C for 3.0 h. 45 mL of dichloromethane was then measured and placed in a 250 mL flat-bottom flask. The flask was placed on a simultaneous distillation extractor with a water bath attached to one side and refluxed at 60 °C for 3.0 h, then cooled to room temperature. The reflux liquid was collected in a 250 mL separatory funnel, dried with anhydrous sodium sulfate, filtered, and extracted. The separatory funnel was rinsed three times with 10 mL of dichloromethane, and the extracts were combined. The extracts were then concentrated to approximately 1.0 mL using a rotary evaporator and analyzed by gas chromatography-mass spectrometry (GC-MS).

[0034] Measurement parameters: Column: fused silica capillary column, 30m × 0.32mm × 0.25um; Injector: 250℃; Carrier gas: helium; Split flow rate: 50ml / min; Injection volume: 2.0ul; Temperature program 1: ramp from 50℃ to 200℃ at 10℃ / min, hold for 2min; ramp from 200℃ to 260℃ at 8℃ / min, hold for 15min. Detector: MS; Transfer line: 230℃; Ion source: 170℃; Scan mode: full scan.

[0035] The results were calculated using the internal standard method for quantification. The β-ionone content was expressed as the relative content of the internal standard-corrected peak area, in μg / g. The results showed that the β-ionone content in the mutant was 11.09 μg / g, while the wild-type β-ionone content was 8.11 μg / g. The mutant β-ionone content was approximately 37% higher than that of the wild-type (control group). Figure 3 , p <0.01).

[0036] While some exemplary embodiments of this application have been described and illustrated, this application is not limited to the disclosed embodiments. Rather, those skilled in the art will recognize that modifications and changes may be made to the described embodiments without departing from the spirit and scope of this application as described in the appended claims.

Claims

1. A CCD4 protein mutant P301L, characterized in that, The mutant, compared to the wild-type CCD4 protein, has the following mutation: p. P301L, the NCBI accession number of the wild-type CCD4 protein is AKO22632.

1.

2. The CCD4 protein mutant P301L according to claim 1, characterized in that, The amino acid sequence of the CCD4 protein mutant P301L is shown in SEQ ID No.

3.

3. A CCD4 gene mutant, characterized in that, The nucleotide sequence of the mutant is the DNA sequence encoding the CCD4 protein mutant P301L as described in any one of claims 1-2.

4. The CCD4 gene mutant according to claim 3, characterized in that, Compared with the wild-type CCD4 gene, the CCD4 gene mutant has the following mutation: g.902C>T; Preferably, the NCBI accession number for the nucleotide sequence of the wild-type CCD4 gene is KM605433.1; Preferably, the nucleotide sequence of the CCD4 gene mutant is shown in SEQ ID No.

4.

5. A biomaterial, characterized in that, The biomaterials include: (A) A vector comprising the CCD4 gene mutant of claim 3; (B) A cell comprising the carrier described in (A); Preferably, the biomaterial is a plant biomaterial, and more preferably a herbaceous plant; Preferably, the plant is selected from one or more of tobacco, wheat, corn, and sorghum.

6. A method for constructing a transgenic plant, characterized in that, The transgenic plant expresses the CCD4 protein mutant P301L as described in any one of claims 1-2.

7. The construction method according to claim 6, characterized in that, The construction method includes introducing the CCD4 gene mutant according to any one of claims 3-4; Preferably, the plant is a herbaceous plant; Preferably, the herbaceous plant is selected from one or more of tobacco, wheat, corn, and sorghum.

8. The application of the CCD4 protein mutant P301L according to any one of claims 1-2, the CCD4 gene mutant according to any one of claims 3-4, and the biomaterial according to claim 5 in pigment degradation.

9. The application according to claim 8, characterized in that, The pigments include carotenoids; Preferably, the carotenoids include β-carotene.

10. The use of the CCD4 protein mutant P301L according to any one of claims 1-2, the CCD4 gene mutant according to any one of claims 3-4, and the biomaterial according to claim 5 in the preparation of β-ionone.

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