Liriodendron chinense LCYE gene and expression protein and application thereof

Abstract

The application discloses a Liriodendron LCYE gene, an expression protein thereof and application, and relates to the technical field of plant genetic engineering.The Liriodendron LCYE gene disclosed by the application comprises LcLCYE gene, or / and LtLCYE gene, or / and variant gene LcLCYE-alpha, or / and variant gene LtLCYE-alpha; and the nucleotide sequences of the genes correspond to SEQ ID NO.1, 3, 5 and 7 respectively.The results of the examples of the application show that the Liriodendron LCYE homologous protein has traditional LCYE enzyme activity, and also has LCYB enzyme activity for catalyzing lycopene to form gamma-carotene and beta-carotene; and the C-terminal structure of the Liriodendron LCYE homologous protein is necessary for the LCYE enzyme activity function, but does not affect the LCYB enzyme activity function.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant genetic engineering, and more particularly relates to a class of Liriodendron LCYE genes, expression proteins thereof and application thereof. BACKGROUND

[0002] Flower color is an important flower trait, and plays a very important role in the reproductive ecology and species evolution of flowering plants, and is an important index for evaluating the ornamental and economic value of plants. Flower pigments are the main factors affecting flower color formation, and mainly include flavonoids, carotenoids and betalains. Among them, flavonoids and carotenoids are two major pigment groups that form the flower color of most plants, and widely exist in most plants, and are involved in the process of flower color formation of plants, thereby making the flower color of plants diverse.

[0003] The biosynthetic pathway of carotenoids has been studied in detail, and is highly conserved in different plants. Among them, GGPP derived from the MEP pathway will be converted into red lycopene under the catalytic action of a series of enzymes such as phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene isomerase (Z-ISO), ζ-carotene desaturase (ZDS) and carotenoid isomerase (CRTISO). Subsequently, lycopene as a substrate will be catalyzed by two lycopene cyclases LCYE and LCYB, which can each catalyze one end of lycopene to form ε ring and β ring. Therefore, lycopene will form ε-carotene under the continuous catalysis of LCYE enzyme, γ-carotene and β-carotene under the continuous catalysis of LCYB enzyme, and α-carotene under the sequential action of LCYE and LCYB enzymes.

[0004] Because carotenoids play a crucial role in plant growth and development as well as human health, research on their regulation has largely focused on plant leaves or crop roots and stems, with relatively few studies on their regulation in flower coloration (DOI: 10.3389 / fpls.2019.01017). In recent years, significant breakthroughs have been made in the study of carotenoid biosynthesis regulation during flower coloration, but these studies are mainly concentrated in a few herbaceous plants such as tobacco, monkey head mushroom, and alfalfa. In tobacco, the F-box protein COI1 mediates the expression regulation of PSY, ZDS, and LCYB genes through a concise regulatory mechanism, thereby affecting the β-carotene content in petals and altering flower color (DOI: 10.3389 / fpls.2019.01017). In monkey head mushroom, a tetrapeptide repeat protein RCP2 can indirectly regulate the expression of multiple genes, including CRISTO and LCYB, thereby altering the coloration pattern at the base of the corolla tube (DOI: 10.1105 / tpc.19.00755). In alfalfa, the R2R3 MYB transcription factor WP1 can directly upregulate the expression of LCYE and LCYB by forming an MBW complex with TT8 and WD40-1, thereby enhancing carotenoid synthesis in alfalfa petals and resulting in yellow petals (DOI: 10.1105 / tpc.19.00480). These results provide a new perspective for the study of carotenoid-mediated flower petal coloration in plants, but in both breadth and depth, they are far less comprehensive than studies on leaves or tubers, especially regarding carotenoid-mediated flower color variation in woody plants and related fundamental research.

[0005] *Liriodendron* belongs to the Magnolia group and includes a pair of sister species with an inter-regional distribution between East Asia and eastern North America: *Liriodendron chinense*, naturally distributed in East Asia, and *Liriodendron tulipifera*, found in eastern North America. *Liriodendron chinense* petals are entirely green, with yellow longitudinal stripes running through them during peak bloom; *Liriodendron tulipifera* petals have a vibrant orange-yellow band near the base. Previous studies have shown that the orange-yellow band in *Liriodendron tulipifera* primarily accumulates carotenoids, especially carotenoids including α-carotene and β-carotene. However, the key gene LCYB, which plays a crucial role in carotenoid synthesis, maintains a stable and low expression level in the petals. Conversely, the expression of LCYE is highly correlated with the coloration of the orange-yellow band in *Liriodendron tulipifera* and the accumulation of carotenoids (DOI: 10.1038 / s41438-020-0287-3).

[0006] In conclusion, strengthening research on the function and genetic regulatory mechanism of the LCYE gene will help to deepen our understanding of the molecular mechanisms of petal coloration and carotenoid synthesis in Liriodendron species, laying a theoretical foundation for breeding new varieties of Liriodendron. Therefore, research on the function of the LCYE gene has significant scientific and applied value. Summary of the Invention

[0007] To address the aforementioned problems in the existing technology, the technical problem to be solved by this invention is to provide a class of LCYE genes from the genus *Liriodendron*. Another technical problem to be solved by this invention is to provide an expression protein of a class of LCYE genes from the genus *Liriodendron*. A further technical problem to be solved by this invention is to provide an application of a class of LCYE genes from the genus *Liriodendron* for regulating carotenoid synthesis.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0009] A class of LCYE genes in the genus Liriodendron includes the LcLCYE gene, and / or the LtLCYE gene, and / or the variant LcLCYE-α, and / or the variant LtLCYE-α;

[0010] The nucleotide sequence of the LcLCYE gene is shown in SEQ ID NO.1;

[0011] The nucleotide sequence of the LtLCYE gene is shown in SEQ ID NO.3;

[0012] The nucleotide sequence of the variant gene LcLCYE-α is shown in SEQ ID NO.5;

[0013] The nucleotide sequence of the variant gene LtLCYE-α is shown in SEQ ID NO.7.

[0014] The expressed proteins of the LCYE gene of the Liriodendron genus include LcLCYE protein, and / or LtLCYE protein, and / or variant protein LcLCYE-α, and / or variant protein LtLCYE-α;

[0015] The amino acid sequence of the LcLCYE protein is shown in SEQ ID NO.2;

[0016] The amino acid sequence of the LtLCYE protein is shown in SEQ ID NO.4;

[0017] The amino acid sequence of the variant protein LcLCYE-α is shown in SEQ ID NO. 6;

[0018] The amino acid sequence of the variant protein LtLCYE-α is shown in SEQ ID NO.8.

[0019] The application of the LCYE gene of the Liriodendron genus in promoting the conversion of lycopene into carotenoids.

[0020] Application of the LcLCYE or LtLCYE gene in Liriodendron spp. in promoting the conversion of lycopene into ε-carotene and γ-carotene.

[0021] The application described herein includes the following specific steps:

[0022] 1) Construct prokaryotic expression vectors for the LcLCYE gene or LtLCYE gene;

[0023] 2) Transform the prokaryotic expression vector into a prokaryotic expression strain;

[0024] 3) Cultivate and screen strains with significantly increased ε-carotene and γ-carotene content.

[0025] The application of the Liriodendron genus variant LcLCYE-α or variant LtLCYE-α in promoting the conversion of lycopene to β-carotene.

[0026] The application described herein includes the following specific steps:

[0027] 1) Construct prokaryotic expression vectors for the variant gene LcLCYE-α or the variant gene LtLCYE-α;

[0028] 2) Transform the prokaryotic expression vector into a prokaryotic expression strain;

[0029] 3) Cultivate and screen strains with significantly increased β-carotene content.

[0030] The application of the LCYE gene expression protein of the Liriodendron genus in promoting the conversion of lycopene into carotenoids.

[0031] Application of LcLCYE protein or LtLCYE protein in promoting the conversion of lycopene into ε-carotene and γ-carotene.

[0032] The use of the LcLCYE-α protein of claim 2 in promoting the conversion of lycopene to β-carotene.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] 1) This invention discloses a class of LCYE genes in the genus Liriodendron, including the LcLCYE gene, and / or the LtLCYE gene, and / or the variant gene LcLCYE-α, and / or the variant gene LtLCYE-α; the nucleotide sequence of the LcLCYE gene is shown in SEQ ID NO.1; the nucleotide sequence of the LtLCYE gene is shown in SEQ ID NO.3; the nucleotide sequence of the variant gene LcLCYE-α is shown in SEQ ID NO.5; and the nucleotide sequence of the variant gene LtLCYE-α is shown in SEQ ID NO.7.

[0035] 2) The LcLCYE gene or LtLCYE gene disclosed in this invention promotes the conversion of lycopene into carotenoids ε-carotene and γ-carotene. The variant gene LcLCYE-α or the variant gene LtLCYE-α promotes the conversion of lycopene into β-carotene.

[0036] 3) In addition to lycopene, ε-carotene and γ-carotene were detected in the pAC-LYC+pET-LcLCYE and pAC-LYC+pET-LtLCYE strains constructed in this invention, indicating that the LCYE homolog of Liriodendron tulipifera not only has the traditional LCYE enzyme activity, but also has the LCYB enzyme activity that catalyzes the formation of γ-carotene from lycopene.

[0037] 4) No ε-carotene was detected in the pAC-LYC+pET-LcLCYE-α and pAC-LYC+pET-LtLCYE-α strains constructed in this invention, but β-carotene was detected, indicating that the C-terminal structure of the LCYE homolog of Liriodendron tulipifera is essential for its LCYE enzyme activity but does not affect its LCYB enzyme activity.

[0038] 5) This invention reveals the function of the LCYE homolog in the synthesis of carotenoids by cloning and prokaryotic expression of the LCYE homolog in Liriodendron tulipifera, providing a new perspective for flower color breeding of Liriodendron genus, and also providing new gene resources for the artificial synthesis of carotenoids. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the structure of the LCYE homologous gene and its variant genes in the genus Liriodendron.

[0040] Figure 2 This is a diagram of the pET-28a vector and the prokaryotic expression vector of the LCYE homolog of the Liriodendron tulipifera gene;

[0041] Figure 3This is a diagram of the vector digestion results (M: DL5000 Marker; 1 is the result of double digestion of pET-28a vector with BamHI and HindIII, 2-4 are undigested pET-28a).

[0042] Figure 4 This is a screening diagram of positive recombinants (M is DL2000 Marker, 1-3 are PCR results of pET-LcLCYE-α positive recombinants, 4-6 are PCR results of pET-LcLCYE positive recombinants, 7-9 are PCR results of pET-LtLCYE positive recombinants, 10-12 are PCR results of pET-AtLCYE positive recombinants, 13-15 are PCR results of pET-LtLCYE-α positive recombinants, and 16-18 are H2O (negative control)).

[0043] Figure 5 This is a diagram showing the prokaryotic expression results of the LCYE homologous gene from the genus Liriodendron and the AtLCYE gene from Arabidopsis thaliana (pET-28a is the empty vector control, pAC-LYC is the precursor lycopene control, LcLCYE and LtLCYE are the LCYE homologous genes from the genus Liriodendron, LcLCYE-α and LtLCYE-α are variants of the LCYE homologous genes from the genus Liriodendron, and AtLCYE is the LCYE gene from Arabidopsis thaliana).

[0044] Figure 6 This is a mass spectrum of carotenoid-targeted sequencing. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0046] The petals of Liriodendron tulipifera and Liriodendron chinense used in this application were collected from the campus of Nanjing Forestry University. After collection, they were quick-frozen in liquid nitrogen and stored in an ultra-low temperature freezer (-80℃).

[0047] Example 1

[0048] 1. Total RNA extraction from petal tissue

[0049] according to The Super Total RNA Extraction Kit instructions specify the extraction of total RNA from the petal tissues of *Liriodendron tulipifera* and *Liriodendron chinense*, respectively. The specific procedures are as follows:

[0050] Quickly place the petal tissues of *Liriodendron tulipifera* and *Liriodendron chinense* into a mortar containing liquid nitrogen and grind them continuously, adding liquid nitrogen as needed to prevent melting, until the tissue is completely ground into powder. Immediately transfer the powdered sample to a nuclease-free EP tube and weigh it. When the remaining liquid nitrogen is almost evaporated, add lysis buffer and diluent (500 μL). Use a pipette to repeatedly pipette until no obvious lumps of tissue remain in the lysate. Incubate at room temperature for 3-5 minutes. Centrifuge at 12000-14000 × g for 5 minutes and carefully aspirate the supernatant. Add 0.5 times the volume of the supernatant in anhydrous ethanol and rapidly pipette 20-25 times until the liquid becomes a pale blue turbidity with white foam (it is recommended to use a 1 mL pipette adjusted to a 500 μL range for rapid pipetting). Remove the centrifuge column / collection tube and transfer the mixture to the column. If the mixture volume is too large, it can be loaded into the column in two separate passes. Centrifuge at 12000-14000×g for 1 min, discard the filtrate. Add 600 μL of RNA wash buffer, centrifuge at 12000-14000×g for 45 s, discard the filtrate. Add 50 μL of DNase I incubation solution to the center of the adsorption membrane, incubate at room temperature for 15 min. Add 600 μL of RNA wash buffer, centrifuge at 12000-14000×g for 45 s, discard the filtrate. Add 600 μL of RNA wash buffer, centrifuge at 12000-14000×g for 45 s, discard the filtrate. Replace the centrifuge column onto the collection tube, centrifuge at 12000-14000×g for 2 min. Transfer the centrifuge column to the elution tube, add 50-200 μL of nuclease-free water to the center of the centrifuge column membrane, incubate at room temperature for 2 min, centrifuge at 12000-14000×g for 1 min, and store the RNA at -70℃.

[0051] 2 μL of RNA was analyzed by 1% agarose gel electrophoresis. The results showed that the 28S and 18S bands were relatively clear. The 28S band was approximately twice as bright as the 18S band, indicating good RNA quality. RNA purity was determined using a Nonodrop 2000, and OD... 260 / OD 280 It is 2.03, OD 260 / OD 230 The value is 2.01, indicating good integrity, and it can be used for reverse transcription.

[0052] 2. Synthesis of first-strand cDNA

[0053] Using total RNA from the petals of *Liriodendron tulipifera* and *Liriodendron chinense* as templates, reverse transcription was performed using the TaKaRa reverse transcription kit with Oligo(dT) as the anchoring primer to synthesize first-strand cDNA. The specific procedures are as follows:

[0054] Prepare a 6 μL mixture in a centrifuge tube according to the following template RNA / primer sequence: 2 μL template, 3 μL Oligo(dT)12-18 Primer (50 μM), and 1 μL RNase-free ddH2O. Incubate at 70 °C for 10 min on a PCR instrument, then quickly cool on ice and let stand for 2 min. Centrifuge at 5000 rpm for 5 s to allow the denatured template RNA / primer solution to collect at the bottom of the centrifuge tube. Prepare a 10 μL reverse transcription reaction solution in the same centrifuge tube: 6 μL RNA / primer denaturation solution, 2 μL 5×M-MLV Buffer, 0.5 μL dNTP Mixture (10 mM), 0.25 μL RNase Inhibitor (40 U / μL), 0.5 μL RTase M-MLV (200 U / μL), and 0.75 μL RNase-free dH2O. Incubate at 42°C for 1 hour in a PCR instrument. Then incubate at 70°C for 15 minutes in a PCR instrument, and then cool on ice to obtain cDNA solution.

[0055] 2. Cloning of the target gene

[0056] Based on the transcriptome sequencing data of *Liriodendron tulipifera*, BLAST homology alignments were performed using LCYE-related gene sequences from other species. Primers were designed using Oligo 7.0 and Prime 5.0. The primer sequences are shown below:

[0057] LcLCYE-F: 5'-ATGGAGTGTTTTGGTGTTCGTCAC-3',

[0058] LcLCYE-R: 5'-TAGGGTGAGATATGTTCTTACCAAGATTGCC-3';

[0059] LcLCYE-α-F: 5'-ATGGAGTGTTTTGGTGTTCGTCAC-3',

[0060] LcLCYE-α-R: 5'-CTATTCGACCTGCTTCCATGCC-3';

[0061] LtLCYE-F: 5'-ATGGAGTGTTTTGGTGTTCGTCAC-3',

[0062] LtLCYE-R: 5'-TAGGGTGAGATATGTTCTTACCAAGATTGCC-3';

[0063] LtLCYE-α-F: 5'-ATGGAGTGTTTTGGTGTTCGTCAC-3',

[0064] LtLCYE-α-R: 5'-CTAAAGATGGCTGTGATTTTCAGTCAACG-3'.

[0065] Using cDNA first strand as a template, the gene for hybridizing *Liriodendron tulipifera* was cloned using high-fidelity Phusion DNA polymerase. The PCR amplification system (50 μL) consisted of: 25 μL 2×Phusion Master Mix, 2 μL Forward Primer, 2 μL Reverse Primer, 3 μL Template DNA, and 18 μL Nuclease-free Water. The PCR program was: 98℃ for 5 min; 98℃ for 30 s, 57℃ for 30 s, 72℃ for 1 min 40 s, 35 cycles; 72℃ for 7 min; 4℃ forever. After the PCR reaction, a small amount of PCR product (approximately 4 μL) was taken for agarose gel electrophoresis. After electrophoresis, the target fragment was cut, and the PCR target amplification product was purified by gel recovery. The target fragment was purified and recovered using an AXYGEN DNA gel recovery kit. 2 μL of the purified product was then analyzed by gel electrophoresis using 1% agarose gel.

[0066] 3. Target gene transformation of Escherichia coli

[0067] Ligation was performed using the pMD19-T Vector (D102A) cloning vector from TaKaRa. The ligation system (10 μL) consisted of 5 μL Solution I, 1 μL pMD19-T Vector, and 4 μL of purified PCR product (50 ng). After gentle mixing, the mixture was centrifuged at low speed and incubated overnight at 16°C in a water bath. Positive recombinant plasmids of the target gene and the pMD19-T cloning vector were extracted using a plasmid miniprep kit (Tiangen Biotech).

[0068] Take a small amount of DH5α strain (Qingke Biotechnology) stored in an ultra-low temperature freezer at -80℃, streak it on LB medium without antibiotics, and incubate it upside down in a 37℃ incubator for 12-16 hours.

[0069] Remove competent DH5α cells from the cryogenic freezer and thaw on ice. Add 5 μL of the overnight ligation product to 100 μL of competent cells; place the centrifuge tube on ice for 30 min; heat shock in a 42°C water bath for 90 s without shaking; immediately place on ice for 2 min; add 800 μL of antibiotic-free liquid culture medium to a clean bench and incubate at 37°C and 180 rpm for 1 h; centrifuge at 4000 rpm for 3 min and aspirate 800 μL of supernatant; resuspend the precipitated bacterial cells and plate on LB agar plates (Amp concentration 100 mg / L), and incubate overnight at 37°C.

[0070] 4. Screening and validation of recombinant plasmids

[0071] Pick a single colony that has grown overnight on LB solid medium containing the antibiotic (Amp) and inoculate it into 750 μL of LB liquid medium containing the same antibiotic. Incubate overnight at 37°C and 250 rpm.

[0072] The PCR amplification system consisted of: 2 μL 10×PCR Buffer, 1.2 μL MgCl2 (25 mM), 0.4 μL dNTP (10 mM), 1 μL M13-F / R, 2 μL bacterial culture, 0.2 μL rTaq, and ddH2O to a final volume of 20 μL.

[0073] The PCR program was as follows: 95℃ for 5 min; 94℃ for 30 s, 55℃ for 15 s, 72℃ for 30 s, 37 cycles; 72℃ for 5 min; 4℃ forever.

[0074] 10 μL of PCR product was aspirated and analyzed by agarose gel electrophoresis. After verification, bacterial culture samples with correct band sizes were sent to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing using universal primers M13F / R. Sequencing results were compared and analyzed on NCBI.

[0075] Based on sequencing results, two homologous genes of the *Liriodendron* genus *LCYE* were finally cloned, named LcLCYE (its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.2) and LtLCYE (its nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.4), as well as two variant genes with C-terminal variations. Figure 1The two genes were named LcLCYE-α (a variant of LcLCYE) (its nucleotide sequence is shown in SEQ ID NO.5, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.6) and LtLCYE-α (a variant of LtLCYE) (its nucleotide sequence is shown in SEQ ID NO.7, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.8), respectively.

[0076] Example 2

[0077] The selected pAC-LYC lycopene synthesis plasmid (addgene) contains three genes from the carotenoid pathway gene cluster of Erwinia herbicola Eho10—crtE, crtI, and crtB. The selected strain is the BL21(DE3) prokaryotic expression strain (Weidi Biotechnology).

[0078] 1. Construction of the pET-28a vector

[0079] The full-length ORF sequence of the target gene was ligated into the prokaryotic expression vector pET-28a to construct the vector. The vector map is shown below. Figure 2 As shown. Positive recombinant plasmids were extracted using a plasmid miniprep kit (Tiangen Biotech).

[0080] 2. Addition of specific restriction enzyme sites

[0081] Using positive recombinant plasmids of the pET-28a vector and the prokaryotic expression vector of the target gene as templates, specific restriction enzyme sites BamHI and HindIII were added to both sides of the target gene via PCR. The primer sequences used are shown below:

[0082] pET-LcLCYE-F:

[0083] 5'-agcaaatgggtcgcggatccATGGAGTGTTTTGGTGTTCGTCAC-3',

[0084] pET-LcLCYE-R:

[0085] 5'-tcgagtgcggccgcaagcttTAGGGTGAGATATGTTCTTACCAAGATTGCC-3';

[0086] pET-LcLCYE-α-F:

[0087] 5'-agcaaatgggtcgcggatccATGGAGTGTTTTGGTGTTCGTCAC-3',

[0088] pET-LcLCYE-α-R:

[0089] 5'-tcgagtgcggccgcaagcttTTCGACCTGCTTCCATGCCAC-3';

[0090] pET-LcLCYE-F:

[0091] 5'-agcaaatgggtcgcggatccATGGAGTGTTTTGGTGTTCGTCAC-3',

[0092] pET-LcLCYE-R:

[0093] 5'-tcgagtgcggccgcaagcttTAGGGTGAGATATGTTCTTACCAAGATTGCC-3';

[0094] pET-LtLCYE-α-F:

[0095] 5'-agcaaatgggtcgcggatccATGGAGTGTTTTGGTGTTCGTCAC-3',

[0096] pET-LtLCYE-α-R:

[0097] 5'-tcgagtgcggccgcaagcttTAAAGATGGCTGTGATTTTCAGTCA-3'.

[0098] PCR reaction system (20 μL): 2 μL 10×PCR Buffer, 1.2 μL MgCl2 (25 mM), 0.4 μL dNTP (10 mM), 1 μL Forward Primer, 1 μL Forward Primer, 1 μL plasmid DNA, 0.2 μL rTaq, 13.2 μL ddH2O.

[0099] PCR reaction program: 94℃ for 5 min; 94℃ for 30 s, 55-59℃ for 30 s, 72℃ for 1 min and 40 s, 36 cycles; 72℃ for 7 min; 4℃ forever.

[0100] The obtained PCR products were separated by 1% agarose gel electrophoresis and purified using the AxyPrep DNA Gel Extraction Kit to obtain gene fragments with added homologous arms.

[0101] 3. Double enzyme digestion reaction

[0102] The empty pET-28a expression vector was digested with the restriction endonucleases BamHI and HindIII.

[0103] The pET-28a digestion reaction system consisted of 2 μL 10×K buffer, 0.5 μL BamHI, 0.5 μL HindIII, 1 μg of recovered product / pET-28a empty vector plasmid, and ddH2O to a final volume of 20 μL. The mixture was incubated at 37°C for 4-6 hours. The digestion reaction was terminated by adding 10× Loading Buffer. The digested products were separated and detected by 1% agarose gel electrophoresis. The digested products were recovered and purified using the AxyPrepDNA Gel Extraction Kit (AXYGEN) and dissolved in 20 μL ddH2O. The results are shown below. Figure 3 As shown, M is the DL2000 Marker, 1 is the linearized pET-28a after enzyme digestion, and 2-4 are the undigested pET-28a.

[0104] 4. Connection reaction

[0105] The recovered gene fragment containing homologous arms and the linearized vector pET-28a after double enzyme digestion were added according to the purity and concentration of the target fragment and vector, and ligated overnight at 16°C. The ratio of target fragment molecules to vector molecules was 3:1 to 5:1. The ligation reaction system consisted of 2 μL T4 DNA ligase buffer (10×), 2 μL of enzyme-digested expression vector, 10 μL of target fragment, 1 μL of T4 DNA ligase, and ddH2O to a final volume of 20 μL. The ligation product of the target fragment and vector pET-28a was then co-transformed with the pAC-LYC plasmid into E. coli BL21(DE3) supersensor cells.

[0106] 5. Identification of recombinants

[0107] Single colonies from the plates were inoculated into LB broth containing antibiotics (kanamycin) and incubated overnight at 37°C with shaking at 250 rpm. Colony PCR was performed using full-length primers for the target gene to screen for positive clones. The screened positive clones were sent to AxyGEN for sequencing. Simultaneously, plasmids were extracted using the AxyPrep Plasmid Miniprep Kit (AXYGEN) and enzyme digestion was performed to verify the consistency of fragment sizes after digestion.

[0108] The results are as follows Figure 4As shown, M is the DL2000 Marker, 1-3 are the PCR results of pET-LcLCYE-α positive recombinant, 4-6 are the PCR results of pET-LcLCYE positive recombinant, 7-9 are the PCR results of pET-LtLCYE positive recombinant, 10-12 are the PCR results of pET-AtLCYE positive recombinant, 13-15 are the PCR results of pET-LtLCYE-α positive recombinant, and 16-18 are H2O (negative control).

[0109] Example 3

[0110] 1. IPTG-induced prokaryotic expression

[0111] Accumulate bacterial count by overnight shaking of successfully transformed *E. coli* BL21(DE3). The required culture medium resistance should correspond to the plasmid resistance (kanamycin, chloramphenicol). Transfer 2 mL of the above bacterial culture to a 250 mL Erlenmeyer flask, add 50 mL of LB medium, and incubate at 28°C and 220 rpm on a shaker for 4-6 hours, until OD reaches [value missing]. 600 Add approximately 0.5 mg of IPTG solution to a final concentration of 0.5 mM, and incubate at 16°C and 160 rpm. Keep the incubation process in the dark. Incubate with shaking for approximately 20 hours to accumulate lycopene. Then transfer the bacterial culture to a centrifuge tube, centrifuge at 5000 rpm for 10 minutes, collect the bacterial cells, and observe and photograph them.

[0112] 2. Phenotypic observation

[0113] Different BL21(DE3) prokaryotic expression strains were sequentially transformed with plasmids pET-28a, pAC-LYC+pET-28a, pAC-LYC+pET-LcLCYE, pAC-LYC+pET-LcLCYE-α, pAC-LYC+pET-LtLCYE, pAC-LYC+pET-LtLCYE-α, and pAC-LYC+pET-AtLCYE, respectively, at 16℃ and 160 rpm for prokaryotic expression. After induction, the bacterial culture was centrifuged to collect the bacterial cells.

[0114] The results are as follows Figure 5 As shown, all five LCYE genes have the function of converting lycopene (red) into carotenoids (orange-yellow). The plaques transformed with the LcLCYE-α and LtLCYE-α genes are the darkest, those transformed with the LcLCYE and LtLCYE genes are slightly lighter, and those transformed with the AtLCYE gene are the lightest. This indicates that the LCYE homologous genes and the AtLCYE gene in the genus *Liriodendron* have different functions.

[0115] 3. Assay of carotenoid-targeted metabolism in prokaryotic expression strains

[0116] The results are as follows Figure 6 As shown, the results of the carotenoid targeted metabolism assay revealed that the main metabolite in the pET-28a+pAC-LYC control was red lycopene, while ε-carotene was clearly detected in the pAC-LYC+pET-AtLCYE strain, indicating that the AtLCYE protein possesses traditional LCYE enzyme activity, meaning it can catalyze the conversion of lycopene to ε-carotene through continuous catalysis. In contrast, in the pAC-LYC+pET-LcLCYE and pAC-LYC+pET-LtLCYE strains, in addition to lycopene, both ε-carotene and γ-carotene were detected, indicating that the Liriodendron tulipifera LCYE homologous protein, besides possessing traditional LCYE enzyme activity, also has LCYB enzyme activity that catalyzes the formation of γ-carotene from lycopene. In addition, ε-carotene was not detected in strains expressing pAC-LYC+pET-LcLCYE-α and pAC-LYC+pET-LtLCYE-α, but β-carotene was detected, indicating that the C-terminal structure of the LCYE homolog of Liriodendron tulipifera is essential for its LCYE enzyme activity but does not affect its LCYB enzyme activity.

[0117] In summary, the LCYE homologs in both Liriodendron tulipifera and Liriodendron chinense have undergone new functionalization. The proteins they encode not only possess the traditional LCYE enzyme activity, which continuously catalyzes the formation of ε-carotene from lycopene, but also possess the LCYB enzyme activity, which catalyzes the formation of γ-carotene from lycopene and further catalyzes the formation of β-carotene.

[0118] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. Application of the variant gene LtLCYE-α, whose nucleotide sequence is shown in SEQ ID NO.7, in promoting the conversion of lycopene to β-carotene.

2. The application according to claim 1, characterized in that, The specific steps include: 1) Construct a prokaryotic expression vector for the variant gene LtLCYE-α; 2) Transform the prokaryotic expression vector into a prokaryotic expression strain; 3) Cultivate and screen strains with significantly increased β-carotene content.

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