Liriodendron LCYE gene as well as expression protein and application thereof

Abstract

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

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically, relates to a class of Liriodendron LCYE genes and their expression proteins and applications. Background Art

[0002] Flower color is an important flower trait. It plays a very important role in the reproductive ecology and species evolution of flowering plants. It is an important indicator for evaluating the ornamental value and economic value of plants. Anthocyanins are the main influencing factors in the formation of plant flower color, mainly including three natural pigments: flavonoids, carotenoids and betalains. Among them, flavonoids and carotenoids are the two major pigment groups that form the flower color of most plants. They are widely present in most plants and participate in the process of plant flower color formation, thus making the flower color of plants diverse.

[0003] The biosynthetic pathway of carotenoids has been studied thoroughly and is highly conserved in different plants. Among them, GGPP formed from the MEP pathway is converted into red lycopene under the catalytic reaction 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 catalyze the formation of ε ring and β ring at one end of lycopene. Therefore, lycopene forms ε-carotene under the continuous catalysis of LCYE enzyme, γ-carotene and β-carotene are formed in sequence under the continuous catalysis of LCYB enzyme, and α-carotene is formed under the sequential action of LCYE and LCYB enzymes.

[0004] Since carotenoids play a very important role in plant growth and development and human health, most of the research on the regulation of carotenoids is focused on plant leaves or crop rhizomes, while there are relatively few studies on the regulation of plant flower coloring (DOI: 10.3389 / fpls.2019.01017). In recent years, there have been major breakthroughs in the regulation of carotenoid biosynthesis in the process of flower coloring, but they are mainly concentrated in a few herbaceous plants such as tobacco, monkey face flower and alfalfa. In tobacco, the F-box protein COI1 mediates the expression regulation of PSY, ZDS and LCYB genes through indirect regulation, thereby affecting the β-carotene content in the petals and changing the flower color (DOI: 10.3389 / fpls.2019.01017). In monkey face flower, a tetrapeptide repeat TPR protein RCP2 can indirectly regulate the expression of multiple genes such as CRISTO and LCYB, thereby changing the coloring 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 the synthesis of carotene in alfalfa petals and making the petals yellow (DOI: 10.1105 / tpc.19.00480). The relevant results provide a new perspective for the study of carotenoid-mediated plant petal coloration, but the breadth and depth are far less than the research in leaves or tubers, especially for carotenoid-mediated flower color variation in woody plants and related basic research.

[0005] Liriodendron belongs to the magnolia family and includes a pair of sister species with interstellar distribution in East Asia and Eastern North America, namely, Liriodendron chinense, which is naturally distributed in East Asia, and Liriodendron tulipifera, which is naturally distributed in Eastern North America. The petals of Liriodendron chinense are green throughout, with yellow vertical stripes running through the petals during the blooming period; the petals of Liriodendron tulipifera have an orange-yellow band near the base, which is brightly colored. Previous studies have shown that the main pigments accumulated in the orange-yellow band area of ​​Liriodendron tulipifera are carotenoids, especially carotene substances including α-carotene and β-carotene. However, LCYB, a key gene that acts on carotene synthesis, maintains a stable and low expression level in petals. Instead, the expression of LCYE is highly correlated with the coloration of the color band area of ​​Liriodendron tulipifera and the accumulation of carotene (DOI: 10.1038 / s41438-020-0287-3).

[0006] In summary, strengthening the study of LCYE gene function and its genetic regulation mechanism will help us to deeply understand the molecular mechanism of petal coloration and carotenoid synthesis in Liriodendron plants and lay a theoretical foundation for breeding new varieties of Liriodendron. Therefore, the study of LCYE gene function has important scientific and application value. Summary of the invention

[0007] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a class of LCYE genes of the genus Liriodendron. Another technical problem to be solved by the present invention is to provide an expression protein of a class of LCYE genes of the genus Liriodendron. Another technical problem to be solved by the present invention is to provide an application of a class of LCYE genes of the genus Liriodendron for regulating carotenoid synthesis.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0009] A class of LCYE genes of the genus Liriodendron, including LcLCYE gene, or / and LtLCYE gene, or / and variant gene LcLCYE-α, or / and variant gene 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 protein of the LCYE gene of the genus Liriodendron includes LcLCYE protein, or / and LtLCYE protein, or / and variant protein LcLCYE-α, or / and 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 Liriodendron LCYE gene in promoting the conversion of lycopene into carotenoids.

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

[0021] The application specifically comprises the following steps:

[0022] 1) constructing a prokaryotic expression vector of the LcLCYE gene or the LtLCYE gene;

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

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

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

[0026] The application specifically comprises the following steps:

[0027] 1) constructing a prokaryotic expression vector of the variant gene LcLCYE-α or the variant gene LtLCYE-α;

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

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

[0030] The application of the expression protein of the Liriodendron LCYE gene 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 LcLCYE-α protein as described in claim 2 or the use of LcLCYE-α protein in promoting the conversion of lycopene into β-carotene.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1) A class of LCYE genes of the genus Liriodendron disclosed in the present invention includes LcLCYE gene, or / and LtLCYE gene, or / and variant gene LcLCYE-α, or / and 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; 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 the present invention promotes the conversion of lycopene into carotenoids ε-carotene and γ-carotene. The variant gene LcLCYE-α or 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 the present invention, indicating that the LCYE homologous protein of Liriodendron chinense has not only the traditional LCYE enzyme activity, but also the LCYB enzyme activity that catalyzes lycopene to form γ-carotene.

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

[0038] 5) The present invention reveals the function of LCYE homologous genes in carotenoid synthesis through cloning and prokaryotic expression of LCYE homologous genes in Liriodendron, providing a new perspective for the flower color breeding of Liriodendron plants, and also providing a new gene resource for the artificial synthesis of carotenoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a schematic diagram of the structure of LCYE homologous genes and their variant genes in the genus Liriodendron;

[0040] Figure 2 It is a map of the pET-28a vector and the prokaryotic expression vector of the LCYE homologous gene of Liriodendron spp.;

[0041] Figure 3It is the result of vector enzyme digestion (M: DL5000 Marker; 1 is the result of double digestion of pET-28a vector with BamHI and HindIII, 2-4 are pET-28a without enzyme digestion);

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

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

[0044] Figure 6 Mass spectra of targeted sequencing of carotenoids. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0046] The petal materials of Liriodendron tulipifera and Liriodendron chinense used in this application were collected on the campus of Nanjing Forestry University, and were quickly frozen in liquid nitrogen and stored in an ultra-low temperature refrigerator (-80°C).

[0047] Example 1

[0048] 1. Total RNA extraction from petal tissue

[0049] according to Super Total RNA Extraction Kit Instructions, extract total RNA from petal tissues of Liriodendron tulipifera and Liriodendron chinense respectively, the specific steps are as follows:

[0050] Quickly put the petal tissues of Liriodendron chinense and Liriodendron truncatum into a mortar with liquid nitrogen added for grinding. Liquid nitrogen should be continuously added during the grinding process to prevent the tissue from melting until the tissue is completely ground into powder. Quickly transfer the ground sample into a nuclease-free EP tube and weigh it. When the remaining liquid nitrogen is about to evaporate, add the lysis solution and diluent (500μL), and repeatedly blow with a pipette until there is no obvious block tissue in the lysate. Leave at room temperature for 3-5min. Centrifuge at 12000-14000×g for 5min, and carefully aspirate the supernatant. Add 0.5 times the volume of supernatant anhydrous ethanol, and blow quickly with a pipette 20-25 times to make the liquid light blue turbid with white foam (it is recommended to use a 1mL pipette and adjust to 500μL range for rapid blowing). Take out the centrifuge column / collection tube and transfer the mixture to the centrifuge column. If the volume of the mixture is too large, it can be loaded into the column twice. Centrifuge at 12000-14000×g for 1min and discard the filtrate. Add 600μL RNA wash solution, centrifuge at 12000-14000×g for 45s, and discard the filtrate. Add 50μL DNase I incubation solution to the center of the adsorption membrane and let it stand at room temperature for 15min. Add 600μL RNA wash solution, centrifuge at 12000-14000×g for 45s, and discard the filtrate. Add 600μL RNA wash solution, centrifuge at 12000-14000×g for 45s, and discard the filtrate. Re-place the centrifuge column on the collection tube and centrifuge at 12000-14000×g for 2min. Transfer the centrifuge column to the elution tube, add 50-200μL nuclease-free water to the center of the centrifuge column membrane, let it stand at room temperature for 2min, centrifuge at 12000-14000×g for 1min, and store the RNA at -70℃.

[0051] 2 μL RNA was drawn and tested on 1% agarose gel electrophoresis. The results showed that the 28S and 18S bands were relatively clear. The brightness of the 28S band was about twice that of the 18S band, indicating that the RNA quality was good. The purity of RNA was tested using Nonodrop 2000. OD 260 / OD 280 OD is 2.03 260 / OD 230 The value is 2.01, which indicates good integrity and can be used for reverse transcription.

[0052] 2. Synthesis of first-strand cDNA

[0053] The total RNA of petals of Liriodendron chinense and Liriodendron tulipifera was used as template, and reverse transcription was performed using TaKaRa reverse transcription kit, and Oligo(dT) was used as anchor primer to synthesize the first-strand cDNA. The specific operation is as follows:

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

[0055] 2. Target gene cloning

[0056] Based on the transcriptome sequencing data of Liriodendron chinense, Blast homology alignment was performed using LCYE-related gene sequences of other species. The corresponding primers were designed using Oligo 7.0 and Prime 5.0. The primer sequences are as follows:

[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] The hybrid Liriodendron gene was cloned using the first strand of cDNA as a template using the ultra-fidelity Phusion DNA polymerase. The PCR amplification system (50 μL) was: 25 μL 2×Phusion Master Mix, 2 μL Forward Primer, 2 μL Reverse Primer, 3 μL Template DNA, 18 μL Nuclease-free Water. The PCR program was: 98°C 5 min; 98°C 30 s, 57°C 30 s, 72°C 1 min 40 s, 35 cycles; 72°C 7 min; 4°C forever. After the PCR reaction was completed, a small amount of PCR product (about 4 μL) was taken for agarose gel electrophoresis detection, and the target fragment was cut after electrophoresis, and the PCR target amplification product was purified by gel recovery. The DNA gel recovery kit of AXYGEN was used to purify and recover the target fragment, and 2 μL of the recovered purified product was taken and detected by gel electrophoresis using 1% agarose.

[0066] 3. Transformation of target gene into Escherichia coli

[0067] The pMD19-T Vector (D102A) cloning vector of TaKaRa was used for the ligation reaction. The ligation system (10 μL) was as follows: 5 μL Solution I, 1 μL pMD19-T Vector, 4 μL PCR purified product (50 ng). After gently pipetting and mixing, centrifuge at low speed and ligate overnight in a water bath at 16°C. The positive recombinant plasmid of the target gene and the cloning vector pMD19-T was extracted using a plasmid extraction kit (Tiangen Biotechnology).

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

[0069] Take out the competent cell DH5α strain from the ultra-low temperature refrigerator and put it on ice to melt. Pipette 5μL of the overnight ligation product and add it to 100μL competent cells; place the centrifuge tube on ice for 30 minutes; water bath in a 42℃ water bath, heat shock for 90s, do not shake during the period; then immediately place it on ice for 2 minutes; add 800μL of antibiotic-free liquid culture medium in the clean bench, shake at 37℃, 180rpm for 1h to revive; centrifuge at 4000rpm for 3min, and remove 800μL of supernatant; resuspend the precipitated bacteria, apply it to LB plate (Amp concentration is 100mg / L), and culture at 37℃ overnight.

[0070] 4. Recombinant plasmid screening and verification

[0071] A single colony grown overnight on LB solid medium containing antibiotics (Amp) was picked and inoculated into 750 μL LB liquid medium containing the same antibiotics. Cultured overnight at 250 rpm and 37°C.

[0072] The PCR amplification system was: 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 solution, 0.2 μL rTaq, and ddH2O supplemented to 20 μL.

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

[0074] 10 μL of PCR product was taken for agarose gel electrophoresis analysis. After verification, the bacterial sample with the correct band size was entrusted to Shanghai Yingjun Company for sequencing, and the sequencing primer was the universal primer M13F / R. The sequencing results were compared and analyzed on NCBI.

[0075] According to the sequencing results, two LCYE homologous genes from the genus Liriodendron were finally cloned and named LcLCYE (the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the expressed protein is shown in SEQ ID NO.2) and LtLCYE (the nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence of the expressed protein is shown in SEQ ID NO.4), as well as two variant genes with mutations in the C-terminus ( Figure 1), named LcLCYE-α (variant gene of LcLCYE) (the nucleotide sequence of which is shown in SEQ ID NO.5, and the amino acid sequence of the expressed protein is shown in SEQ ID NO.6) and LtLCYE-α (variant gene of LtLCYE) (the nucleotide sequence of which is shown in SEQ ID NO.7, and the amino acid sequence of the 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 of the carotenoid pathway gene cluster of Erwinia herbicola Eho10—crtE, crtI and crtB. The selected strain material is the BL21 (DE3) prokaryotic expression strain (Weidi Biotechnology).

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

[0079] The full-length ORF sequence of the target gene was connected to the prokaryotic expression vector pET-28a to construct the vector. The vector map is shown in Figure 2 The positive recombinant plasmid was extracted using a plasmid extraction kit (Tiangen Biochemical Technology).

[0080] 2. Addition of specific enzyme cutting sites

[0081] Using pET-28a vector and the positive recombinant plasmid of the prokaryotic expression vector constructed with the target gene as templates, specific restriction sites BamH I and Hind III were added to both sides of the target gene by PCR technology. The primer sequences used are as follows:

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

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

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

[0101] 3. Double enzyme digestion reaction

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

[0103] pET-28a restriction enzyme reaction system: 2μL 10×K buffer, 0.5μL BamH I, 0.5μL HindIII, 1μg recovery product / pET-28a empty vector plasmid, ddH2O supplemented to 20μL. 37℃ water bath, enzyme digestion for 4-6h. Add 10×LoadingBuffer buffer to terminate the enzyme digestion reaction. The enzyme digestion products were separated and detected by 1% agarose gel electrophoresis, and the enzyme digestion products were recovered and purified using the AxyPrepDNA Gel Extraction Kit (AXYGEN), and dissolved in 20μL ddH2O. The results are as follows Figure 3 As shown, M is DL2000 Marker, 1 is linearized pET-28a after enzyme digestion, and 2-4 are pET-28a without enzyme digestion.

[0104] 4. Ligation reaction

[0105] The recovered gene fragment containing homology arms and the linearized vector pET-28a after double enzyme digestion were added with various reagents according to the connection system according to the detected purity and concentration, and connected at 16°C overnight. Among them, the number of target fragment molecules: the number of vector molecules = 3:1-5:1, and the connection reaction system is: 2μL T4 DNA ligase buffer (10×), 2μL of enzyme-digested expression vector, 10μL target fragment, 1μL T4 DNA ligase, and ddH2O supplemented to 20μL. The product after the target fragment and the vector pET-28a were connected and co-transfected with the pAC-LYC plasmid into E. coli BL21 (DE3) super-sense cells.

[0106] 5. Identification of recombinants

[0107] Pick a single colony on the plate and inoculate it into LB liquid medium containing antibiotics (kanamycin), and culture it overnight at 37°C and 250rpm. Use the full-length primers of the target gene to perform bacterial liquid PCR to screen positive clones. The screened positive clones are sent to Qingke for sequencing. At the same time, use AxyPrep Plasmid Miniprep Kit (AXYGEN) to extract the plasmid and perform enzyme digestion verification to determine whether the fragment size is consistent after enzyme digestion.

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

[0109] Example 3

[0110] 1. IPTG-induced prokaryotic expression

[0111] The successfully transformed E. coli BL21 (DE3) was shaken overnight to accumulate the number of bacteria. The required culture medium resistance should correspond to the resistance of the plasmid (kanamycin, chloramphenicol). Take 2mL of the above bacterial solution into a 250mL Erlenmeyer flask, add 50mL LB culture medium, and culture on a shaker at 28℃ and 220rpm for 4-6h until the OD 600 The concentration of lycopene is about 0.5, add IPTG solution with a final concentration of 0.5mM, 16℃, 160rpm. Keep away from light throughout the process. Oscillating culture for about 20h to accumulate lycopene. Then transfer the bacterial solution to a centrifuge tube, centrifuge at 5000rpm for 10min, collect the bacteria, observe and take pictures.

[0112] 2. Phenotypic observation

[0113] 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 plasmids were respectively transferred into different BL21 (DE3) prokaryotic expression strains at 16°C and 160 rpm for prokaryotic expression. After induction, the bacterial solution was centrifuged to collect the cells.

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

[0115] 3. Targeted metabolism assay of carotenoids in prokaryotic expression strains

[0116] The results are as follows Figure 6 As shown in the figure, the results of targeted metabolic determination of carotenoids showed that the main metabolite contained in the pET-28a+pAC-LYC control was red lycopene, while ε-carotene was clearly detected in the expressed pAC-LYC+pET-AtLCYE strain, indicating that the AtLCYE protein has the traditional LCYE enzyme activity, that is, it can convert lycopene into ε-carotene through continuous catalysis. In comparison, in addition to lycopene, ε-carotene and γ-carotene were detected in the expression pAC-LYC+pET-LcLCYE and pAC-LYC+pET-LtLCYE strains, indicating that the LCYE homologous protein of Liriodendron chinense has not only the traditional LCYE enzyme activity, but also the LCYB enzyme activity that catalyzes lycopene to form γ-carotene. In addition, no ε-carotene was detected in the 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 homologous protein in Liriodendron chinense is essential for its LCYE enzymatic activity but does not affect its LCYB enzymatic activity.

[0117] In summary, the LCYE homologous genes in Liriodendron chinense and Liriodendron tulipifera have undergone new functionalization. In addition to the traditional LCYE enzyme activity, which continuously catalyzes lycopene to form ε-carotene, the proteins they encode also have LCYB enzyme activity, which catalyzes lycopene to form γ-carotene and further catalyzes the formation of β-carotene.

[0118] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A class of LCYE genes of the genus Liriodendron, including LcLCYE gene, or / and LtLCYE gene, or / and variant gene LcLCYE-α, or / and 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; The nucleotide sequence of the variant gene LtLCYE-α is shown in SEQ ID NO.

7.

2. The expressed protein of the LCYE gene of Liriodendron spp. according to claim 1, comprising LcLCYE protein, or / and LtLCYE protein, or / and variant protein LcLCYE-α, or / and variant protein LtLCYE-α; The amino acid sequence of the LcLCYE protein is shown in SEQ ID NO.2; The amino acid sequence of the LtLCYE protein is shown in SEQ ID NO.4; The amino acid sequence of the variant protein LcLCYE-α is shown in SEQ ID NO.6; The amino acid sequence of the variant protein LtLCYE-α is shown in SEQ ID NO.

8.

3. Use of the Liriodendron LCYE gene of claim 1 in promoting the conversion of lycopene into carotenoids.

4. Use of the LcLCYE gene or LtLCYE gene of the genus Liriodendron according to claim 1 in promoting the conversion of lycopene into ε-carotene and γ-carotene.

5. Use of the variant gene LcLCYE-α or variant gene LtLCYE-α of the genus Liriodendron according to claim 1 in promoting the conversion of lycopene into β-carotene.

6. The use according to claim 4, characterized in that: The specific steps include: 1) constructing a prokaryotic expression vector of the LcLCYE gene or the LtLCYE gene; 2) transforming the prokaryotic expression vector into a prokaryotic expression strain; 3) Cultivate, screen and obtain strains with significantly increased ε-carotene and γ-carotene contents.

7. The use according to claim 5, characterized in that: The specific steps include: 1) constructing a prokaryotic expression vector of the variant gene LcLCYE-α or the variant gene LtLCYE-α; 2) transforming the prokaryotic expression vector into a prokaryotic expression strain; 3) Cultivate, screen and obtain strains with significantly increased β-carotene content.

8. Use of the expression protein of the Liriodendron LCYE gene according to claim 2 in promoting the conversion of lycopene into carotenoids.

9. Use of the LcLCYE protein or LtLCYE protein according to claim 2 in promoting the conversion of lycopene into ε-carotene and γ-carotene.

10. The LcLCYE-α protein according to claim 2 or use of the LcLCYE-α protein in promoting the conversion of lycopene into β-carotene.

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