Carotenoid oxygenase and its application

Abstract

A protease with a function of degrading carotenoids of scallop is provided, which belongs to the technical field of genetic engineering. In particular, the present invention relates to the sequence and application of a carotenoid oxidase gene of scallop. The present invention analyzes the gene sequence, and verifies that the gene has function of degrading carotenoids of scallop scallop by RNA interference technology, and can be used to increase the carotenoid content of the scallop muscle of scallop scallop. The gene verified that the gene expression products can degrade carotenoids accumulated in bacteria. In addition, the RNA interference method provides a powerful tool for the study of scallop functional genes; the prokaryotic expression and protein purification methods provided can efficiently prepare oxidative lyases of carotenoid in scallop, so as to provide necessary experimental tools for in-depth research of the mechanism of shell carotene metabolism.

Claims

1. A protease, comprising: (1) a protease with an amino acid sequence of SEQ ID NO: 1; (2) a homologous protease which is substituted, deleted, or added with one or more amino acids in the amino acid sequence of (1) and has protease activity of the protease in (1); and (3) an allele enzyme of the protease with the amino acid sequence of the SEQ ID NO: 1, which is screened from Patinopecten yessoensis.

2. A method for cleaving carotenoids comprising introducing the protease according to claim 1.

3. A method for increasing carotenoids content in animals, through reducing a content of the protein as recited in claim 1.

4. The method, as recited in claim 3, which is implemented by reducing an expression amount of a gene encoding the protease according to claim 1.

5. The method, as recited in claim 4, wherein a nucleotide sequence of the encoded gene is SEQ ID NO: 2.

6. The method, as recited in claim 3, wherein the animal is an aquatic organism.

7. The method, as recited in claim 6, wherein the aquatic organism is a mollusk, an arthropod, an echinoderm or algae.

8. The method, as recited in claim 7, wherein the mollusk is shellfish, the arthropod is shrimp and crab, and the echinoderm is sea urchin.

9. The method, as recited in claim 8, wherein the shellfish is bivalve shellfish.

10. The method, as recited in claim 9, wherein the bivalve shellfish is scallop.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1: HPLC detection of carotenoids in the adductor muscle of the experimental and control groups. A: experimental group with dsRNA injection for inhibiting carotenoid oxygenase expression, B: control group.

[0026] FIG. 2: SDS-PAGE analysis of prokaryotic expression of the fusion protein. M: Protein Marker; 1: total protein from the cells without induction of carotenoid oxygenase expression; 2: supernatant of lysis of E. coli cultured at 20 C. for inducing carotenoid oxygenase expression; 3: pellets of lysis of E. coli cultured at 20 C. for inducing carotenoid oxygenase expression; 4: supernatant of lysis of E. coli cultured at 37 C. for inducing carotenoid oxygenase expression; 5: pellets of lysis of E. coli cultured at 37 C. for inducing carotenoid oxygenase expression.

[0027] FIG. 3: Color change of -carotene accumulating strains caused by expression of carotenoid oxygenase, (C): -carotene accumulating strains with empty plasmid pET-28a; (C+): -carotene accumulating strains with recombinant plasmid for expressing carotenoid oxygenase.

[0028] FIG. 4: SDS-PAGE analysis of fusion protein purified with nickel-agarose affinity chromatography. M: Protein marker; 1: loading; 2: elution; 3-4: 20 mM Imidazole-eluted fraction; 5: 50 mM Imidazole eluted fraction; 6: 500 mM Imidazole-eluted fraction.

[0029] FIG. 5: SDS-PAGE analysis of the final purified protein. M: Protein marker; 1: target fusion protein.

[0030] FIG. 6: Expression of carotenoid oxygenase genes in striated muscle with dsRNA injection;

[0031] FIG. 7: Color change of scallop adductor muscle with dsRNA injection. A: control group, injected with 1PBS solution; B: experimental group, injected with dsRNA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0032] The protease of the present invention is described in detail below with reference to the examples.

Example 1: Screening for Proteases

[0033] The applicant has discovered through multiple omics studies such as whole- genome linkage analysis, association analysis, and expression profiling that the reason for the accumulation of carotenoids in scallop with orange adductor muscles is that the expression of carotenoid oxygenase is down-regulated, which reduces the degradation of carotenoids.

[0034] The sequence of carotenoid oxygenase gene was obtained from the scallop genome and transcriptome database. The gene is 9107 bp in length and the corresponding cDNA is 2682 bp in length. The CDS length is 1566 bp (SEQ ID NO: 2) and encodes 521 amino acids (SEQ ID NO: 1). The predicted molecular weight of the protein is 59.2 KDa, and the isoelectric point is 6.54. The 5 UTR and 3 UTR length is 510 bp and 609 bp, respectively.

[0035] dsRNA injection was performed to inhibit the expression of carotenoid oxygenase. qRT-PCR was used to detect the effect of interference. By observing the changes in the color of the adductor muscle and the following HPLC analysis, carotenoids accumulation in the muscles of scallops with carotenoid oxygenase gene interference was determined (FIG. 1), that is, the gene identified has the function of degrading carotenoids.

[0036] Though suppressing the expression of carotenoid oxygenase gene by RNA interference (RNAi), and detecting the change in the expression of carotenoid oxygenase gene and carotenoids content of in the adductor muscle of scallop after carotenoid oxygenase gene interference, function of the analyzed gene in carotenoid cleavage is identified, and the gene encodes a carotenoid oxygenase of scallop.

Example 2

[0037] The prokaryotic expression of the scallop carotenoid oxygenase protein in the invention includes the following steps:

[0038] a) amplification of carotenoid oxygenase coding sequence;

[0039] b) construction of expression plasmids;

[0040] c) prokaryotic expression of carotenoid oxygenase;

[0041] d) Verification of carotenoid oxygenase activity in prokaryotic systems.

[0042] The specific operations are as follows:

[0043] a) Amplification of carotenoid oxygenase coding sequence. Using the scallop striated muscle cDNA as the template, using primers containing the corresponding restriction sites and protective bases of the pET-28a vector:

TABLE-US-00001 5-CCGGAATTCGTCCCACCAGC-3; 5-TCCCAAGCTTTCAAGGGATGTTAAAGAACC-3

[0044] Amplify the target fragment. The reaction system (20 L) is:

[0045] Template cDNA 2.5 L

[0046] 5Phusion HF Buffer 4.0 L

[0047] 10 mM dNTPs 0.4 L

[0048] Forward primer (10 mM) 1.0 L

[0049] Reverse primer (10 mM) 1.0 L

[0050] Phusion DNA Polymerase 0.2 L

[0051] ddH2O add to 20 L

[0052] The PCR reaction program used for the amplification was: denaturation at 98 C. for 30 s; denaturation at 98 C. for 10 s, annealing at 62 C. for 30 s, extension at 72 C. for 50 s, 30 cycles; extension at 72 C. for 10 min.

[0053] b) Construction of expression plasmid. Digestion of the amplified target fragment and pET-28a vector, the digestion system is:

[0054] CutSmart Buffer (10) 5L

[0055] DNA 1 g

[0056] Ecor I 1 L

[0057] Hind III 1 L

[0058] ddH2O add to 50 L

[0059] Digestion was performed at 37 C. for 15 min. The digestion products were purified by QIAquick PCR Purification Kit.

[0060] The purified fragment was ligated with the vector using T4 ligase. The ligation system (25 L) was:

[0061] T4 DNA Ligase Buffer 2.5 L

[0062] ATP 2 L

[0063] Purified digestion fragment 100 ng

[0064] Vector 300 ng

[0065] T4 DNA Ligase 1 L

[0066] Ligation was carried out with PCR instrument at 16 C. overnight. After obtaining the recombinant plasmid through transformation and extraction, sequencing was performed to verify the correctness of the introduced sequence.

[0067] c) Prokaryotic expression of carotenoid oxygenase. The recombinant plasmid was transferred into Rosetta (DE3) competent cells and spread on LB solid medium (50 g/mL kanamycin). Single clones were picked and cultured in a liquid medium containing antibiotics. When the OD value reached 0.6, the inducer IPTG was added and the cells werecultured at 20 C. for 16 h or 37 C. for 4 h. The negative control was cultured without the inducer. Then the bacterial cells were collected by centrifugation, and the supernatant was discarded. Buffer A was added to the collected bacterial cells, and the cells were fully broken using a sonicator. Centrifugation was performed again, and the precipitate after centrifugation was dissolved in buffer B. The supernatant and the precipitate were collected separately, and subjected to SDS-PAGE detection. The optimal expression conditions were determined based on the detection results (FIG. 2).

[0068] d) Verification of carotenoid oxygenase activity in prokaryotic systems. The recombinant plasmid was transformed into E. coli which could synthesize -carotene and is in orange color. Then the E. coli cells were spread on LB solid medium (50 g/mL kanamycin). Pick a single clone and culture it in liquid medium (50 g/mL kanamycin, 25 g/mL chloramphenicol) until the OD600 reaches 0.6. After adding the inducer IPTG, the culture was continued at 28 C. for 16 hours in dark. At the same time, the empty pET-28a plasmid was also transformed into E. coli which could synthesize -carotene, and the cells were then cultured and induced in the same way as above. Cells were collected by centrifugation, and the color of the bacteria was observed. It was found that the color of the strain expressing the carotenoid oxygenase protein was obviously lighter than the strain with the empty pET-28a plasmid, which proved that the prokaryotically expressed enzyme has carotenoid oxygenase function (FIG. 3).

Example 3

[0069] Purification of scallop carotenoid oxygenase protein in the present invention includes the following steps:

[0070] a) large amount expression of carotenoid oxygenase protein;

[0071] b) protein purification;

[0072] c) purification test;

[0073] d) dialysis concentration.

[0074] The specific operations are as follows:

[0075] a) Large amount expression of carotenoid oxygenase protein. Bacteria were cultured in a liquid medium containing 50 g/mL kanamycin. When the OD value reached 0.6, 0.5 mM inducer IPTG was added, and the cells were cultured at 20 C. for 16 h to express a large number of cells.

[0076] b) Protein purification. The bacterial cells were lysed with buffer C and then sonicated, and the supernatant crude protein was collected by centrifugation. Take 5 mL of Ni-NTA and wash the equilibration column with 5 times the bed volume of Binding buffer. The flow rate is 5 mL/min. The crude protein was incubated with the equilibrated column packing for 1 h, and then the incubated product was loaded onto the column and collected. Then wash the equilibration column with Binding buffer, wash the column with Washing buffer, and collect the effluent. Finally the column was washed with Elution buffer and the effluent was collected.

[0077] c) Purification test. Crude protein, washed-out and eluted effluent were collected and prepared for SDS-PAGE detection (FIG. 4).

[0078] d)Dialysis concentration. Fraction 6 which showed higher purity was dialyzed into a protein storage buffer (1PBS, 20% Glycerol, 2 mM DTT, 0.1% SKL, pH=8.0). At the end of dialysis, the protein was concentrated with PEG20000, filtered through a 0.22 m filter membrane, and then packed and stored at 80 C. Finally, 5 mg of carotenoid oxygenase recombinant protein with a purity of >85% was obtained (FIG. 5).

[0079] The above buffer solution is formulated as follows:

TABLE-US-00002 Name of buffer Formulas Buffer A 50 mM Tris, 300 mM NaCl, pH = 8.0 Buffer B 8M Urea, 50 mM Tris-HCl, 300 mM NaCl, pH = 8.0 Buffer C 7M Gua-HCl, 50 mM Tris, 300 mM NaCl, 0.1% Triton X-100, pH = 8.0 Binding buffer 8M Urea, 50 mM Tris, 300 mM NaCl, pH = 8.0 Washing buffer 8M Urea, 50 mM Tris, 300 mM NaCl, 20/50 mM Imidazole, pH = 8.0 Elution buffer 8M Urea, 50 mM Tris, 300 mM NaCl, 500 mM Imidazole, pH = 8.0

Example 4: Improving Carotenoids Content in Scallop by RNAi Interference

[0080] The specific operation of RNAi is as follows:

[0081] a) Primer design. Use siDirect version 2.0 (http://sidirect2.rnai.jp/) to predict and screen RNAi target sequences of genes. According to the basic principles of RNAi primer design, corresponding primers are designed for the target sequence. The T7 promoter is introduced at the 5 end of each primer. The primer sequence is:

TABLE-US-00003 5-TAATACGACTCACTATAGGGCTCCCTTCGATGTAGCTGAAAAATT- 3; 5-TAATACGACTCACTATAGGGAGTTTGGTGACAGAGAACGAATG-3.

[0082] b) Preparation of RNAi template. Using the scallop muscle cDNA of Yesso scallop as the template, the Phusion high-fidelity DNA polymerase was used to amplify the PCR product with T7 promoter. After the product was purified by QIAquick PCR Purification Kit, its quality was detected by 1.5% agarose gel electrophoresis, and was stored at 20 C. as a template for subsequent synthesis of dsRNA.

[0083] c) In vitro transcription and synthesis of dsRNA. The MEGAscript RNAi Kit is used for in vitro transcription reaction to prepare dsRNA. The reagents and equipment used are processed to avoid nuclease. The reaction system (20 L) is as follows: template DNA 3 g, T7 Reaction Buffer (10) 2 L, ATP solution (75 mM) 2 L, CTP solution (75 mM) 2 L, GTP solution (75 mM) 2 L, UTP solution (75 mM) 2 L, T7 Enzyme Mix 2 L, Nuclease-free Water for supplement. Add all reagents on ice, and mix slightly after centrifugation. The mixture was then incubated at 37 C. for 6 h, and then at 75 C. for 5 min, followed by cooling to room temperature to maximize the amount of product. The DNA and ssRNA were digested with nuclease. The reaction system was: 20 L of the product from the previous step, 5 L of Digestion Buffer (10), 2 L of DNase I, 2 L of RNase, and supplemented with nuclease-free Water to 50 uL. The above reagents were added in the tube on ice, mixed and centrifuged, and incubated at 37 C. for 1 h. DsRNA was purified using water-saturated phenol and chloroform: isoamyl alcohol (24: 1, v/v). Precipitate dsRNA with isopropanol (20 C. pre-cooled) and sodium acetate solution (4 C. pre-cooled), then wash the precipitate twice with 75% ethanol (pre-cooled at 20 C.). 50 L of 1PBS solution (Nuclease-free) was used to dissolve the pellet. The quality of dsRNA was measured by 1.5% agarose gel electrophoresis, and its concentration was measured by Nanoview (GE).

[0084] d) Injection of dsRNA for interfereing the expression of carotenoid oxygenase gene. Scallops were divided into two groups, namely the experimental group and the control group. Dilute dsRNA to 1 g/L with 1PBS. Use a micro syringe to inject 100 L of the diluted dsRNA solution into the adductor muscle of the scallop in the experimental group. Inject 100 L 1PBS into the control scallop in the same way. In the adductor muscle. The above experimental steps were performed once a week for a total of four injections. Throughout the experiment, two groups of scallops were reared under exactly the same conditions, and Chlorella was fed twice a day. After the experiment, the color of the adductor muscle was observed. The adductor muscle of the experimental group turned orange (FIG. 6).

[0085] e) The effect of qRT-PCR on interference detection. Two groups of adductor muscle of scallops were obtained by dissection, and frozen in liquid nitrogen and stored at 80 C. RNA was extracted and reverse-transcribed to obtain cDNA which was diluted 20-fold as the template. Design primers for qRT-PCR experiments of the carotenoid oxygenase gene, and select Ubiquitin (UBQ) as the internal reference gene. The primer sequence is as follows:

TABLE-US-00004 Targetgeneupstreamprimer: 5-GATGCCAGGCTCTAAAGCAAC-3; Targetgenedownstreamprimer: 5-CTGAACCCGTACGAGTAACGATACT-3; Ubiquitin-s 5-TCGCTGTAGTCTCCAGGATTGC-3; Ubiquitin-a 5-TCGCCACATACCCTCCCAC-3.

[0086] The qRT-PCR reaction system is as follows: cDNA template 2 L, primer-s (2 M) 4 L, primer-a (2 M) 4 L, SYBR Green I Real-time PCR Master Mix (2) 10 L. Each sample was set up in triplicate, and the reaction was performed on the LightCycler 480 Real-time PCR System. The program was set to: 50 C., 2 min; 94 C., 10 min; 94 C., 15 s, and 62 C. 1 min, 40 cycles. After the amplification reaction is complete, a fusion curve analysis is performed to exclude the effects of non-specific amplification. The test results showed that the expression level of the target gene in the scallop muscle of the experimental group was significantly reduced (FIG. 7).

[0087] f) Determination of carotenoid content by HPLC. Accurately weigh the adductor muscle of scallop, and the same weight of muscle was lyophilized for each sample. To prevent carotenoids from being oxidized, 0.1% BHT was added to methanol as an extraction solution. The extract was added to the lyophilized tissue, and the mixture was homogenized, centrifuged for 1 min (13,000 rpm, 4 C.) and the supernatant was collected. The above extraction steps were repeated three times, and the supernatants were combined and then subjected to rotary evaporation. Dry the residual liquid with nitrogen, then use 2 mL of mobile phase to dissolve the carotenoids, pass through a 0.22 pm filter, and load them into an autosampler vial. The above steps are performed in dark conditions, taking care to keep low temperature. LaChrom C18 (4.6250 mm, 5 m ID) column (HITACHI, Japan) was used to analyze the samples. The mobile phase was acetonitrile: methanol: dichloromethane (50: 46: 4, v/v/v) and 0.1. % BHT. The HPLC parameters are as follows: injection volume is 10 L, column temperature is 25 C., flow rate is 1.0 mL/min, monitoring wavelength is 450 nm, and separation time is 10 min.

[0088] The chromatographic peaks of pectenolone and pectenoxanthin were identified based on the retention time. The results showed that carotenoids were detected in the RNAi group samples, while no carotenoid accumulation was found in the control group.

[0089] The above results indicate that by reducing the amount of the protease having the amino acid sequence of SEQ ID NO: 1, the carotenoid content in scallop can be improved.

[0090] One skilled in the art will understand that the embodiment of the present invention as shown in the drawings and described above is exemplary only and not intended to be limiting.

[0091] It will thus be seen that the objects of the present invention have been fully and effectively accomplished. Its embodiments have been shown and described for the purposes of illustrating the functional and structural principles of the present invention and is subject to change without departure from such principles. Therefore, this invention includes all modifications encompassed within the spirit and scope of the following claims.