Mcic000159 gene of CYP450 family gene participating in cantharidin biosynthesis and application of Mcic000159 gene

Abstract

The invention provides an Mcic000159 gene of a CYP450 family gene participating in cantharidin biosynthesis, the nucleotide sequence of the Mcic000159 gene is as shown in SEQ ID NO. 1, and the coded amino acid sequence is as shown in SEQ ID NO. 2. When the expression of the Mcic000159 gene of the CYP450 family gene in the Eye spot Brassica rapa larva is interfered, the content of juvenile hormone III in the Eye spot Brassica rapa larva is not influenced, the content of cantharidin is reduced, and the relative expression quantity of the upstream genes McicHMGR, McicFPPS and McicJHEH for synthesizing the cantharidin is up-regulated. The Mcic000159 gene of the CYP450 family gene acts between the downstream metabolite of the juvenile hormone III and the cantharidin, so that the blank of the stage from the juvenile hormone III to the cantharidin in the synthetic route of the cantharidin is filled.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cantharidin biosynthesis, and particularly relates to a Mcic000159 gene of a CYP450 family gene participating in cantharidin biosynthesis and application thereof. BACKGROUND

[0002] The stage from juvenile hormone III to cantharidin in the current cantharidin synthesis route has not been resolved, and it is not clear whether juvenile hormone III can be directly metabolized and synthesized into cantharidin or whether juvenile hormone III metabolites further synthesize cantharidin. SUMMARY

[0003] The technical problem to be solved by the present application is to provide a Mcic000159 gene of a CYP450 family gene participating in cantharidin biosynthesis and application thereof in view of the above-mentioned deficiencies of the prior art, the Mcic000159 gene of the CYP450 family gene acts on the metabolites downstream of juvenile hormone III to cantharidin, and when the Mcic000159 gene of the CYP450 family gene is interfered, the content of cantharidin is reduced.

[0004] To solve the above-mentioned technical problem, the technical scheme adopted by the present application is as follows: a Mcic000159 gene of a CYP450 family gene participating in cantharidin biosynthesis, the nucleotide sequence of the Mcic000159 gene of the CYP450 family gene is shown in SEQ ID NO. 1, and the amino acid sequence encoded by the Mcic000159 gene of the CYP450 family gene is shown in SEQ ID NO. 2.

[0005] The present application also provides application of the above-mentioned CYP450 gene participating in cantharidin biosynthesis, the Mcic000159 gene of the CYP450 family gene acts on the metabolites downstream of juvenile hormone III to cantharidin, and when the Mcic000159 gene of the CYP450 family gene is interfered, the content of cantharidin is reduced.

[0006] Preferably, when the expression of the Mcic000159 gene of the CYP450 family gene in the eye-spotted budworm larvae is interfered, the content of juvenile hormone III in the eye-spotted budworm larvae is not affected, the content of cantharidin is down-regulated, and the relative expression amounts of the upstream genes McicHMGR, McicFPPS and McicJHEH of cantharidin synthesis are up-regulated.

[0007] Compared with the prior art, the present application has the following advantages: The application utilizes a gene family identification technology to screen out the Mcic000159 gene of the CYP450 family gene involved in the synthesis of cantharidin to more accurately determine the functional position in the synthesis pathway. When the expression of the Mcic000159 gene in the larva of the Callosa marginata is interfered, the content of juvenile hormone III in the larva of the Callosa marginata has no influence, while the content of cantharidin decreases, and the relative expression amount of the upstream genes McicHMGR, McicFPPS and McicJHEH of cantharidin synthesis is up-regulated, which indicates that the Mcic000159 gene is in the stage from juvenile hormone III to cantharidin in the synthesis of cantharidin. The application fills the blank of the stage from juvenile hormone III to cantharidin in the synthesis route of cantharidin, and further provides a scientific basis for the route analysis between juvenile hormone III and cantharidin.

[0008] The application will be further described in detail below in combination with the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 is a CYP450 family gene differential expression weinberg chart of the Callosa marginata involved in the synthesis of cantharidin in the embodiment 1 of the application, wherein A is a CYP450 differential expression gene during the adjacent two ages of the Callosa marginata from 1 to 5 ages; B is a CYP450 differential expression gene during the Callosa marginata from 1 to 2 ages, 2 to 3 ages and 3 to 5 ages; C is a CYP450 differential expression gene during the Callosa marginata from 1 to 3 ages and 3 to 5 ages.

[0010] Figure 2 is a Mcic000159 gene function verification chart of the Callosa marginata larva after injecting dsRNA in the embodiment 2 of the application, wherein A is a relative expression level of the Mcic000159 gene; B is a content of cantharidin; C is a content of juvenile hormone III; D is a relative expression level of the McicHMGR, McicFPPS and McicJHEH genes; * indicates P<0.05 significant difference; ** indicates P<0.01 significant difference; and ns indicates non-significance. DETAILED DESCRIPTION

[0011] Embodiment 1 In this embodiment, the Mcic000159 gene of the CYP450 family gene is obtained. S1, extraction of the CYP450 family gene of the Callosa marginata: According to the NCBI homologous alignment and the hidden Markov model (HMM) retrieval, all CYP450 gene sequences in the transcriptome sequencing of the Callosa marginata larva are extracted, and the redundant sequences are removed to obtain the CYP450 family gene of the Callosa marginata; S2, screening of the CYP450 family and the cantharidin synthesis related gene: Analysis of the differential expression of CYP450 family genes in the larval stage of the spotted blister beetle at different instars, such as Figure 1 As shown in Figure A, Venn diagrams are used to visualize differentially expressed genes between adjacent age groups from 1 to 5 years. Figure 1 As shown in Figures B and C, further analysis of the cantharidin content change trend during the larval stage reveals a significant downregulation in the 1st-2nd instar, a significant upregulation in the 2nd-3rd instar, and a gradual accumulation in the 3rd-5th instar. Venn diagrams were used to visualize the differentially expressed CYP450 genes among the 1st-2nd, 2nd-3rd, and 3rd-5th instars, as well as the differentially expressed CYP450 genes among the 1st-3rd and 3rd-5th instars. By integrating the differentially expressed CYP450 genes obtained from each group and removing duplicates, a set of differentially expressed CYP450 genes was obtained. Combining the trends in the expression levels of differentially expressed genes during the larval stage with the trends in cantharidin content change, the differentially expressed gene Mcic000159 of the CYP450 family genes was found to conform to the change trend.

[0012] Example 2 This example demonstrates the application of the Mcic000159 differentially expressed gene of the CYP450 family genes obtained in Example 1: (a) Synthesis of double-stranded RNA (dsRNA) interfered with by the Mcic000159 gene: Green fluorescent protein (GFP) was used as the control group for the RNA interference experiment.

[0013] (1) Design of dsRNA primers for the Mcic000159 gene: According to T7 RiboMAX TM The Express RNAi System kit follows the principle of dsRNA primer design, designing and synthesizing specific upstream primers Mcic000159-F and T7Mcic000159-F, and specific downstream primers Mcic000159-R and T7Mcic000159-R for the Mcic000159 gene dsRNA; and designing and synthesizing specific upstream primers GFP-F and T7GFP-F, and specific downstream primers GFP-R and T7GFP-R for the GFP gene dsRNA. The nucleotide sequence of the specific upstream primer Mcic000159-F is shown as SEQ ID NO. 3; the nucleotide sequence of the specific upstream primer T7Mcic000159-F is shown as SEQ ID NO. 4; the nucleotide sequence of the specific downstream primer Mcic000159-R is shown as SEQ ID NO. 5; the nucleotide sequence of the specific downstream primer T7Mcic000159-R is shown as SEQ ID NO. 6; the nucleotide sequence of the specific upstream primer GFP-F is shown as SEQ ID NO. 7; the nucleotide sequence of the specific upstream primer T7GFP-F is shown as SEQ ID NO. 8; the nucleotide sequence of the specific downstream primer GFP-R is shown as SEQ ID NO. 9; and the nucleotide sequence of the specific downstream primer T7GFP-R is shown as SEQ ID NO. 10.

[0014] (2) Total RNA of the Calomastix guttata larvae was extracted by Trizol method: 30 mg of the Calomastix guttata larvae was weighed in a 1.5 mL desenzyming EP tube and placed on ice, a steel ball and 1 mL of Trizol reagent were added to the desenzyming EP tube, and the mixture was homogenized in a high-throughput homogenizer at a temperature of 4°C and a frequency of 45 Hz for 100 s; the tissue homogenate was transferred to another clean 1.5 mL desenzyming EP tube, mixed well, and centrifuged at a temperature of 4°C and a speed of 12000 g for 5 min; the supernatant was transferred to a new EP tube; 0.2 mL of chloroform was added to the new EP tube, vortexed for 15 s, and placed on ice for 10 min; the mixture was centrifuged at a temperature of 4°C and a speed of 12000 g for 15 min, and the liquid in the EP tube was separated into three layers; about 0.5 mL of the supernatant was transferred to a new EP tube, 0.5 mL of isopropanol was added, and the mixture was mixed well by inverting for 10 times; the mixture was placed at room temperature for 15 min and centrifuged at a temperature of 4°C and a speed of 12000 g for 10 min; the supernatant was discarded, and 1 mL of 75% ethanol (diluted with DEPC water) was added for washing; the mixture was centrifuged at a temperature of 4°C and a speed of 12000 g for 5 min, and the supernatant was discarded; the EP tube with the discarded supernatant was inverted and placed in a clean bench to dry the RNA; after drying, an appropriate amount of DEPC water was added to dissolve the RNA, and the total RNA of the Calomastix guttata larvae was obtained; the total RNA concentration and OD value were detected by an enzyme marker, and the total RNA was stored in a -80°C refrigerator for later use.

[0015] (3) Synthesis of cDNA template: The cDNA template was prepared using PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) reverse transcription kit, and the reaction steps were as follows: ① The reaction system for eliminating genomic DNA was as follows: gDNA Eraser 1 μL, total RNA of Callosobruchus maculatus larvae 2 μL, 5×gDNA Eraser Buffer 2 μL, RNase Free dH2O 5 μL; the reaction condition was 42°C for 2 min; ② The reaction system for reverse transcription reaction was as follows: RT Primer Mix 1 μL, PrimeScript RT Enzyme Mix 1 μL, 5×primescript Butter (for Real time) 4 μL, RNase Free dH2O 4 μL, and 10 μL of the reaction solution of step ①; the reaction condition was 37°C for 15 min, 85°C for 5 s to terminate the reaction, and the obtained cDNA template was stored in a refrigerator at -20°C for standby.

[0016] (4) Amplification and detection of candidate genes: The PCR amplification reaction system of candidate genes Mcic000159 and GFP was as follows: 2×Taq Master Mix 25 μL, upstream primer Mcic000159-F / GFP-F 1 μL, downstream primer Mcic000159-R / GFP-R 1 μL, cDNA template 4 μL, and ddH2O 19 μL; vortex mixing, centrifugation, and the PCR reaction condition was 95°C for 3 min; 95°C for 15 s, 60°C for 30 s, 72°C for 30 s, 38 cycles, 72°C for 5 min, and 4°C storage; the PCR product was detected by 1.5% agarose gel electrophoresis and gel imaging system.

[0017] (5) Purification and recovery of PCR products: The product with correct band size was purified and recovered using TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0 kit.

[0018] (6) Connection and transformation of cloning vector: 1 μL T-Vector pMD19, 1 μL purified PCR product, 3 μL ddH2O, and 5 μL solution l were added to a 200 μL centrifuge tube, and fully mixed, and the connection of the cloning vector was completed at 16°C for 30 min.

[0019] The obtained cloning vector was added to 100 μL of E. coli competent cells, mixed gently, and then placed in ice for 30 min, heated at 42°C for 45 s, quickly placed in ice for 1 min, added with 890 μL of SOC medium, and cultured at 37°C for 1 h. 50 μL of the transformation liquid was coated on LB solid medium with an Amp final concentration of 10 ug / mL, and placed in a carbon dioxide incubator for overnight culture at 37°C. A white single colony was selected, and each single colony was picked in 500 μL of sterilized water as a PCR template. PCR amplification was performed, and the PCR product was detected by 1.5% agarose gel electrophoresis. The PCR product with correct band size was selected and sent to Shenguo Bioengineering Co., Ltd. for sequencing.

[0020] (7) Extraction of plasmid: The bacterial liquid with correct sequencing was cultured for expansion, and the TIANprep Mini Plasmid Kit plasmid extraction kit was used to extract the plasmid, to obtain T-Vector pMD19-Mcic000159 plasmid and T-Vector pMD19-GFP plasmid, respectively, which were stored in a -20°C refrigerator for standby use.

[0021] (8) Preparation of dsRNA: The dsRNA was prepared by using T7 RiboMAX TMExpress RNAi System kit synthesis, the main steps are as follows: ① synthesis system of sense strand single-stranded RNA (ssRNA) template as follows: 2 x Rapid Taq Master Mix 25 μL, DEPC water 19 μL, T-Vector pMD19-Mcic000159 plasmid / T-Vector pMD19-GFP plasmid 4 μL, upstream primer T7Mcic000159-F / T7GFP-F 1 μL, downstream primer Mcic000159-R / GFP-R 1 μL; ② synthesis system of antisense strand single-stranded RNA (ssRNA) template as follows: 2 x Rapid Taq Master Mix 25 μL, DEPC water 19 μL, T-Vector pMD19-Mcic000159 plasmid / T-Vector pMD19-GFP plasmid 4 μL, upstream primer Mcic000159-F / GFP-F 1 μL, downstream primer T7Mcic000159-R / T7GFP-R 1 μL; vortex mix and centrifuge, PCR reaction conditions are: 95℃ 3min; 95℃ 15s, 60℃ 30s, 72℃ 30s, 38 cycles; 72℃ 5min, 4℃ storage; 1.5% agarose gel electrophoresis and gel imaging system are used to detect PCR products; ③ recovery of PCR products: single, bright, and correct length bands corresponding to PCR products are selected for purification and recovery; ④ transcription synthesis ssRNA: respectively synthesize sense strand and antisense strand ssRNA, PCR synthesis system is: RiboMaX TMExpress T7 2x Buffer 10 μL, sense strand single-stranded RNA template / antisense strand single-stranded RNA template 8 μL, Enzyme Mix T7 Express 2 μL; 37 °C incubation for 4 h, synthesis of ssRNA; ⑤ Synthesis of dsRNA: equal volume of sense strand ssRNA and antisense strand ssRNA were mixed in a new PCR tube, 70 °C incubation for 10 min, room temperature standing for 20 min, annealing to obtain dsRNA, 2 μL of 1:200 diluted RNase A solution and 2 μL of RNase-Free DNase were added to the obtained dsRNA, 37 °C water bath for 30 min, for removing residual single-stranded RNA and template in the reaction solution; ⑥ Purification of dsRNA: 4.4 μL of 3 M Sodium Acetate with pH 5.2 and 110 μL of 95% ethanol were added to the synthesized dsRNA, gently mixed, and stood on ice for 5 min; centrifuged at 16,000 g for 10 min, after discarding the supernatant, 500 μL of pre-cooled 70% ethanol was added, centrifuged at 16,000 g for 10 min, after discarding the supernatant, the PCR tube was inverted on the clean bench and dried at room temperature for 5-8 min, then 50 μL of DEPC-treated water was added to fully dissolve the precipitate, and the concentration of dsMcic000159 and dsGFP was 1 ug / μL.

[0022] (II) Injection of dsMcic000159 and dsGFP into eye-spotted dragonfly larvae: Take 3rd instar eye-spotted dragonfly larvae for injection, take dsGFP as control group, and take dsMcic000159 as experimental group, set 5 biological replicates for each treatment, inject 1 μg of dsRNA into the side of the fifth and sixth abdominal segments of the eye-spotted dragonfly larvae, and then put them back into the incubator; after each injection, clean the needle with anhydrous ethanol and deionized water; sample after 24 h, take 1 / 4 sample for RNA interference effect detection, and freeze the rest for cantharidin and juvenile hormone III content detection.

[0023] RNA interference effect detection was performed on the 3rd instar eye-spotted dragonfly larvae injected with dsRNA, β-actin was used as an internal reference gene, and the qPCR reaction system was as follows: 2x SYBR ®Premix Ex Taq TM II 10μL, upstream primer Mcic000159-qPCR-F / β-actin-qPCR-F 0.5μL, downstream primer Mcic000159-qPCR-R / β-actin-R 0.5μL, cDNA template 2μL, ddH2O 7μL; qPCR amplification reaction stage conditions as follows: 95℃, 30s; 95℃, 5s, 60℃, 30s, 39 cycles; qPCR dissolution reaction stage conditions as follows: 95℃, 10s, 65℃, 5s, 95℃, 5s; The nucleotide sequence of the upstream primer Mcic000159-qPCR-F is shown as SEQ ID No. 11; the nucleotide sequence of the downstream primer Mcic000159-qPCR-R is shown as SEQ ID No. 12; the nucleotide sequence of the upstream primer β-actin-qPCR-F is shown as SEQ ID No. 13; and the nucleotide sequence of the downstream primer β-actin-qPCR-R is shown as SEQ ID No. 14.

[0024] The results are shown in Table 1. Figure 2 As shown in Table 1A, the expression amount of the Mcic000159 gene was significantly down-regulated by 54.19%.

[0025] (Three) cantharidin content detection: The cantharidin was extracted by acid hydrolysis method: the sample after freeze-drying was ground into powder, weighed, and then hydrochloric acid aqueous solution with pH of 1, chloroform and 1 mL of n-hexadecane were added, and the mixture was placed at room temperature for 24h-36h, then filtered to obtain the lower organic phase, which was dried and made up to 1 mL. The sample was detected together with the mixed standard of cantharidin and n-hexadecane, and the content X of cantharidin in the freeze-dried cantharidin sample was calculated according to the internal standard method formula: X= (R2 x M1) / (R1 x M2) R1: the peak area ratio of cantharidin to n-hexadecane internal standard in the mixed standard solution; R2: the peak area ratio of cantharidin to n-hexadecane internal standard in the extracted sample; M1: the mass of cantharidin standard (mg); M2: the mass of the extracted cantharidin sample (g).

[0026] The cantharidin content of the 3rd instar middle eye cantharis larvae injected with dsRNA was detected, and the results are shown in Table 1B. Figure 2 As shown in Table 1B, the inhibition of the expression of the Mcic000159 gene led to a significant down-regulation of the cantharidin content by 19.11%.

[0027] (Four) juvenile hormone III (JHIII) content detection of eye cantharis larvae: According to the insect JHIII enzyme-linked immunosorbent assay kit, samples of mid-3rd instar blister beetle larvae injected with dsRNA were weighed for JHIII content determination; Figure 2 As shown in Figure C, the content of juvenile hormone III did not change significantly, indicating that the inhibitory effect of the Mcic000159 gene on cantharidin synthesis occurs between juvenile hormone III and cantharidin.

[0028] (v) Detection of upstream genes in cantharidin synthesis: The relative expression levels of the upstream genes for cantharidin synthesis, 3-hydroxy-3-methylglutaryl-CoA reductase (McicHMGR), farnesyl pyrophosphate synthase (McicFPPS), and juvenile hormone epoxide hydrolase (McicJHEH), were detected in samples of mid-3rd instar blister beetle larvae injected with dsRNA. The qPCR reaction system was 2×SYBR. ® Premix Ex Taq™ II 10 μL, upstream primer McicHMGR-F / McicFPPS-F / McicJHEH-F 0.5 μL, downstream primer McicHMGR-R / McicFPPS-R / McicJHEH-R 0.5 μL, cDNA template 2 μL, ddH2O 7 μL; qPCR amplification reaction conditions were as follows: 95℃, 30 s; 95℃, 5 s, 60℃, 30 s, 39 cycles; qPCR melting reaction conditions were as follows: 95℃, 10 s, 65℃, 5 s, 95℃, 5 s; The nucleotide sequence of the upstream primer McicHMGR-F is shown in SEQ ID No. 15; the nucleotide sequence of the downstream primer McicHMGR-R is shown in SEQ ID No. 16; the nucleotide sequence of the upstream primer McicFPPS-F is shown in SEQ ID No. 17; the nucleotide sequence of the downstream primer McicFPPS-R is shown in SEQ ID No. 18; the nucleotide sequence of the upstream primer McicJHEH-F is shown in SEQ ID No. 19; and the nucleotide sequence of the downstream primer McicJHEH-R is shown in SEQ ID No. 20.

[0029] The results are as follows Figure 2 As shown in Figure D, all upstream genes were significantly upregulated, further suggesting that the Mcic000159 gene may act between the downstream metabolite of juvenile hormone III and cantharidin.

[0030] (vi) Molecular docking verification of substrate binding: The amino acid sequence encoded by the Mcic000159 gene is submitted to the Swiss-model website for 3D protein structure homology modeling; JHIII and downstream metabolites are downloaded from the Pubchem database; the protein is subjected to active pocket prediction using the Prank web site, and the docking process is performed using AutoDock software, and the specific operation steps are as follows: ①Import the Mcic000159 protein structure obtained by homology modeling in AutoDock, remove water molecules, add hydrogen, remove nonpolar hydrogen atoms, and output in PDBQT format; ②Import the small molecule structure, calculate the charge, and output in PDBQT format; ③Select the docking region, and the docking region is set according to the active region set by the Prank web site for active pocket prediction of the protein, and output in GPF file format; ④Select docking parameters, select the target protein molecular structure as the receptor, JHIII and juvenile hormone acid diol (JHAD) as the small molecule ligand, select genetic algorithm as the docking parameter, select Lamarckian GA as the output result, and output the file in DPF format; ⑤Run AutoDock, and select 10 as the output result; ⑥Visualize the docking results in Pymol software, and compare the binding of Mcic000159 protein.

[0031] The docking results are shown in Table 1, and juvenile hormone III and juvenile hormone acid diol have high binding energy.

[0032] Table 1 Verification of molecular docking to bind substrates The above is only a preferred embodiment of the present application, and does not limit the present application. Any simple modification, change and equivalent change made according to the technical essence of the application to the above embodiment are still within the protection scope of the technical solution of the present application.

Claims

1. A gene of Mcic000159 of a CYP450 family gene involved in the biosynthesis of cantharidin, characterized in that, The nucleotide sequence of the Mcic000159 gene of the CYP450 family gene is shown as SEQ ID NO. 1; the amino acid sequence encoded by the Mcic000159 gene of the CYP450 family gene is shown as SEQ ID NO.

2.

2. Use of the gene Mcic000159 of the CYP450 family gene involved in the biosynthesis of cantharidin according to claim 1, characterized in that, The Mcic000159 gene of the CYP450 family gene acts on the downstream metabolite of juvenile hormone III to cantharidin, and when the Mcic000159 gene of the CYP450 family gene is interfered, the content of cantharidin is reduced.

3. Use according to claim 2, characterized in that, When the expression of the Mcic000159 gene of the CYP450 family gene in the body of the Callosobruchus maculatus larvae is interfered, the content of juvenile hormone III in the body of the Callosobruchus maculatus larvae has no effect, the content of cantharidin is down-regulated, and the relative expression amounts of the genes McicHMGR, McicFPPS and McicJHEH upstream of cantharidin synthesis are up-regulated.

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