Mcic000159 gene of a cyp450 family gene involved in cantharidin biosynthesis and application thereof

Abstract

The application provides a CYP450 family gene Mcic000159 gene involved in cantharidin biosynthesis, the nucleotide sequence of the Mcic000159 gene is shown as SEQ ID NO. 1, and the coded amino acid sequence is shown as SEQ ID NO. 2. When the expression of the CYP450 family gene Mcic000159 gene in the application is interfered in the body of the L. decemnotata larva, the content of juvenile hormone III in the body of the L. decemnotata larva has no influence, the content of cantharidin is reduced, and the relative expression amount of the upstream genes McicHMGR, McicFPPS and McicJHEH of cantharidin synthesis is up-regulated. The CYP450 family gene Mcic000159 gene in the application acts on the metabolic product downstream of juvenile hormone III to cantharidin, and fills the blank of the stage from juvenile hormone III to cantharidin in the cantharidin synthesis route.

Description

Technical Field

[0001] This invention belongs to the field of cantharidin biosynthesis technology, specifically relating to the Mcic000159 gene of the CYP450 family of genes involved in cantharidin biosynthesis and its application. Background Technology

[0002] The current synthesis route of cantharidin has not been elucidated in the stage from juvenile hormone III to cantharidin. It is unclear whether the synthesis of cantharidin is directly metabolized from juvenile hormone III or whether the metabolites of juvenile hormone III are further synthesized into cantharidin. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a CYP450 family gene Mcic000159 gene involved in cantharidin biosynthesis and its application. The CYP450 family gene Mcic000159 gene acts between the downstream metabolites of juvenile hormone III and cantharidin. When the CYP450 family gene Mcic000159 gene is interfered with, the content of cantharidin decreases.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a Mcic000159 gene of the CYP450 family gene involved in cantharidin biosynthesis, wherein 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 invention also provides the application of the CYP450 gene involved in cantharidin biosynthesis, wherein the Mcic000159 gene of the CYP450 family acts between the downstream metabolites of juvenile hormone III and cantharidin. When the Mcic000159 gene of the CYP450 family is interfered with, it is used to reduce the content of cantharidin.

[0006] Preferably, when the expression of the Mcic000159 gene of the CYP450 family is interfered with in the larvae of the spotted blister beetle, it has no effect on the content of juvenile hormone III in the larvae of the spotted blister beetle, the content of cantharidin is downregulated, and the relative expression levels of the upstream genes for cantharidin synthesis, McicHMGR, McicFPPS and McicJHEH, are upregulated.

[0007] Compared with the prior art, the present invention has the following advantages: This invention utilizes gene family identification technology to screen for the Mcic000159 gene, a CYP450 family gene involved in cantharidin biosynthesis, and to pinpoint its functional location within the synthetic pathway. When the expression of the Mcic000159 gene was interfered with in the larvae of the blister beetle, it had no effect on the juvenile hormone III content, but the cantharidin content decreased. Furthermore, the relative expression levels of upstream genes involved in cantharidin synthesis, McicHMGR, McicFPPS, and McicJHEH, were upregulated, indicating that the Mcic000159 gene's role in cantharidin synthesis occurs between juvenile hormone III and cantharidin. This invention fills the gap in the cantharidin synthesis pathway from juvenile hormone III to cantharidin, providing further scientific evidence for elucidating the pathway between juvenile hormone III and cantharidin.

[0008] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0009] Figure 1 This is a Venn diagram showing the differential expression of CYP450 family genes involved in cantharidin synthesis in *Blephalothorax glabra* in Example 1 of this invention; wherein, A represents the differentially expressed CYP450 genes between adjacent instars of *Blephalothorax glabra* from 1 to 5 years old; B represents the differentially expressed CYP450 genes between 1 to 2 years old, 2 to 3 years old, and 3 to 5 years old; and C represents the differentially expressed CYP450 genes between 1 to 3 years old and 3 to 5 years old.

[0010] Figure 2 This is a diagram verifying the function of the Mcic000159 gene after injecting dsRNA into blister beetle larvae in Example 2 of this invention. In this diagram, A represents the relative expression level of the Mcic000159 gene; B represents the cantharidin content; C represents the juvenile hormone III content; D represents the relative expression levels of the McicHMGR, McicFPPS, and McicJHEH genes; * indicates a significant difference of P < 0.05; ** indicates a significant difference of P < 0.01; ns indicates no significant difference. Detailed Implementation

[0011] Example 1 This example demonstrates the acquisition of the Mcic000159 gene from the CYP450 family of genes: S1. Extraction of the CYP450 family gene from *Echinops latifolius*: All CYP450 gene sequences in the transcriptome sequencing of *Brachys thunbergii* larvae were extracted using two methods: NCBI homology alignment and Hidden Markov Model (HMM) retrieval. Redundant sequences were removed to obtain the CYP450 family genes of *Brachys thunbergii*. Screening of S2 and CYP450 family genes related to cantharidin synthesis: 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 in SEQ ID NO.3; the nucleotide sequence of the specific upstream primer T7Mcic000159-F is shown in SEQ ID NO.4; the nucleotide sequence of the specific downstream primer Mcic000159-R is shown in SEQ ID NO.5; the nucleotide sequence of the specific downstream primer T7Mcic000159-R is shown in SEQ ID NO.6; the nucleotide sequence of the specific upstream primer GFP-F is shown in SEQ ID NO.7; the nucleotide sequence of the specific upstream primer T7GFP-F is shown in SEQ ID NO.8; the nucleotide sequence of the specific downstream primer GFP-R is shown in SEQ ID NO.9; and the nucleotide sequence of the specific downstream primer T7GFP-R is shown in SEQ ID NO.10.

[0014] (2) Total RNA extraction from larvae of the spotted blister beetle using the Trizol method: Weigh 30 mg of *Brachys oryzae* larvae into a 1.5 mL enzyme-free EP tube, place it on ice, and add a steel ball and 1 mL of [unspecified substance] to the EP tube. Trizol reagent was used to homogenize the tissue in a high-throughput homogenizer for 100 s at 4°C and 45 Hz for 100 s. The homogenate was then transferred to a clean 1.5 mL enzyme-free EP tube, mixed, and incubated on ice for 5 min. The homogenate was then centrifuged at 12000 g for 5 min at 4°C, and 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 incubated on ice for 10 min. The homogenate was then centrifuged at 12000 g for 15 min at 4°C. The EP tube showed three layers. Approximately 0.5 mL of the uppermost supernatant was transferred to a new EP tube, and 0.5 mL of isopropanol was added. The mixture was inverted 10 times, incubated at room temperature for 15 min, and then centrifuged at 12000 g for 10 min at 4°C. The supernatant was discarded, and 1 mL of isopropanol was added to the new tube. Wash with 75% ethanol (diluted with DEPC water), centrifuge at 4℃ and 12000g for 5 min, and discard the supernatant; invert the EP tube with discarded supernatant in a clean bench to dry the RNA, and add an appropriate amount of DEPC water to dissolve the RNA to obtain total RNA of blister beetle larvae. Detect the total RNA concentration and OD value using an ELISA reader and store at -80℃ for later use.

[0015] (3) cDNA template synthesis: The cDNA template was prepared using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) reverse transcription kit. The reaction steps were as follows: ① The reaction system for eliminating genomic DNA was as follows: 1 μL gDNA Eraser, 2 μL total RNA from *Berberis thunbergii* larvae, 2 μL 5× gDNA Eraser Buffer, and 5 μL RNase-Free dH2O; the reaction conditions were: 42℃ for 2 min. ② The reaction system for the reverse transcription reaction was as follows: 1 μL RT Primer Mix, 1 μL PrimeScriptRT Enzyme Mix, 4 μL 5× primescript Butter (for Real time), 4 μL RNase-Free dH2O, and 10 μL of the reaction solution from step ①; the reaction conditions were: 37℃ for 15 min, and the reaction was terminated at 85℃ for 5 s. The obtained cDNA template was stored at -20℃ for later use.

[0016] (4) Amplification and detection of candidate genes: The PCR amplification reaction system for candidate genes Mcic000159 and GFP was as follows: 2×Taq Master Mix 25μL, upstream primer Mcic000159-F / GFP-F 1μL, downstream primer Mcic000159-R / GFP-R 1μL, cDNA template 4μL, ddH2O 19μL; vortexed, centrifuged, and the PCR reaction conditions were: 95℃ for 3min; 95℃ for 15s, 60℃ for 30s, 72℃ for 30s, 38 cycles, 72℃ for 5min, and stored at 4℃; the PCR products were detected using 1.5% agarose gel electrophoresis and gel imaging system.

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

[0018] (6) Ligation and transformation of cloning vectors: Add 1 μL T-Vector pMD19, 1 μL purified PCR product, 3 μL ddH2O, and 5 μL solution l to a 200 μL centrifuge tube, mix thoroughly, and incubate at 16℃ for 30 min to complete the ligation of the cloning vector.

[0019] The cloning vector obtained above was added to 100 μL of E. coli competent cells, gently mixed, and placed on ice for 30 min. It was then heat-shocked at 42℃ for 45 s, quickly placed on ice for 1 min, and 890 μL of SOC medium was added. The cells were incubated at 37℃ with shaking for 1 h. 50 μL of the transformation solution was spread onto LB solid medium with a final Amp concentration of 10 μg / mL and incubated overnight at 37℃ in a CO2 incubator. White single colonies were selected, and a single colony was picked from every 500 μL of sterile water as a PCR template for PCR amplification. PCR products were detected by 1.5% agarose gel electrophoresis. PCR products with the correct band size were sent to Sangon Biotech Co., Ltd. for sequencing.

[0020] (7) Plasmid extraction: The correctly sequenced bacterial culture was amplified and cultured. Plasmids were extracted using the TIANprep Mini Plasmid Kit to obtain T-Vector pMD19-Mcic000159 and T-Vector pMD19-GFP plasmids, which were then stored at -20°C for later use.

[0021] (8) Preparation of dsRNA: dsRNA uses T7 RiboMAX TMThe main steps for synthesizing the Express RNAi System template are as follows: ① The synthesis system for the sense strand single-stranded RNA (ssRNA) template is as follows: 2×Rapid Taq Master Mix 25μL, DEPC water 19μL, T-Vector pMD19-Mcic000159 plasmid / T-Vector pMD19-GFP plasmid 4μL, upstream primer T7Mcic000159-F / T7GFP-F 1μL, and downstream primer Mcic000159-R / GFP-R 1μL; ② The synthesis system for the antisense strand single-stranded RNA (ssRNA) template is as follows: 2×Rapid Taq Master Mix 25μL, DEPC water 19μL, T-Vector pMD19-Mcic000159 plasmid / T-Vector pMD19-GFP plasmid 4μL, and upstream primer Mcic000159-F / GFP-F 1 μL of downstream primer T7Mcic000159-R / T7GFP-R; vortex and centrifuge; PCR reaction conditions: 95℃ for 3 min; 95℃ for 15 s, 60℃ for 30 s, 72℃ for 30 s, 38 cycles; 72℃ for 5 min, store at 4℃; PCR products were detected using 1.5% agarose gel electrophoresis and gel imaging system; ③ Recovery of PCR products: Single, bright, and correctly lengthed bands were selected to purify and recover the corresponding PCR products; ④ Transcription and synthesis of ssRNA: Sensitive and antisense ssRNA were synthesized separately; the PCR synthesis system was RiboMaX. TM⑤ Synthesis of dsRNA: Equal volumes of positive and negative ssRNA were mixed in a new PCR tube, incubated at 70°C for 10 min, and allowed to stand at room temperature for 20 min to anneal and obtain dsRNA. 2 μL of 1:200 diluted RNase A solution and 2 μL of RNase-Free DNase were added to the obtained dsRNA, and the mixture was incubated at 37°C for 30 min to remove residual single-stranded RNA and template from the reaction solution. ⑥ Purification of dsRNA: 4.4 μL of 3 MS sodium acetate at pH 5.2 and 110 μL of [unclear - possibly a specific solution or solution] were added to the synthesized dsRNA. Mix gently with 95% ethanol and let stand on ice for 5 min. Centrifuge at 16000g for 10 min, discard the supernatant, add 500 μL of pre-chilled 70% ethanol, centrifuge at 16000g for 10 min, discard the supernatant, invert the PCR tube in a clean bench and dry at room temperature for 5 min–8 min, then add 50 μL of DEPC-treated water to fully dissolve the precipitate, obtaining dsMcic000159 and dsGFP at a concentration of 1 μg / μL.

[0022] (ii) Injecting dsMcic000159 and dsGFP into the larvae of the bladderwort: Third-instar mid-stage blister beetle larvae were injected with dsGFP as the control group and dsMcic000159 as the experimental group. Five biological replicates were set up for each treatment. 1 μg of dsRNA was injected into the side of the fifth and sixth abdominal segments of the blister beetle larvae. After injection, the larvae were returned to the incubator. The needles were cleaned with anhydrous ethanol and deionized water after each injection. Samples were taken after 24 hours. One-quarter of the samples were used to detect the RNA interference effect, and the remaining samples were freeze-dried for the detection of cantharidin and juvenile hormone III content.

[0023] The RNA interference effect was detected in mid-3rd instar blister beetle larvae injected with dsRNA, using β-actin as an internal reference gene. The qPCR reaction system was as follows: 2×SYBR... ®Premix Ex Taq™ II 10 μL, upstream primer Mcic000159-qPCR-F / β-actin-qPCR-F 0.5 μL, downstream primer Mcic000159-qPCR-R / β-actin-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 Mcic000159-qPCR-F is shown in SEQ ID No. 11; the nucleotide sequence of the downstream primer Mcic000159-qPCR-R is shown in SEQ ID No. 12; the nucleotide sequence of the upstream primer β-actin-qPCR-F is shown in SEQ ID No. 13; and the nucleotide sequence of the downstream primer β-actin-qPCR-R is shown in SEQ ID No. 14.

[0024] The results are as follows Figure 2 As shown in Figure A, the expression level of the Mcic000159 gene was significantly downregulated by 54.19%.

[0025] (III) Cantharidin content detection: Cantharidin was extracted using acid hydrolysis: The freeze-dried sample was ground into powder, weighed, and then mixed with hydrochloric acid aqueous solution (pH 1), chloroform, and 1 mL of n-hexadecane. The mixture was allowed to stand at room temperature for 24-36 hours. After standing, the lower organic phase was filtered, dried, and brought to a final volume of 1 mL. This mixture, along with a mixed standard of cantharidin and n-hexadecane, was analyzed. The cantharidin content (X) in the freeze-dried cantharidin sample was calculated using the internal standard method formula: X = (R2 × M1) / (R1 × M2) R1: The ratio of peak areas of cantharidin to n-hexadecane internal standard in the mixed standard solution; R2: The ratio of peak areas of cantharidin to n-hexadecane internal standard in the extracted sample; M1: Mass of cantharidin standard (mg); M2: Mass of extracted cantharidin sample (g).

[0026] The cantharidin content of mid-3rd instar blister beetle larvae injected with dsRNA was detected, such as... Figure 2 As shown in Figure B, inhibition of Mcic000159 gene expression led to a significant downregulation of cantharidin content by 19.11%.

[0027] (iv) Detection of juvenile hormone III (JHIII) content in larvae of the bladderwort *Bladinium glomeratum*: 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 was submitted to the Swiss-model website for 3D protein structure homology modeling; JHIII and downstream metabolites were downloaded from the Pubchem database; the active pocket of the protein was predicted using the Prankweb website, and the docking process was performed using AutoDock software. The specific operation steps are as follows: ① Import the Mcic000159 protein structure obtained from homology modeling into AutoDock, remove water molecules, add hydrogen, remove nonpolar hydrogen atoms, and output in PDBQT format; ② Import small molecule structures, calculate charges, and output in PDBQT format; ③ Select the docking region. The docking region is set according to the active pocket prediction of the protein by the Prankweb website, and the output is in GPF file format. ④ Select docking parameters, select the target protein molecular structure as the docking receptor, JHIII and juvenile hormone diol (JHAD) as small molecule ligands, select genetic algorithm for docking parameters, select Lamarckian GA for output results, and the output file is in DPF format; ⑤ Run AutoDock and select 10 output results; ⑥ Visualize the docking results in Pymol software and compare the binding of the Mcic000159 protein.

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

[0032] Table 1. Molecular docking verification binding substrates The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

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

2.

2. A CYP450 family gene involved in cantharidin biosynthesis as described in claim 1. Mcic000159 The application of genes is characterized by, The CYP450 family genes Mcic000159 The gene acts between the downstream metabolites of juvenile hormone III and cantharidin, when interference with the CYP450 family genes... Mcic000159 During gene expression, the content of cantharidin decreases; The CYP450 family genes Mcic000159 When gene expression in *Brachys fasciatus* larvae is disrupted, it has no effect on the juvenile hormone III content in the larvae, but the cantharidin content is downregulated, and the upstream gene for cantharidin synthesis is also affected. McicHMGR , McicFPPS and McicJHEH Relative expression levels were upregulated.

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