CN115161306B

CN115161306B - Apolygus lucorum RNA degrading enzyme, encoding gene, vector, strain and application thereof

Info

Publication number: CN115161306B
Application number: CN202210756221.6A
Authority: CN
Inventors: 谭永安; 张杰钰; 肖留斌; 赵静
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2022-06-29
Filing date: 2022-06-29
Publication date: 2023-09-15
Anticipated expiration: 2042-06-29
Also published as: CN115161306A

Abstract

The invention discloses a lygus lucorum RNA degrading enzyme, the amino acid sequence of which is shown as SEQ ID NO. 4. The invention also discloses a nucleic acid or a gene for encoding the lygus lucorum RNA degrading enzyme, and the nucleotide sequence of the nucleic acid or the gene is shown as SEQ ID NO. 3. The invention also discloses an expression cassette, a recombinant vector, a recombinant cell or a recombinant strain and application thereof. The invention firstly obtains the amino acid and the gene sequence of the lygus lucorum RNA degrading enzyme, constructs the pFastBac1 expression vector, and obtains the recombinant rod-shaped plasmid transfected SF9 cells by transformation identification in the escherichia coli DH10Bac to obtain the P1-P3 generation virus, wherein the concentration of the purified protein is 0.6mg/ml. The purified RNA degrading enzyme protein can provide a preliminary preparation for the functional verification of in-vitro degradation of the recombinant protein, and provides a certain experimental support for the subsequent exploration of the capability of the recombinant protein to degrade dsRNA.

Description

Apolygus lucorum RNA degrading enzyme, encoding gene, vector, strain and application thereof

Technical Field

The invention relates to the technical fields of agricultural science and biology, in particular to lygus lucorum RNA degrading enzyme, a coding sequence, a vector, a strain and recombinant protein and application thereof.

Background

Apolygus lucorum (Hemiptera: miridae) belongs to Apolygus lucorum, has wide distribution range, and is an important pest on various economic crops such as vegetables, fruit trees, etc. RNA interference (RNAinterference, RNAi) is the process of initiating silencing of endogenous complementary messenger RNA (mRNA) by exogenous double-stranded RNA (dsRNA). dsRNA-mediated RNA interference is successfully applied to insects such as hemiptera, diptera and lepidoptera, but RNAi efficiency of different insects is quite different, wherein the most important factor is that a class of RNA degrading enzyme (dsRNase) exists in the bodies of the insects, and RNAi efficiency is reduced by degrading double-stranded RNA.

Therefore, research on the coding structure of the lygus lucorum AldsRNase gene, in-vitro recombinant protein thereof and functional verification of degradation after recombinant protein purification, and research on the ability of the recombinant protein AldsRNase thereof to degrade dsRNA can provide a certain technical support for improving RNAi efficiency.

Disclosure of Invention

The invention aims to: the invention aims to solve the technical problem of providing a preparation method of lygus lucorum RNA degrading enzyme protein.

The technical scheme is as follows: in order to solve the technical problems, the invention provides the lygus lucorum RNA degrading enzyme, and the amino acid sequence of the lygus lucorum RNA degrading enzyme is shown as SEQ ID NO. 4.

The invention also comprises a nucleic acid or a gene for encoding the lygus lucorum RNA degrading enzyme, and the nucleotide sequence of the nucleic acid or the gene is shown as SEQ ID NO. 3.

The invention also comprises a primer pair for amplifying the nucleic acid or the gene of the lygus lucorum RNA degrading enzyme, wherein the primer pair has a sequence shown as SEQ ID NO.1 and SEQ ID NO. 2.

The invention also includes an expression cassette, recombinant vector, recombinant cell or recombinant strain comprising said nucleic acid or gene.

Wherein the recombinant vector is pFastbacI-AldsRNase, and other vectors capable of being used for the gene expression are also applicable.

Wherein the recombinant strain includes, but is not limited to, recombinant E.coli.

The invention also discloses application of the nucleic acid or gene, the expression cassette, the recombinant vector, the recombinant cell or the recombinant strain in preparation of lygus lucorum RNA degrading enzyme protein.

The invention also discloses a preparation method of the lygus lucorum RNA degrading enzyme protein, which comprises the following steps:

1) Obtaining the lygus lucorum RNA degrading enzyme protein gene: designing PCR amplification primers according to the known gene sequences of the kindred species, and amplifying the gene of the lygus lucorum RNA degrading enzyme;

2) Constructing and identifying an expression vector of the lygus lucorum RNA degrading enzyme protein recombinant baculovirus;

3) Transfection and harvesting of lygus lucorum RNA degrading enzyme protein recombinant baculovirus;

4) Purification of lygus lucorum RNA degrading enzyme protein.

The construction and identification steps of the lygus lucorum RNA degrading enzyme protein recombinant baculovirus in the step 2) are that the lygus lucorum RNA degrading enzyme gene is introduced into a plasmid pFastBac1, and the recombinant baculovirus expression vector is obtained by induction transformation and screening of blue white spots in competent cells of escherichia coli DH10 Bac.

The transfection step of the lygus lucorum RNA degrading enzyme protein recombinant baculovirus in the step 3) is to transfect and culture the P1 generation virus with SF9, and then amplify and harvest the P2 and P3 generation viruses.

Wherein the protein purification step in step 4) is to purify the virus obtained in step 3) by Ni column affinity chromatography.

The invention also provides a method for measuring the protein activity of the lygus lucorum RNA degrading enzyme.

The specific operation method of the invention can be divided into the following 4 steps:

1. regulating the content of the RNA degrading enzyme protein to a range of 0ng to 400 ng;

2. adjusting the pH of the RNA degrading enzyme protein solution to a range of ph=3 to ph=12;

3. adjusting the incubation time of the RNA degrading enzyme protein solution to a range of 0min to 10min;

4. the incubation temperature of the RNA degrading enzyme protein solution is adjusted to a range of 10 ℃ to 60 ℃.

The degradation condition of the lygus lucorum RNA degrading enzyme protein is determined by incubating the lygus lucorum RNA degrading enzyme protein under the conditions of different enzyme amounts, different pH values, different reaction times and different temperatures. Degradation of 1,000 ng dsGFP was accomplished by incubating 10. Mu.l of a Berry-Robinson Buffer solution (Britton-Robinson Buffer) containing 400ng of pH=5 at 30℃for 10min with dsGFP as a reference gene. The AldsRNase degrading enzyme has remarkable degradation effect on dsRNA, restricts the application of dsRNA in RNAi pest control, plays a key role in the RNAi action process and efficiency influence, and provides a certain experimental basis for improving the stability of dsRNA.

The beneficial effects are that: compared with the prior art, the invention has the following advantages: the invention firstly obtains the amino acid and the gene sequence of lygus lucorum RNA degrading enzyme (AldsRNase), constructs the pFastBac1 expression vector, converts in escherichia coli DH10Bac, obtains recombinant rod-shaped plasmid by utilizing a blue-white spot screening method, and transfects SF9 cells after positive identification by PCR to obtain the P1-generation virus and the P2-generation virus. The protein expression condition is detected by Western Blot, P3 generation cells are obtained by expansion culture, and the result after the detection by WesternBlot shows that the target protein is expressed in secretion supernatant and cell lysis supernatant, and the target protein is obtained by Ni column affinity chromatography purification. The molecular weight of the obtained protein is about 45KD, and the protein concentration is 0.6mg/ml. The purified RNA degrading enzyme protein can provide a preliminary preparation for the functional verification of in-vitro degradation of the recombinant protein, and provides a certain experimental support for the subsequent exploration of the capability of the recombinant protein AldsRNase to degrade dsRNA.

Drawings

FIG. 1 PCR amplification electrophoresis of the full-length sequence of the lygus lucorum RNA degrading enzyme protein gene. Lane 1: the total length of the lygus lucorum RNA degrading enzyme protein gene; lane 2: the DNA molecular mass standard, the band size is 100bp, 250bp, 500bp, 750bp, 1 000bp, 1 500bp, 2 000bp, 3 000bp, 5 000bp from bottom to top respectively;

FIG. 2BamHI and HindIII double cut pFastBac1 electrophoreses. Lane 1: pFastBac1 plasmid; lane 2: the pFastBac1 plasmid is digested in BamHI-HindIII sites; lane M: the DNA molecular weight standard, the size of the band is 250bp, 500bp, 750bp, 1 000bp, 1 500bp, 2 000bp, 2 500bp, 3 000bp, 4 000bp, 5 000bp, 6 000bp, 8 000bp and 12 000bp from bottom to top respectively;

FIG. 3 PCR identification analysis of recombinant baculovirus expression vector pFastBac1-dsRNase gene. Lane 1: a negative control; lanes 2-6: recombinant gene dsRNase1-5; lane M: the DNA molecular mass standard, the band size is 1 000bp, 2 000bp, 3 000bp, 4 000bp, 5 000bp, 6 000bp, 8 000bp, 10 000bp from bottom to top respectively;

FIG. 4 identification analysis of recombinant protein expression WesternBlot. Lane 1: cell lysis pellet (negative); lane 2: cell lysis supernatant (negative); lane 3: secretion medium (negative); lane 4: cell lysis pellet (dsRNase); lane 5: cell lysis supernatant (dsRNase); lane 6: secretion medium (dsRNase); lane M: protein molecular mass standard;

FIG. 5 SDS-PAGE analysis of purified proteins from secreted supernatants. Lane 1: cell secretion supernatant samples; lane 2: outflow; lanes 3-7: eluting; lane M: protein molecular mass standard;

FIG. 6 shows an identification pattern of SDS-PAGE for protein purification. Lane 1: BSA; lane 2: purifying protein AldsRNase; lane M: protein molecular mass standard;

FIG. 7 Western Blot electrophoresis characterization of protein purification. Lane 1: purifying protein AldsRNase; lane M: protein molecular mass standard.

FIG. 8 graphs and gel photographs of the enzyme amount (A), pH condition (B), reaction time (C) and temperature condition (D) dependent activities of protein purified AldsRNase. A. The lower graph of the B, C, D gel graphs shows the optical density of the strips in the gel graphs, which is automatically calculated by GIS digital gel image analysis software. Results are mean ± standard deviation.

Detailed Description

The invention is further illustrated and explained below with reference to examples. The illustrated embodiments are only some, but not all, embodiments of the invention. The foregoing is merely a preferred embodiment of the present invention, and it should be apparent that: the invention and the embodiments are not limited to the inventive work, but can be modified by those skilled in the art without departing from the scope of the invention.

The experimental procedures, instruments, etc. used in the following examples are conventional procedures and instruments unless otherwise specified.

Reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified.

Example 1: acquisition of AldsRNase Gene

Extracting total RNA of lygus lucorum by adopting a TRIzol method, selecting proper total RNA, purifying, synthesizing first-strand cDNA by using RevertAid First Strand cDNA Synthesis Kit reverse transcription of Fermentas company, and a PCR reaction system is as follows: 1. Mu.g total RNA, 1. Mu.L of 100. Mu.M oligo (dT) primer (5'-TTTTTTTTTTTTTTTTTT-3') (SEQ ID NO. 5), and water was added to a volume of 12. Mu.L, and the mixture was treated at 65℃for 5 minutes and allowed to stand on ice for 1 minute. Then, 4. Mu.L of 5X reaction buffer, 1. Mu. L RiboLock RNase Inhibitor (20U/. Mu.L), 2. Mu.L of 10mM dNTP mix, 1. Mu.L of Reverted air M-MuLV Reverse Transcriptase (200U/. Mu.L) were added in this order, and the mixture was incubated at 42℃for 1 hour and 5 minutes at 70℃to give cDNA. According to the known gene sequence of the kindred species, the primer AldsRNase-F (5'-ATGTCGGTTCTGGCGCTCTG-3') (SEQ ID NO. 1) and AldsRNase-R (5'-TTATTTCTTCTTGAATTGGAGGGTG-3') (SEQ ID NO. 2) suitable for PCR amplification are designed to obtain the conserved sequence of the lygus lucorum RNA degrading enzyme gene, wherein the conserved sequence is shown as SEQ ID NO.3 in the sequence table, and the PCR amplification system is as follows: 15 mu L ddH ₂ O, 25. Mu.L 2X PCR Buffer for KOD FX Neo, 1. Mu.L dNTP Mix (10 mM), 5. Mu.L cDNA, 1. Mu.L KOD FX Neo (1U/. Mu.L), 1.5. Mu.L AldsRNase-F, 1.5. Mu.L AlsRNase-R; the PCR amplification procedure was: pre-denaturation at 98 ℃ for 5min, denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min for 30s, circulation for 35 times, extension at 72 ℃ for 10min; separating the PCR product by 1% agarose gel electrophoresis, recovering and purifying target DNA by gel, carrying out T4 enzyme connection with a pMD-18Vector to obtain pMD-18Vector-AldsRNase, transferring into escherichia coli JM109, selecting single colony for culture, identifying positive bacterial liquid by colony PCR, sequencing by biological engineering Limited company, and carrying out the next insect cell expression Vector construction with correct sequence determination.

Conclusion: the full length 1227bp (figure 1) of the ORF sequence of the lygus lucorum RNA degrading enzyme gene is obtained through amplification, recovery, sequencing and splicing, the gene codes for 408 amino acids, and a 19aa signal peptide exists.

Example 2: construction of AldsRNase Gene recombinant baculovirus expression vector (Bacmid)

The target gene AldsRNase and the Vector pFastBacl which are correctly sequenced and connected into the pMD-18Vector are digested with restriction enzymes BamHI and HindIII (FIG. 2), and then the ligation product pFastBac1-AldsRNase is transformed into E.coli T1 E.coli competent cells, and positive clones are screened by PCR. After transformation of the identified correct recombinant plasmid pFastbacI-AldsRNase into E.coli DH10Bac E.coli competent cells, S.O.C. medium (2% (W/V) Tryptone,0.5% (W/V) Yeast Extract,0.05% (W/V) NaCl,2.5mM KCl,10mM MgCl was added ₂ 20mM glucose), 37℃and 225r/min for 4h; the mixture was spread on LB plates containing 50. Mu.g/mL kanamycin, 7. Mu.g/mL gentamicin, 10. Mu.g/mL tetracycline, 24mg/mL IPTG and 20mg/mL X-gal, and cultured in an inverted state at 37℃for 48 hours. The transformed plates were inoculated with a selection of 4mL of LB medium containing 50. Mu.g/mL kanamycin, 7. Mu.g/mL gentamicin, and 10. Mu.g/mL tetracycline by shaking. Positive clone bacteria were initially screened by bacterial liquid PCR, plasmids were extracted in small amounts to obtain pFastbacI-AldsRNase recombinant plasmids, and the PCR was run on a 1% agarose gel for amplification and concentration detection (FIG. 3).

Conclusion: through the identification of the recombinant rod-shaped plasmid pFastbacI-AldsRNase, the purity OD260/OD280 value of the recombinant rod-shaped plasmid pFastbacI-AldsRNase is 1.90, the volume is 0.18mL, and the concentration is 719 ng/. Mu.L; and selecting the positive bacteria of the No.2 lane for seed conservation according to the PCR result.

Example 3: transfection and harvesting of recombinant rod-shaped plasmid pFastbacI-AldsRNase

SF9 cells were cultured using SF900II medium (GIBCO, 10902-088), and transfection experiments were performed using SF9 cells in log phase and with > 95% cell viability. Inoculation of 5X 10 in 6 well plates ⁵ Cells were attached to each well by culturing at 27℃for 1 hour per 2 mL/well. Preparation of bacmid and transfection reagent complexes: mu.g of recombinant rod-shaped plasmid pFastbacI-AldsRNase was diluted with 200. Mu.L of SF900II medium without double antibody, FBS, and the transfection reagent was usedIncubation is carried out at room temperature for 30min, 5 mu L of the recombinant rod-shaped plasmid pFastbacI-AldsRNase is taken and mixed gently with diluted recombinant rod-shaped plasmid pFastbacI-AldsRNase, and incubation is carried out at room temperature for 45min. Adding the well incubated mixture into a hole, incubating for 5d in an incubator at 27 ℃, and sucking the supernatant to obtain the P1 generation virus. 400. Mu.L of P1 virus was added to 20mL of SF9 cells. After 5d incubation in an incubator at 27℃the P2 virus was harvested as the supernatant and the cells were sampled separately for Western Blot detection (FIG. 4). 1mL of P2 virus was added to 200mLSF9 cells. After culturing in an incubator at 27 ℃ for 5 days, the P3 virus is harvested, and a purification component is selected according to the detection condition of the Western Blot of the P2 generation and is used for subsequent protein purification.

Conclusion: transfection and harvesting by pFastbacI-AldsRNase was found to be predominantly present in cell lysis supernatant and secretion supernatant.

Example 4: aldsRNase gene recombinant protein purification

The Tris buffer (ph=8.0) equilibrated Ni column was added to the post-P3 generation secretion supernatant cell culture medium and incubated at 4 ℃ for 2h on a rotary incubator. The mixture of the dialyzed cell culture medium and the Ni column after incubation is slowly added into the purification empty column by using a low-pressure chromatography system. The Ni column was equilibrated with Balane Buffer (50mM Tris,300mM NaCl,20mM Imidazole,pH8.0) Buffer at a flow rate of 0.5mL/min until the effluent OD280 reached baseline; eluting the target protein with a Washing Buffer (50 mM imidazole, 50mM Tris,300mM NaCl,pH8.0) at a flow rate of 1mL/min, and collecting the effluent; the target protein was eluted with an Elutation Buffer (500 mM imidazole, 50mM Tris,300mM NaCI,pH8.0) at a flow rate of 1mL/min, the effluent was collected, and the eluted and collected proteins were detected by 12% SDS-PAGE (FIG. 5). The Western Blot detection method is as follows: transferring the film after sample loading and glue running, and keeping the constant pressure for 100V and 1.5h; washing with PBS for 4 times, each time for 5min, and sealing in 5% skimmed milk powder sealing solution at 37deg.C for 1 hr; diluting the primary antibody by using a sealing solution, and washing the membrane for 4 times after the membrane reacts in the primary antibody diluent at 37 ℃ for 1h, wherein each time is 5min; diluting the secondary antibody with a blocking solution containing 5% milk, and reacting the membrane in the secondary antibody at 37 ℃ for 1h; and (5) film washing ECL development. Purified protein results were identified by 12% SDS-PAGE electrophoresis and Western Blot electrophoresis (FIG. 6; FIG. 7).

Conclusion: by example 3, the cell secretion supernatant was selected as the purification component, and the protein was mainly present in the eluate, and after purification, an AldsRNase protein was successfully obtained, having a size of about 47KD, at a concentration of 0.6mg/mL.

Example 5: aldsRNase protease Activity assay

The degradation of dsGFP by recombinant enzyme proteins was determined in vitro under various enzyme amounts, pH conditions, reaction times, and temperature conditions, according to the protein concentrations determined in example 4. To test the dsGFP degrading activity (a) of AldsRNase, 1 000ng dsGFP was dissolved in 5 μl of nuclease-free water, 10 μl of recombinase solution diluted in Britton-Robinson buffer with ph=5 at different AldsRNase doses (0, 10, 50, 100, 200, 400 ng) prepared in example 4 was added and incubated at 28 ℃ for 10min; to determine the optimal reaction pH (B), 1,000 ng dsGFP was dissolved in 5. Mu.L nuclease-free water and incubated with 10. Mu.L of Briton-Robinson Buffer at various pH values (3, 4, 5, 6, 7, 8, 9, 10, 11, 12) for 28℃for 10min with 400ng of AldsRNase enzyme prepared in example 4; to determine the optimal reaction time (C), 1.000 ng dsGFP was dissolved in 5. Mu.L of ultrapure water and incubated with 10. Mu.L of the solution containing 400ng of AldsRNase enzyme prepared in example 4, britton-Robinson Buffer at pH=5 at 28℃for various times (0, 1, 2, 5, 10 min); to determine the optimal reaction temperature (D), 1.000 ng dsGFP was dissolved in 5. Mu.L of ultrapure water and incubated with 10. Mu.L of the solution containing 400ng of the AldsRNase enzyme prepared in example 4 at a pH=5 of Britton-Robinson Buffer at different temperatures (10 ℃,20 ℃,30 ℃,40 ℃,50 ℃,60 ℃) for 10min.

After incubation, the samples were run on a 1% agarose Gel containing Gel Stain nucleic acid dye. dsGFP was visualized under UV light with a Tanon-1600 gel image processing system. And automatically calculating the optical density value of the strip by using GIS digital gel image analysis software.

Conclusion: when mixed with dsGFP, aldsRNase showed high RNase activity, requiring only 200ng to completely degrade dsGFP (FIG. 8-A); aldsRNase exhibits high activity at optimum pH conditions at ph=5 (fig. 8-B); aldsRNase degraded very rapidly, completely degrading dsRNA within 2 minutes of incubation (FIG. 8-C); the maximum temperature activity occurs at 30 ℃ (fig. 8-D).

Sequence listing

<110> academy of agricultural sciences in Jiangsu province

<120> lygus lucorum RNA degrading enzyme, encoding gene, vector, strain and application thereof

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Asp Leu Asn Thr Gly Glu Glu Phe Ile Val Ala Cys Pro Ser Asn Lys

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Asn Val Ile Val Glu Thr Gly Lys Gly Glu Ser Val Ala Lys Cys Val

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Ser Gly Ser Thr Val Ser Ile Gly Gly Lys Thr Val Asp Thr Lys Thr

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Leu Asp Cys Lys Ser Lys Ala Asp Ser Asp Val Ile Lys Thr Asp Arg

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Asn Cys Ala Asn Gly Lys Gly Thr Val Leu Glu Ile Gly Phe Lys Val

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Ala Tyr Leu Asp Ser Lys Ala Thr Lys Phe Leu Ala Arg Gly His Leu

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Tyr Tyr Val Asn Ala Ala Pro Gln Trp Gln Ser Ile Asn Ala Gly Asn

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Trp Leu Arg Thr Glu Thr Asn Ser Arg Thr Val Ala Ala Ala Leu Asn

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Val Asp Leu Ala Val Val Thr Gly Thr Leu Gly Val Ser Lys Met Val

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Asp Asp Lys Gly Gln Glu Lys Glu Ile Phe Met Glu Gly Lys Ser Arg

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325 330 335

Asn Ala Cys Ile Ala Ile Val Ala Thr Asn Asn Pro Phe Leu Lys Ser

340 345 350

Ala Pro Lys Pro Val Cys Lys Asp Val Cys Glu Lys Asn Gly Trp Pro

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Thr Tyr Gln Asp Asp Leu Phe Lys Gly Tyr Val Tyr Cys Cys Glu Tyr

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Lys Asp Leu Lys Ser Val Val Pro His Met Pro Glu Ile Asn Cys Lys

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Ser Thr Leu Gln Phe Lys Lys Lys

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<210> 5

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<212> DNA

<213> oligo (dT)(Artificial Sequence)

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Claims

1. The application of the lygus lucorum RNA degrading enzyme protein in degrading dsRNA is characterized in that the amino acid sequence of the lygus lucorum RNA degrading enzyme protein is shown as SEQ ID NO.4, and the nucleotide sequence of the lygus lucorum RNA degrading enzyme protein is shown as SEQ ID NO. 3.