CN106701805A - Coding gene of endogenous beta-glucosidase, coded protein, and expression vector and application of coding gene - Google Patents

Coding gene of endogenous beta-glucosidase, coded protein, and expression vector and application of coding gene Download PDF

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CN106701805A
CN106701805A CN201611242880.9A CN201611242880A CN106701805A CN 106701805 A CN106701805 A CN 106701805A CN 201611242880 A CN201611242880 A CN 201611242880A CN 106701805 A CN106701805 A CN 106701805A
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glucosidase
coding gene
coptotermes formosanus
beta
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CN106701805B (en
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李志强
吴文静
张世军
柯云玲
刘小琳
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Institute of Zoology of Guangdong Academy of Sciences
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Guangdong Institute of Applied Biological Resources
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
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Abstract

The invention discloses a coding gene of Coptotermes formosanus endogenous beta-glucosidase, and the sequence of the coding gene is represented by SEQ ID NO:1. The sequence of a protein encoded by the coding gene is represented by SEQ ID NO:2. The invention also discloses a prokaryotic expression vector of the coding gene of the Coptotermes formosanus endogenous beta-glucosidase. The coding gene of the Coptotermes formosanus endogenous beta-glucosidase is slightly sensitive to the temperature, and experiments confirm that low temperature and high temperature stress has no influences on the expression of the gene. The coding gene of the Coptotermes formosanus endogenous beta-glucosidase responses to pesticide stress, and can be used for researching the expression regulation mechanism of Coptotermes formosanus cellulase gene; beta-glucosidase encoded by the gene can be used for degrading used for celluloses; and the gene and expressed enzymes can be used for development application of the cellulose ethanol fuel industry, the food industry and the feed industry.

Description

The protein and its table of a kind of encoding gene of endogenous beta-glucosidase, coding Up to carrier and application
Technical field
The present invention relates to technological field of biochemistry, and in particular to a kind of Workers of Coptotermes formosanus Shiraki endogenous beta-glucosidase Encoding gene, the protein of the gene code, corresponding expression vector and application.
Technical background
Energy shortage is the serious problems that the whole world faces, but the most ethanol used in fuel combination come from Fermented grain, there is a problem of striving grain with people.Lignocellulosic is renewable biomass resources most abundant on the earth（Gardens are green Change solid manure of rubbish, grain straw, weeds, wood chip and some animals etc.）If, with the biodegradable wooden fibre of cellulase Dimension element produces reduced sugar, and further fermentation produces ethanol, provide not only a large amount of cellulosic ethanol fuel, and contribute to life The protection of state environment（Deng Tianfu etc., 2010）.
Additionally, cellulase has application in the industries such as food, weaving, feed, detergent and Chinese herbal medicine extraction or field It is extensive：The quality of food is improved, shortened alcohol, sauce fermentation time, improve juice extraction rate and is promoted juice clarification（Yan instructs Friend etc., 2004）；Cellulase can significantly improve the nutritive value of feed as feed addictive, strengthen the enzymolysis of digestive system Function, promotes animal growth（Hu Yi and Sun Bo, 2006）；Improve the feel and outward appearance of textile industry bafta, and base Originally the problems such as solving environmental pollution present in stone mill washing process（Wu great Fu etc., 2007）.Cellulose is by D-Glucose With the polysaccharide that β-Isosorbide-5-Nitrae glycosidic bond is constituted, including endoglucanase（endo-β-1,4-glucanases, EGs, EC 3.2.1.4）, exoglucanase（exo-β-1,4-glucanases, C1）And beta-glucosidase（β-glucosidases, BGs, EC 3.2.1.21）Three kinds of main types.Exoglucanase hydrocellulose produces cellobiose and glucose, and Cellobiose, a small amount of glucose and cellotriose, beta-glucosidase is produced to act on fibre after most endo-glucanase enzyme effects The non-reducing end of the cell-oligosaccharide of dimension disaccharides and low-molecular-weight, hydrolysis generation glucose（Klesov, 1991; Watanabe et al., 2010）.Beta-glucosidase is the crucial enzyme system of degraded cellulose.
The cellulase of high activity is found from nature at present, it is to solve cellulase efficiency and drop to carry out biodegradation The effective way of low cost.The particularly wooden feeding habits insect of some plant-feed insects（xylophagous insects）Can be by fibre The plain enzyme efficient degradation cellulose substances of dimension, and meet the need for own growth develops（Watanabe and Tokuda, 2001; 2010）, such as eat kalotermitid, roundheaded borer, carpenter bee larva and lepidopterous larvae.Wherein, termite（Isoptera Isoptera, existing evidence supports that it is incorporated to Blattaria Blattodea）It is the maximally effective insect assembly of digest cellulose, is recognized To be high-efficient biological reactor minimum on the earth（Ohkuma, 2003）, the cellulose substances total amount of digestion is huge every year.
Workers of Coptotermes formosanus Shiraki（Coptotermes formosanusShiraki）Category Rhinotermitidae （Rhinotermitidae）, formosanes category（Coptotermes）, serious harm is caused to agricultural trees, building structure（Lin, 1987；Rust and Su, 2012）, there is degradation efficiency very high to cellulose substances.Have in Workers of Coptotermes formosanus Shiraki body rich Fiber-rich element enzyme and its gene, current multiple Workers of Coptotermes formosanus Shiraki endogenous cellulose enzyme genes have been cloned and have obtained full-length cDNA sequence Row, wherein EG enzymes have including eight including CfEG1, CfEG1b, CfEG2, CfEG3, CfEG3a, CfEG3b, CfEG4, CfEG5 Homologous gene（Nakashima et al., 2002b; Zhang et al., 2009; Zhang et al., 2011）, and Beta-glucosidase gene only has tetra- homologous genes of Glu1A, Glu1B, Glu1C, Glu1D（Zhang et al., 2010; Zhang et al., 2012a ）.
At present with the technique of cellulose degraded cellulose, difficulty is that cellulase efficiency is low, will to environmental condition Ask harsh, and high cost, new cellulose enzyme gene is excavated from termite body and is used, be solve this problem effective One of approach.In addition cellulose enzyme gene, as termiticide target, is also the developing direction of following termite biological control.
The content of the invention
It is an object of the invention to provide a kind of coding of the beta-glucosidase for stablizing expression relatively at different temperatures Gene.
Another object of the present invention is the protein for providing above-mentioned encoding gene coding.
It is also an object of the present invention to provide the prokaryotic expression carrier of above-mentioned encoding gene.
It is also an object of the present invention to provide the application of above-mentioned encoding gene.
Above-mentioned purpose of the present invention is achieved by the following technical programs：
A kind of encoding gene of Workers of Coptotermes formosanus Shiraki endogenous beta-glucosidase, its sequence such as SEQ ID NO:Shown in 1.
Amino acid sequence such as SEQ ID coded by the encoding gene of above-mentioned Workers of Coptotermes formosanus Shiraki endogenous beta-glucosidase NO:Shown in 2.
A kind of prokaryotic expression carrier, the encoding gene for expressing Workers of Coptotermes formosanus Shiraki endogenous beta-glucosidase.
The temperature sensitive property of encoding gene of Workers of Coptotermes formosanus Shiraki endogenous beta-glucosidase of the present invention is low, through experiment Confirm, low temperature does not interfere with the expression of the gene with high temperature stress.Workers of Coptotermes formosanus Shiraki endogenous beta-glucosidase of the present invention The encoding gene of enzyme responds to pesticide stress, is experimentally verified that, Workers of Coptotermes formosanus Shiraki takes food gene expression after imidacloprid Start to make within three days and obvious response to, the gene expression amount is significantly raised within the 4th day.The gene can be used to study termite cellulase Gene expression regulation mechanism；The beta-glucosidase of the gene code can be used for cellulose degradation；The gene and its expression enzyme Can be used for the development and application of cellulosic ethanol fuel, food and feed industry；The gene and its expression enzyme can be used as termite The target of preventing and treating.
Compared with prior art, the present invention has the advantages that：
The invention provides a kind of encoding gene of new beta-glucosidase, primer and carrier.Can be used for cellulose degraded, The development and application of cellulosic ethanol fuel, food and feed industry, the encoding gene and its expression enzyme of the beta-glucosidase Class can be used as the target of termite control.
Brief description of the drawings
Fig. 1 is geneCfBG-Ib3 ' RACE and 5 ' RACE PCR results（M is D2000 DNA Maker, and 1 is gene 5 ' RACE PCR amplifications, 2 is 3 ' RACE PCR amplifications of gene）.
Fig. 2 is geneCfBG-IbThe signal amino acid sequence peptide prediction of coding.
Fig. 3 is the three-dimensional structure of CfBG-Ib（Note：The three-dimensional structure of the CfBG-Ib albumen built based on template, template is covered The amino acid of lid 84%, confidence level is more than 90%）.
Fig. 4 is the prokaryotic expression of recombinant protein（1st, 2,4 is the pET-32a- of inductionCfBG-IbProkaryotic expression, 3 is not The pET-32a- of inductionCfBG-IbProkaryotic expression）.
Fig. 5 is after temperature stress processes 24hCfBG-IbGene expression dose（All data are mean+SD, no Significant difference is represented with letter（P<0.05））.
Fig. 6 is after temperature stress processes 24hGlu1BGene expression dose（All data are mean+SD, different Letter represents significant difference（P<0.05））.
Fig. 7 is after imidacloprid is processedCfBG-IbGene expression dose（All data are mean+SD, different words Matrix shows significant difference（P<0.05））.
Fig. 8 is after imidacloprid is processedGlu1BGene expression dose（All data are mean+SD, different letters Represent significant difference（P<0.05））.
Specific embodiment
With reference to embodiment, the present invention is further elaborated.But embodiment only expresses several implementations of the invention Mode, its description is more specific and detailed, but therefore can not be interpreted as the limitation to the scope of the claims of the present invention.It should be understood that , for the person of ordinary skill of the art, without departing from the inventive concept of the premise, some changes can also be made Shape and improvement, these belong to protection scope of the present invention.
Embodiment 1
1. the extraction of termite total serum IgE
The extraction of Workers of Coptotermes formosanus Shiraki total serum IgE is according to TaKaRa MiniBEST Universal RNA Extraction Kit （TaKaRa companies）Operation instruction is carried out.
2. the first chain cDNA synthesis
The synthesis of the first chain cDNA uses PrimeScriptTMII 1st Strand cDNA Synthesis Kit （TaKaRa）.
3. degenerate primer design
Using CODEHOP（Consensus Degenerate Hybrid Oligonuleotide Primers）Online software journey Sequence designs the degenerate primer of beta-glucosidase, can obtain several degenerate primer sequences to by being made up of the nucleotide chain of degeneracy Row（Wherein R=A/G, Y=C/T, M=A/C, K=G/T, S=C/G, W=A/T, H=A/C/T, B=C/G/T, V =A/C/G, D=A/G/T, N=A/C/G/T）.
After PCR amplifications, take 5 μ l products carries out electrophoresis detection on 1.0% Ago-Gel, and remaining product is used after detection Make gel extraction target DNA fragment.
PCR primer is connected by pMD TM 18-T Vector Cloing Kit。
The conversion of plasmid
The preparation of 4.1 competent escherichia coli cells：
1) it is connected in the test tube of 3 ml LB fluid nutrient mediums from one single bacterium colony of picking on bacillus coli DH 5 alpha flat board, 37 DEG C are shaken Bed overnight incubation；
2) take the above-mentioned bacterium solutions of 0.5 ml to be transferred in the conical flask containing 50 mL LB fluid nutrient mediums, 250 rpm at 37 DEG C Shaking table culture 2-3 h, determine OD550It is 0.6 or so；
3) bacterium solution is transferred in 50 ml centrifuge tubes, 10 min is placed on ice, then in 4 DEG C, 5000 rpm are centrifuged 5 min；
4) centrifuge tube is inverted to use up supernatant, the 0.1 mol/L CaCl for adding 25 ml ice-cold2Solution, is being vortexed immediately Mixed on blender, after placing 30 min in insertion ice, 4 DEG C, 5000 rpm are centrifuged 5 min；
5) after abandoning supernatant, with the 0.1 mol/L CaCl that 2.5 ml are ice-cold2Solution is resuspended, and every part of 100 μ l dispense cell, can To be directly used as conversion.
4.2 thermal shock methods convert competent escherichia coli cell：
1) the 100 μ l DH5 α competent cells that two pipes system is got ready are taken, wherein a pipe adds 10 μ l connection products, another pipe to make Negative control, two pipes place 30 min on ice after gently mixing；
2) 45 s carry out heat-shock transformed in 42 DEG C of water-baths, then put back in ice rapidly, stand 3 min；
3) gently mix to being separately added into 400 μ l LB fluid nutrient mediums in above-mentioned pipe, 37 DEG C of h of concussion and cultivate 1；
4) the μ l of above-mentioned conversion mixed liquor 100 are taken in superclean bench, is dripped to containing ampicillin（100 ug/ml）Solid On LB flat boards, after the coated with glass rod coating burnt with alcolhol burner is uniform, sealed membrane seals culture dish；
5) marked on the culture dish for coating, 37 DEG C of inversion incubated overnights.
Transformant screening and checking
5.1 bacterium solution PCR are identified
5 single bacterium colonies of picking in superclean bench, are seeded to 5 ml LB fluid nutrient mediums respectively（It is blue or green containing 100 μ g/ml ammonia benzyls Mycin）10ml shake tube, 37 DEG C, 250 rpm shake bacterium incubated overnight, to OD550It is 0.6 or so.Take 1 μ l bacterium solution conducts CDNA templates, enter performing PCR identification.
Following system is added in PCR pipe：
Component Usage amount
Premix Taq 10 μl
pMD 18T-simple primers 0.5+0.5 μl
CDNA templates 1 μl
dH2O up to 20 μl
Above-mentioned bacterium colony PCR reaction systems are mixed, after entering performing PCR amplification, 5 μ l products is taken and is reflected with 1.0% agarose gel electrophoresis Fixed, the identified bacterium solution extracting recombinant plasmid being positive, 4 DEG C of part bacterium solution is saved backup.
Plasmid enzyme restriction is identified
The extraction and purification of DNA uses Plasmid Mini Kit I（Omega Bio-Tek）, comprise the following steps that：
1) 1.5-5 ml are collected with centrifuge tube and identify the bacterium solution being positive through bacterium colony PCR, 12000 rpm, room temperature is centrifuged 1 min, Supernatant discarded, collects thalline is in 1.5 ml centrifuge tubes；
2) Solution I of 250 μ l precoolings is added（Plus RNase A）, abundant suspension thalline precipitation；
3) 250 μ l Solution II are added, gently fully turn upside down centrifuge tube, to liquid clarification, room temperature places 2 min；
4) 350 μ l Solution III are added, gently fully turn upside down centrifuge tube 8-9 times, to white precipitate no longer shape Into 12000 rpm, room temperature is centrifuged 10 min；
5) a clean HiBind DNA Minicolumn are placed in one 2 ml collecting pipes, by the transfer of above-mentioned centrifuged supernatant Into HiBind DNA Minicolumn, 12000 rpm, room temperature is centrifuged 1 min；
6) filtrate is abandoned, 500 μ l Buffer HB are added, 12000 rpm, room temperature is centrifuged 1 min；
7) filtrate is abandoned, 750 μ l Wash Buffer are added（Plus absolute ethyl alcohol）, 12000 rpm, 1 min of room temperature centrifugation；
8) step 7 is repeated；
9) filtrate is abandoned, 12000 rpm, room temperature is centrifuged 1 min；
10) HiBind DNA Minicolumn are placed in a new centrifuge tube, add 50-100 μ l sterile deionized waters Or TE Buffer to Silica films center, 12000 rpm, room temperature centrifugation 1 min wash-out DNAs.
Plasmid double digestion reaction system is as follows：
Component Usage amount
DNA x μl
QuickCut EcoR I 1 μl
QuickCut Sal I 1 μl
10× QuickCut Green Buffer 5 μl
dH2O up to 20 μl
Brief centrifugation after gently mixing, 37 DEG C of insulation 15min.Digestion products are separated with 1.0% agarose gel electrophoresis and identified.
Sequencing identification
Hua Da gene is sent to be sequenced the bacterium solution that digestion identification is positive, sequence goes carrier to be committed to NCBI after splicing to be carried out BLAST compares analysis.
The clone of cellulose enzyme gene full length sequence
The structure of 6.1 cDNA libraries
In order to obtain the full length sequence of termite cellulase cDNA, using Rapid amplification of cDNA ends（RACE）Method.
Using SMARTer RACE cDNA Amplification Kit（Clontech companies）Construction cDNA total length text Storehouse, the template as RACE PCR, comprises the following steps that：
1) in PCR pipe（A is managed）It is middle to add following system (Mixture B)：
Component Usage amount
5×First-Strand Buffer 4 μl
DTT （100mM） 0.5 μl
dNTPs（20mM） 1 μl
Total 5.5 μl
Brief centrifugation after gently mixing, room temperature is placed standby；
2) following system is added in another PCR pipe（Opening for 5 '-RACE and 3 '-RACE points is carried out）：
5’-RACE（B1 is managed） 3’-RACE（B2 is managed）
5 μl RNA 5 μl RNA
1 μl 5’-CDS Primer A 1 μl 3’-CDS Primer A
5 μl RNase free dH2O 6 μl RNase free dH2O
Total 11 μl Total 12 μl
Brief centrifugation after gently mixing；
3) B1 pipes and B2 pipes are placed in PCR instrument, 72 DEG C, 3min；42 DEG C, 2min, 14000g after cooling are centrifuged 10s；
4) 1 μ l SMARTer II A Oligonucletide are added in B1 pipes；
5) following reaction system is prepared：
5’-RACE cDNA 3’-RACE cDNA
5.5 μl Buffer Mix from tube A 5.5 μl Buffer Mix from tube A
12 μl Buffer Mix from tube B1 12 μl Buffer Mix from tube B2
0.5 μl RNase Inhibitor（40U/μl） 0.5 μl RNase Inhibitor（40U/μl）
2.0 μl SMARTScribe Reverse Transcriptase 2.0 μl SMARTScribe Reverse Transcriptase
Total 20 μl Total 20 μl
Brief centrifugation after gently mixing；
6) it is placed in PCR instrument, 42 DEG C, 90min；70 DEG C, 10min；
7) the Tricine-EDTA Buffer of 90 μ l are added to dilute above-mentioned product RACE cDNA, -20 DEG C of preservations after packing.
The design of 6.2 gene specific primers
In the intermediate segment sequence inputting Genbank that degenerate primer amplification is obtained, sequence alignment is carried out, select and contain cellulose The sequence of enzyme gene conserved sequence, according to the Kit of SMARTer RACE 5 '/3 '（Clontech companies）The design of primers of offer Illustrate, the gene specific primer GSPs and NGSPs of design 5 '-RACE and 3 ' RACE.The gene of the RACE of 5 '-RACE and 3 ' Special primer GSPs and NGSPs title and corresponding sequence are as shown in the table：
Primer Primer sequence（5’-3’）
GSP-BG12R GATTACGCCAAGCTTCCACAACGATTTCAGGGACACACAAGC
GSP-BG11F GATTACGCCAAGCTTAGCTCGAATTGCACGGCTCTCTCC
GSP-BG22 R GATTACGCCAAGCTTGCCGATGCCTGGTGACTTGATTGC
GSP-BG23F GATTACGCCAAGCTTTCAACGAACCGCTTACTTTCATGGGAGG
NGSP-BG11R GATTACGCCAAGCTTGCCAGAACAATGCTGCTTGCTCACG
NGSP-BG13F GATTACGCCAAGCTTGCTGAGGCAAATCCCAGTGGTACATCG
NGSP-BG21R GATTACGCCAAGCTTCGATGCCCGCCAGGTTCACTTTATTG
NGSP-BG22F GATTACGCCAAGCTTAATGTACGCTCACCCCATCTTCAGCAC
UPM Long TAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT
UPM Short CTAATACGACTCACTATAGGGC
6.3 RACEPCR
With the cDNA of above-mentioned synthesis as RACE PCR template, using SMARTer®RACE 5’/3’ Kit（Clontech Company）Operating instruction carries out RACE PCR, comprises the following steps that：
1) in PCR pipe（C is managed）It is middle to add following system (Master Mix)：
Component Usage amount
PCR-Grade H2O 15.5 μl
2×SeqAmp Buffer 25 μl
SeqAmp DNA Polymerase 1 μl
Total 41.5 μl
Brief centrifugation after gently mixing, places standby；
2) following system (Mixture C) is added in PCR pipe：
5’-RACE 3’-RACE
2.5 μl 5’-RACE cDNA 2.5 μl 3’-RACE cDNA
10×UPM Long 10×UPM Long
5’GSP（10μM） 3’GSP（10μM）
41.5 μl Master Mix from tube C 41.5 μl Master Mix from tube C
Total 50 μl Total 50 μl
3) after above-mentioned PCR reaction systems gently being mixed into brief centrifugation, performing PCR amplification is entered.
If 4) RACE pcr amplification products do not have obvious band or product smear occur, nested is carried out PCR。
A. 5 μ l RACE pcr amplification products are taken, it is standby after being diluted with the Tricine-EDTA Buffer of 245 μ l；
B. repeat step 1-3, wherein, the RACE PCR amplified productions after 5 μ l dilutions replace RACE cDNA, UPM Short Instead of UPM Long, NGSP replaces GSP, PCR response procedures to be set to：94 DEG C of denaturation 30s, 68 DEG C of annealing 30s, 72 DEG C of extensions 3min, circulates 20 times.
The splicing of full length gene
The intermediate segment that Seqman programs in application software Lasergene are obtained to degenerate pcr, and 5 ' RACE and 3 ' RACE 5 ' and 3 ' ends that PCR is obtained are spliced, final to obtain the cDNA full length sequences of cellulose enzyme gene, and log on to NCBI numbers According in storehouse.
Bioinformatic analysis
Using SingalP4.1 online software prediction signal peptide sequences, analyze N- with NetOGlyc and connect glycosylation site, use Phyre 2 predicts protein three-dimensional model.22 are chosen from BLAST nucleic acid databases and comes from β-glucosyl enzym, utilized MEGA5.1 analyzes above-mentioned 24 β-glucosyl enzym amino acid sequences and 1 β-glucosyl enzym of this research, the amino acid of CfBG-Ib Sequence, phylogenetic tree construction.
The prokaryotic expression of gene
The structure of 1 prokaryotic expression carrier
The design of 1.1 special primers
According to the β-glucosyl enzym gene that clone obtainsCfBG-IbCDNA, choose gene ORF as prokaryotic expression piece Section, designs specific primer.Simultaneously according to prokaryotic expression carrier pET-32a (+) the collection of illustrative plates sequence chosen, in the specificity of design 5 ' the suitable restriction enzyme sites of end addition of primerSac I andXhoI（Underscore is indicated）, and protection base, Primer and sequence Row are as follows：
Primer Primer sequence（5’-3’）
Ex-bgF CGAGCTCGTCATGTTAAGTGGAGCAG
Ex-bgR CCACTCGAGTCATAACGGAATCGTCGG
The PCR amplifications of 1.2 prokaryotic expression purpose fragments, gel extraction, connection, conversion and sequencing analysis
The double digestion reaction of 1.3 prokaryotic expression purpose fragments and prokaryotic expression carrier pET-32a (+)
Double digestion reaction will be respectively carried out through correct purpose fragment and pET-32a (+) empty carrier is sequenced, to obtain with identical The fragment of cohesive end.
Double digestion reaction system is as follows：
Component Usage amount
Genes of interest fragment/pET-32a (+) empty carrier x μl
QuickCut Sac I 1 μl
QuickCut Xho I 1 μl
10× QuickCut Green Buffer 5 μl
dH2O up to 20 μl
Brief centrifugation after gently mixing, 37 DEG C of insulation 30min.After double digestion reaction completely, digestion products carry out Ago-Gel Identification is separated by electrophoresis, then gel extraction.
Connection and the screening of positive recombinant plasmid
Purpose fragment and pET-32a (+) carrier connection with identical cohesive end by above-mentioned gel extraction, linked system is such as Under：
Component Usage amount
Band cohesive end genes of interest fragment 10 μl
PET-32a (+) carrier with cohesive end 4 μl
T4 DNA Ligase 1 μl
10 × T4 DNA connect buffer solution 2 μl
dH2O up to 20 μl
Brief centrifugation after gently mixing, 16 DEG C of connections are overnight；Connection product is converted to bacillus coli DH 5 alpha competent cell again In, screening positive clone is reacted with bacterium solution PCR and double digestion, and be sequenced, step is with reference to 2.2.1.3.6.Sequencing identification is correct Recombinant plasmid transformed to Bacillus coli expression type bacterial strain Rosetta (DE3) competent cell in, and screen positive monoclonal.
The extraction of the induced expression and total bacterial protein of recombinant protein
Induced expression：
1）In drawing bacterium solution to the 10mL fresh LB fluid nutrient mediums that 10 μ l sequence verifications are crossed（Containing 100 μ g/ml ammonia benzyl moulds Element）
37 DEG C, 250 rpm shake bacterium incubated overnight；
2）In bacterium solution to the 100mL fresh LB fluid nutrient mediums of absorption 1mL incubated overnights（Containing 100 μ g/ml ammonia benzyl moulds Element）, 37 DEG C, 250 rpm shake bacterium culture, to OD550It is 0.6 or so；
3）To addition IPTG to final concentration of 1mM, 23 DEG C of induced expression 16h in above-mentioned bacterium solution；
The extraction of total bacterial protein：
The preparation of protein sample is extracted using Bacterial Protein Extraction kit, is comprised the following steps that：
1）L ml bacterium solutions are taken, 10 min are centrifuged at 4 DEG C, under the conditions of 5000 × g, abandon supernatant, collects thalline；
2）The ratio of 20mL Bacterial Protein Extraction reagents is added according to every gram of bacterial sediment, to adding extract in bacterial sediment, Fully it is vortexed completely resuspended to thalline；
3）After resuspended, 10min is incubated at room temperature；
4）It is centrifuged 5min under the conditions of 15000 × g, in transfer supernatant to new centrifuge tube, as total bacterial protein.
Electrophoresis
1）12% separation gel is prepared, is added in gel mold, then gently cover one layer of water of 1-5cm on sol solution is separated
Layer, makes gel surface keep smooth, stands 45min, occurs one between glue to be separated and water layer clearly behind interface, goes Except the water layer being covered on separation gel；
2）5% concentration glue is prepared, is added to above separation gel, until gel solution reaches the top of front glass panel, comb inserted Enter in gel, it is to avoid produce bubble, after after gel polymerisation, carefully pull out comb；
3）Take 10 μ l protein samples, add 5 × SDS-PAGE sample-loading buffers, boiling water heat denatured 5min is cool but to room temperature Afterwards, 30s is centrifuged under conditions of 3000rpm, takes appropriate supernatant and add in PAGE gel well；
4）Switch on power, after 80V electrophoresis 30min first, protein sample is entered concentration glue, then voltage is changed into 120V Constant pressure electrophoresis can stop electrophoresis until bromophenol blue is reached at the 0.5-1cm of gel bottom.
5）After by the gel excision concentration glue after electrophoresis, it is put into container, appropriate distilled water is added, after being heated to boiling Stop；
6）Distilled water is discarded, appropriate ProteinShow-G250 albumen rapid dye liquor is added, boiling is heated to, continued de- 5min is shaken on color shaking table；
7）Discard dyeing liquor, add appropriate distilled water, stop after being heated to boiling, discard distilled water, repeat this step decolourize to Clear background；
Result and analysis
1. the extraction of termite total serum IgE
It is total Workers of Coptotermes formosanus Shiraki to be extracted according to kit TaKaRa MiniBEST Universal RNA Extraction Kit The value of RNA, micro-spectrophotometer detection OD260/OD280 has reached the requirement of reverse transcription, for the between 1.8-2.2 The synthesis of one chain cDNA.
The clone of beta -glycosidase full length gene sequence
Using Multalin sequence alignment programs, the intermediate segment that will be obtained with cloned the Workers of Coptotermes formosanus Shiraki β-glucoside for obtaining Enzyme gene（Glu A,Glu B,Glu C,Glu D）Nucleic acid sequence alignment is carried out, discovery has significant difference nucleotide sequence, should β-glucosyl enzym gene intermediate segment 817bp long respectively, is respectively designated asCfBG-Ib。
3 ' RACE and 5 ' RACE PCR（Fig. 1）AmplificationCfBG-IbIntermediate segment, obtain Workers of Coptotermes formosanus Shiraki β-glucosyl enzym The complete cDNA of gene, this cDNA total length is by including polyadenylic acid tail（poly A）1966 base compositions.It is open Reading frame（ORF）Originate in the 115th base, terminate at the 1806th base, length is 1692bp, encode 563 amino acid. Wherein there are 26 signal peptide sequences for amino acid of prediction using SingalP4.1 software discoverys（Fig. 2）.Using http:// The online tool of www.expasy.org/ websites predicts that the protein molecular weight of the gene code is 64.02kDa, and isoelectric point is 6.41。
The sequence analysis of β-glucosyl enzym
According to protein sequence, with reference to the β-glucosyl enzym three-dimensional structure of other known species, thus it is speculated that CfBG-Ib tomographs （Fig. 3）.Tomograph display CfBG-Ib has the typical structure of glycosyl hydrolase family 1,8（β/α）The bucket that structure is surrounded Shape structure, 2 catalytic active centers：Nucleophilic center（nucleophilic residue）Glu244 and acid-base catalysis center （acid/base residue）Glu453,2 cis peptide bonds（cis-peptide bond）：Ala259-Pro260 and Trp495-Ser496。
Based on blast search（http://blast.ncbi.nlm.nih.gov/Blast.cgi）Albumen is searched and shownCfBG-IbEncoding proteins belong to glycoside hydrolase Families 1（GHF1）, while in the presence of corresponding catalytic site and Binding Capacity position Point.Homology analysis equally show geneCfBG-IbBelong to GHF1 families.
The prokaryotic expression of beta -glycosidase gene
1. recombinant prokaryotic expression vector pET-32a-CfBG-IbStructure
Expanded by PCR, reclaim the double digestion reaction of purpose fragment and pET-32a empty carriers, the mesh with identical cohesive end Fragment and carrier connection, after the series reaction such as the conversion of recombinant plasmid, verified by bacterium solution PCR, the PCR fragment of amplification Size meets expected size.Sequencing result display recombinant prokaryotic expression vector in genes of interest fragment withCfBG-IbGene ORF consensus nucleic acid sequences, and the protein sequence predicted is also consistent, the phenomenon do not undergone mutation with reading frame frameshit.Therefore, Recombinant prokaryotic expression vector pET-32a-CfBG-IbSuccessfully construct, can be used for the induced expression of further recombinant protein.
Recombinant prokaryotic expression vector pET-32a-CfBG-IbInduced expression
After Escherichia coli Rosetta 2 (DE3) after conversion recombinant expression carrier is induced through 1 mM IPTG,CfBG-Ibβ-Portugal Polyglycoside enzyme gene realizes expression.10% SDS-PAGE electrophoresis detections, the control group different from having converted empty carrier pET-32a is thin Bacterium total protein, has converted recombinant prokaryotic expression vector pET-32a-CfBG-IbBacterial cell total protein show one substantially Band, the fusion protein of relative molecular mass about 82kD（Fig. 4）.And the β-glucosyl enzym gene predictedCfBG-IbCoding egg White molecular mass is 64.02 kDa, is due to carrying molecular weight about Trx-tag, S- of 18kD on empty carrier pET-32a Tri- amalgamation and expression labels of tag and His-tag.
Beta-glucosidase gene differential expression under the environment stress of embodiment 2
1 Temperature Treatment
Take Workers of Coptotermes formosanus Shiraki worker ant and 4 DEG C, 27 DEG C, the 38 DEG C temperature stress treatment of three thermogrades are set, in relative humidity 75% 24h is cultivated in growth cabinet.
Chemicals treatment
Nature enemy for trying termite：The glass culture dish of a diameter of 150mm is taken, Workers of Coptotermes formosanus Shiraki worker ant 120, soldier ant is put into 10,27 DEG C of temperature is put into, hungry culture 24h in the growth cabinet of relative humidity 75%.
The treatment of imidacloprid different time：The filter paper of 2 × 2cm is taken, is respectively 0.1mg/L's with the acetone and concentration of 60 μ L Imidacloprid soaks, and places one day at room temperature, acetone is fully volatilized.The glass culture dish of a diameter of 90mm is taken, every group is put into third Ketone fully volatilize after filter paper 2, every group is put into termite after Nature enemy, in 27 DEG C of temperature, the artificial climate of relative humidity 75% 4d is cultivated in case, 24h is often processed, 10 worker ants is taken and is extracted RNA.
Quantitative fluorescent PCR
The extraction of 3.1 termite total serum IgEs
Extract the termite total serum IgE of each environment stress treatment group and control.
First chain cDNA synthesizes
The synthesis of the first chain cDNA uses PrimeScriptTM RT reagent Kit with gDNA Eraser （TaKaRa）, comprise the following steps that：
1）The removal of genomic DNA
Following system is added in PCR pipe（Mixture D）：
Component Usage amount
Total RNA x μl
5 ×gDNA Eraser Buffer 2 μl
gDNA Eraser 1 μl
RNase free dH2O up to 10 μl
X is calculated according to the concentration of surveyed Total RNA, and usage amount is in below 1 μ g.
After 42 DEG C of 5 min of reaction, 4 DEG C save backup.
2）Reverse transcription reaction
By in the above-mentioned system Mixture D of following reagent addition：
Component Usage amount
Mixture D 10 μl
5×PrimeScript Buffer 4 μl
PrimeScript RT Enzyme Mix I 1 μl
RT Primer Mix 1 μl
RNase free dH2O up to 20 μl
Slowly shake up, after 15 min are reacted at 37 DEG C, 85 DEG C of s of terminating reaction 5.Product is the first chain cDNA, is centrifuged in short-term After deposit at -20 DEG C, for qPCR reaction.
The determination of dilution factor
Effective quantitative PCR result its enter plateau Ct values preferably between 15-30.Diluted using serial dilution method CDNA, so that its Ct value falls between 15-30.The concentration gradient of dilution is 1,1/10,1/100,1/1000,1/10000, five Concentration gradient.
The design of quantification PCR primer
By above-mentioned Stress treatment, the gene for obtaining is cloned with fluorescence quantitative PCR detectionCfBG-IbDifferential expression situation, Referring concurrently to the Workers of Coptotermes formosanus Shiraki beta-glucosidase gene deliveredGlu1BExpression comparative analysis.For fluorescent quantitation The specific primer and internal control primer of PCR detections are as follows：
Primer Amplification gene Primer sequence（5’-3’）
qPCR-BGF CfBG-Ib GCCTTTCCTCTGCATTCACTG
qPCR -BGR CfBG-Ib CCTCAGCCCCTTCAAGACATT
qPCR –Glu1B-F Glu1B TTGCCTCGTCGTCTTTGTGA
qPCR –Glu1B-R Glu1B CCTTTCCATCCGCATCCCAT
18S-F 18S CGAGATTGAGCAATAACAGGTC
18S-R 18S ACGTAATCAACGCGAGCTTATG
3.5 quantitative PCRs react
1）Following system is added in PCR pipe（Operate on ice）：
Component Usage amount
cDNA 1 μl
SYBR Premix Ex TaqTM(2×) 10 μl
PCR Forward Primer (10 μM ) 0.5 μl
PCR Reverse Primer (10 μM ) 0.5 μl
RNase free dH2O up to 20 μl
2）Reacted using Stratagene Mx3000P operations PCR, response procedures are as follows：
95 DEG C, 30s, 1 circulation；95 DEG C, 10s, 55 DEG C, 10s, 72 DEG C, 20s, 40 circulations.In the extension stage that each is circulated Fluorescence signal is collected, real-time monitoring is carried out.
3）After reaction terminates, 95 DEG C of 1min are first to heat to, often raise 1 DEG C, record first order fluorescence signal, show that amplification is produced The solubility curve of thing.
The data analysis of quantitative fluorescent PCR
After reaction terminates, amplification curve and solubility curve are confirmed, using 2-△△CtMethod carry out the relative expression quantity of genes of interest Analysis.
Result and analysis
Workers of Coptotermes formosanus Shiraki beta-glucosidase gene differential expression result under 1 temperature stress
The total serum IgE of the Workers of Coptotermes formosanus Shiraki of Extracting temperature Stress treatment group and control group, reverse transcription be cDNA after, carried out as template Quantitative fluorescent PCR.With 18S genes as reference gene, beta-glucosidase gene is detectedCfBG-IbWithGlu1BIn different temperatures Expression under stress.
Real-time fluorescence quantitative PCR result shows（Fig. 5 ~ 6）, temperature stress treatment, three genes of beta-glucosidaseCfBG-IbWithGlu1BRelative expression quantity trend it is consistent.Under low temperature and high temperature stress, geneCfBG-IbRelative expression quantity In the absence of significant difference.
The lower Workers of Coptotermes formosanus Shiraki beta-glucosidase gene differential expression result of imidacloprid stress
Extract the total serum IgE of the Workers of Coptotermes formosanus Shiraki of imidacloprid Stress treatment group and control group, after reverse transcription is cDNA, enter as template Row quantitative fluorescent PCR.With 18S genes as reference gene, beta-glucosidase gene is detectedCfBG-IbWithGlu1BIn imidacloprid Expression under stress.
Real-time fluorescence quantitative PCR result shows（Fig. 7 ~ 8）, three genes of imidacloprid Stress treatment beta-glucosidaseCfBG-IbWithGlu1BRelative expression quantity trend it is basically identical.First 3 days, under three relative expression quantities of gene and control group Relative expression quantity is more or less the same, the 4th day for the treatment of, relative expression of three relative expression quantities of gene obviously higher than control group Amount.
SEQ ID NO:1
GAATTAGTTGAAGTTGGCGTCATATTTGACAAGTTTACAACTTGACGGCTTCCCATGGCGACTGGATAGGACGCAGATT
GTGTAAGATTGAGAATATCACCGGCAACTCAAGTCATGTTAAGTGGAGCAGGTGCTTGGAGATGTTTCTTACTTTCAGTA
CTGTTGCTGAATTTATCAGCTACTGCCAGCGGTCGCCTTATTTCTAATATTTTGAATCCCATTTTAAGTAGTCTCGGCCT
AAACTCCCAAAGTACCAAGAACACTGTTGCCCCTTCCCAATATAATGTTGTTACTACTCCCAAGAACACTGTTGCCCCTT
CCCAAAGTAACACCGATCCTACCAAGAAGAACTTCACTTTTCCCGAAGACTTCTACTTTGCAGCTGCAACGGCGGCTTAC
CAAGCCGAAGGAGGTTGGAACGCAGACGGGAAAGGTCCTAATATCTGGGACACACTGACACACAACCATCCAGACTACAT
ATCCGACTTCTCGAATGGTGACGTAGCAGCGGACTCCTACCACAAGTACACTGAAGACATCAAATTACTCAAGGACATTG
GGGTGCAGTTTTACAGATTTTCCATATCCTGGTCACGGATACTTCCTAAGGGAAGTGTGGCAGCCATTAATCAGGCTGGT
GTCGACTACTATAACAACATCATTAATGGTCTGCTAGCCGTCGGAATTCAGCCCATGGTTACGATGTACCACTGGGATTT
GCCTCAGCCCCTTCAAGACATTGGAGGATGGGCCAATGATTCCATAGCCGATTTCTTCAAGGACTACGCCAGGCTTCTAT
ATAGATTTTTCGGCGATAGAGTGAAATGGTGGATACCAATAAATGAGCCTTTCATGATAGCTAACGGATACAGTGAATGC
AGAGGAAAGGCGCCGTCCCTCTGCCAGCCTGGGACGGCAGATTACCTGGCAGCCAGAACAATGCTGCTTGCTCACGCAAA
AGCCTATCACGTCTACCACAACGATTTCAGGGACACACAAGCAGGTAAAGTGAGCACCTCGTTTAGCATCGACTGGCACG
AACCGCGCACCAACAGTACGGCAGATATTTTAGCAGCGGAGAGAGCCGTGCAATTCGAGCTAGGGATGTTCGCTCATCCC
ATCTACAGTACCTCGGGGGACTATCCACCGGAAGTAAGAGCCAGAGTCGATAACAACAGCAGAGCTGAGGGCTACAATAC
TTCCCGCCTGCCGAAATTCACCCAAGAGGAGATAGATTACATTAAAGGGACGTGGGACTTCTTCGCATTAAATCATTACA
CAACTTATTGGGCCCAGGATGGACTGCAAGGGCCGGACCCTTCCCGACAGCGCGATTCGGGTGTCATGAAATCGCAAGAC
CCCAGCTGTCCTGAGACCAGCTCTCCGTGGTTTAGGGTTGTTCCCTGGGGATTCAGGAAGATACTGCGTTGGGTGAAGAA
AGAATACAACAATCCCCCAATATTCATCACAGAGTCCGGTTACTCTGACGATGGTCGTCTCCAGGATACGGGACGGATTA
AGTATTACGTAGACTACATCCGTGAGCTGCTGAAGGCAAAATACGAAGATGGATGTCAAATAATTGGCTACACTGCTTGG
AGTCTAATTGACAATTTTCAATGGGAAGAAGGCTATGAGAGTAAGTTTGGGCTGGTCTACGTCAACTTCAGTGACCCTGC
AAGAACTCGGATCATCAAGCAGTCAGCAAGGTTGTACAGCGAGATCATCCGCACAAGAAAAGTTCCGGACCGCTACCCGC
AGTACAGCTATGACACGCAGGAATGTCACCCGACGATTCCGTTATGAAAGCTATATGAATCATAGTTCCTCACTATCGAT
GTTTACGAAAGAGCGCGTGCCTATGGAATTACGATGGGGAAGAATTTTTCTATTGCGTGTAAACGATTCAGACTATTAAG
TGTATTATGAGAACAGAAAAATCATGAAAATATCGAAAAAAAAAAAA
SEQ ID NO:2
MLSGAGAWRCFLLSVLLLNLSATASGRLISNILNPILSSLGLNSQSTKNTVAPSQYNVVTTPKNTVAPSQSNTDPTKKNF
TFPEDFYFAAATAAYQAEGGWNADGKGPNIWDTLTHNHPDYISDFSNGDVAADSYHKYTEDIKLLKDIGVQFYRFSISWS
RILPKGSVAAINQAGVDYYNNIINGLLAVGIQPMVTMYHWDLPQPLQDIGGWANDSIADFFKDYARLLYRFFGDRVKWWI
PINEPFMIANGYSECRGKAPSLCQPGTADYLAARTMLLAHAKAYHVYHNDFRDTQAGKVSTSFSIDWHEPRTNSTADILA
AERAVQFELGMFAHPIYSTSGDYPPEVRARVDNNSRAEGYNTSRLPKFTQEEIDYIKGTWDFFALNHYTTYWAQDGLQGP
DPSRQRDSGVMKSQDPSCPETSSPWFRVVPWGFRKILRWVKKEYNNPPIFITESGYSDDGRLQDTGRIKYYVDYIRELLK
AKYEDGCQIIGYTAWSLIDNFQWEEGYESKFGLVYVNFSDPARTRIIKQSARLYSEIIRTRKVPDRYPQYSYDTQECHPT
IPL

Claims (4)

1. a kind of encoding gene of Workers of Coptotermes formosanus Shiraki endogenous beta-glucosidase, its sequence such as SEQ ID NO:Shown in 1.
2. the protein sequence described in claim 1 coded by the encoding gene of Workers of Coptotermes formosanus Shiraki endogenous beta-glucosidase Such as SEQ ID NO:Shown in 2.
3. a kind of protokaryon for expressing the encoding gene of the Workers of Coptotermes formosanus Shiraki endogenous beta-glucosidase described in claim 1 Expression vector.
4. the application of the encoding gene of Workers of Coptotermes formosanus Shiraki endogenous beta-glucosidase described in claim 1, it is characterised in that institute The beta-glucosidase for stating the encoding gene coding of Workers of Coptotermes formosanus Shiraki endogenous beta-glucosidase is used for degraded cellulose.
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