CN107043776B - A kind of green plant bug nuclear hormone receptor E75, its coded sequence, carrier and bacterial strain - Google Patents
A kind of green plant bug nuclear hormone receptor E75, its coded sequence, carrier and bacterial strain Download PDFInfo
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Abstract
The present invention discloses the DNA sequence dna for encoding green plant bug nuclear hormone receptor E75.Invention additionally discloses green plant bug nuclear hormone receptor E75, for encoding recombinant expression carrier, transgenic cell system or the transgenosis recombinant bacterium of the DNA sequence dna of green plant bug nuclear hormone receptor E75, the preparation method of the application and green plant bug nuclear hormone receptor E75 of the recombinant expression carrier, transgenic cell system or transgenosis recombinant bacterium in production green plant bug nuclear hormone receptor E75.Utilize green plant bug nuclear hormone receptor E75 gene, construct its pCzn-E75 recombinant plasmid, it is transferred to TOP10 clone strain and the inclusion body of the albumen can be generated after IPTG is induced in Arctic Express expression bacterial strain, the albumen can be formed through denaturation, renaturation again, and is purified and is made.Molecular weight of albumen obtained is about 75KD, and protein concentration is 0.4 mg/ml.
Description
Technical field
The present invention relates to technical field of agriculture science, and in particular to a kind of green plant bug nuclear hormone receptor E75, its coding
Sequence, carrier and bacterial strain.
Background technique
Green plant bug Apolygus lucorum (Hemiptera:Miridae) belong to Semiptera Miridae, be cotton, vegetables,
Important pests in the multiple kinds of crops such as fruit tree, herbage.In recent years, the large area plantation and agricultural industry due to Bt transgenic cotton
The reasons such as the adjustment of structure, green plant bug population quantity steeply rise, and rely on chemical pesticide control for a long time in addition, cause green plant bug
Drug resistance increasingly enhances, and the green plant bug prevention and control in agricultural production face a severe challenge at present, anxious to make wait open up reduction chemical pesticide
Novel non-pollution preventions.
Steroid hormone 20- hydroxyecdysone (20-hydroxyecdysone, 20E) is the main work of insect moulting hormones
Property form, plays important regulating and controlling effect during the development by metamorphosis of insect.Nuclear hormone receptor E75 is moulting hormone
Action target is modulin important in insect bodies, has become one of the important target of novel pesticide at present.E75 is subordinate to
In insect nuclear receptor, 4 modular structural domains: the domain A/B, the domain C, the domain D and the domain E are typically contained.Wherein the domain C and the domain D are relatively conservative, C
2 zinc rouge structures are contained in domain, and there are a ligand binding pockets in the domain E.It studies green plant bug AlE75 gene structure and obtains its and recombinate egg
It is white, it is expected to provide the theoretical basis of early period by the novel pesticide of target of ecdysone receptor.
Summary of the invention
Goal of the invention: technical problem to be solved by the invention is to provide for encoding green plant bug nuclear hormone receptor
The DNA sequence dna of E75, as shown in SEQ ID NO:1.
Also there is provided green plant bug nuclear hormone receptor E75, amino acid sequences for technical problems to be solved by the present invention
As shown in SEQ ID NO:2.
The present invention also technical problems to be solved are to provide for containing for encoding green plant bug nuclear hormone receptor E75
DNA sequence dna recombinant expression carrier, transgenic cell system or transgenosis recombinant bacterium.
The present invention also technical problems to be solved are there is provided the recombinant expression carrier, transgenic cell system or turn
Application of the gene recombination bacterium in production green plant bug nuclear hormone receptor E75.
Also there is provided the preparations of green plant bug nuclear hormone receptor E75 recombinant protein for technical problems to be solved by the present invention
Method.
Technical solution: to solve the above-mentioned problems, it is thin for encoding green plant bug that the technical solution of the present invention is to provide one kind
The DNA sequence dna of karyon hormone receptor E75, base sequence is as shown in SEQ ID NO:1.
The content of present invention further includes a kind of green plant bug nuclear hormone receptor E75, amino acid sequence such as SEQ ID NO:2
It is shown.
The content of present invention further includes the recombination table containing the DNA sequence dna for encoding green plant bug nuclear hormone receptor E75
Up to carrier, transgenic cell system or transgenosis recombinant bacterium.
Wherein, above-mentioned recombinant expression carrier further includes that above-mentioned DNA sequence dna is inserted into coli expression carrier
The recombinant expression carrier of obtained expression green plant bug nuclear hormone receptor E75.
Wherein, above-mentioned coli expression carrier is pCzn1.
Wherein, above-mentioned recombinant bacterium is to import above-mentioned recombinant expression carrier in Escherichia coli, and screening obtains transgenosis weight
Group bacterium.
Wherein, above-mentioned Escherichia coli are Arctic Express.
The content of present invention further includes by above-mentioned DNA sequence dna, recombinant expression carrier, transgenic cell system or transgenosis weight
Application of the group bacterium in production green plant bug nuclear hormone receptor E75.
The content of present invention further includes the preparation method of green plant bug nuclear hormone receptor E75 a kind of, and being that culture is above-mentioned turns
The green plant bug nuclear hormone receptor E75 that gene recombination bacterium obtains.
The contents of the present invention further include the preparation method of green plant bug nuclear hormone receptor E75 a kind of, comprising the following steps:
1), for encoding the amplification of green plant bug nuclear hormone receptor E75 gene, the E75 gene order such as SEQ ID
Shown in NO:1；
2), the Prokaryotic expression vector construction of green plant bug nuclear hormone receptor E75 gene；
3), the denaturation and renaturation of green plant bug nuclear hormone receptor E75；
4), the purifying of green plant bug nuclear hormone receptor E75.
The utility model has the advantages that the present invention compared with the existing technology, has the advantage that the present invention obtains green plant bug cell for the first time
Nuclear hormone receptor E75 gene order, and its pCzn1 prokaryotic expression carrier is constructed, and in Escherichia coli Arctic Express
The inclusion body of the albumen can be generated after IPTG is induced, then the albumen can be formed through denaturation, renaturation, and purified system
?.Molecular weight of albumen obtained is about 75KD, protein concentration 0.4mg/ml.The nuclear hormone receptor E75 being purified into can be
The Research foundation of early period is provided by the novel pesticide of target of ecdysone receptor.
Detailed description of the invention
The end Fig. 1 green plant bug nuclear hormone receptor E75 gene 5' and 3' PCR amplification electropherogram；Swimming lane 1: green plant bug cell
Nuclear hormone receptor E75 gene 5'(A) and the end 3' (B) PCR fragment, the DNA maker of swimming lane 2:2000bp；
Fig. 2 Ndel and Xbal double digestion carrier T electrophoretogram；M:Maker；Swimming lane 1: plasmid before digestion；Swimming lane 2: after digestion
Plasmid；
The SDS-PAGE analytical electrophoresis figure of Fig. 3 pCzn1-E75 inducing expression；M: protein Maker；Swimming lane 1: without IPTG
Induce sample；Swimming lane 2: room temperature induces sample；IPTG induces supernatant at 3:11 DEG C of swimming lane；IPTG induction is outstanding at 4:11 DEG C of swimming lane
Supernatant liquid；
The pCzn1-E75 protein SDS-PAGE of Fig. 4 purifying detects electrophoretogram；Swimming lane 1: unpurified inclusion body；Swimming lane 2:
The not foreign protein in conjunction with Ni column；Swimming lane 3: the destination protein in conjunction with Ni column；M: Protein Marker object.
Specific embodiment
The present invention will be further explained with reference to the accompanying drawing.
Embodiment 1: the acquisition of green plant bug nuclear hormone receptor E75 gene (AlE75 gene)
Green plant bug total serum IgE is extracted using TRIzol method, select electrophorogram good and OD260/OD280 value 1.8~
Total serum IgE sample between 2.0 merges the purifying for carrying out mRNA, and reverse transcription synthesizes the first chain cDNA, PCR reaction system are as follows: 10 μ l
Total serum IgE, 4.0 μ l 5X reaction buffers (Sigma company), 2 μ l 10mmol dNTP, 1 μ l ribonucleotide acid inhibitor (Sigma
Company), 2 μ l reverse transcriptase (Sigma company) add ddH2O to reaction volume be 20 μ l.PCR response parameter are as follows: 42 DEG C of warm bath
60min, 72 DEG C of warm bath 10min.According to other known insects (such as Papilio xuthus, Glossina morsitans,
Aedes aegypti) nuclear hormone receptor E75 conserved sequence design be suitable for PCR amplification primer AlE75-F (5 '-
CATTATGGWGTYTAYAGYTGTG-3 ') and AlE75-R (5 '-AGHAGTTCATTCCAVCCTGCTC-3 '), above-mentioned primer be
Degenerate primer obtains green plant bug nuclear hormone receptor E75 conserved sequence, and the conserved sequence is referring to the SEQ ID in sequence table
NO:3, PCR reaction system are as follows: 12.5 μ l2 × GC Buffer I (Sigma company), 0.5 μ l AlE75-F, 0.5 μ l AlE75-
R, 4 μ l dNTP, 6.3 μ l ddH2O, 1 μ l cDNA, 0.5 μ l Taq enzyme (Sigma company)；PCR response parameter are as follows: 95 DEG C of pre- changes
Property 4min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, 72 DEG C of reparations extend 7min, global cycle 40 times；In basis
The conserved sequence design of the AlE75 gene of acquisition is suitable for the amplimer at 5 ' and 3 ' ends of RACE method, 5 '-E75A-F (5 '-
GCTTGACGGTTCCTCG-3 '), 5 '-E75A-R (5 '-GGGCAAGTGGAGCTCGAC-3 '), 3 '-E75A-F (5 '-
ATCGTCAACAACACTCCCAGTCCTG-3 '), 3 '-E75A-R (5 '-CAATCCAACTCTCCCTGCCCCTCCT-3 ') amplification
Method is expanded referring specifically to the method for rapid amplifying (RACE) kit of the end cDNA of precious biological Dalian Co., Ltd,
PCR response parameter are as follows: 95 DEG C of 180s denaturation, 94 DEG C of 30s, 60 DEG C of 12s, 72 DEG C of 600s, 72 DEG C are continued to extend 10min.Electrophoresis knot
Fruit shows that PCR amplification respectively obtains the band being consistent with expected size 2241bp.By PCR product through 1.2% Ago-Gel electricity
Swimming separation and glue recovery purifying target DNA, are attached with pGEM-TEasy carrier, are transferred to bacillus coli DH 5 alpha, select single bacterium
Culture is fallen, positive bacterium solution and sequencing is accredited as through bacterium colony PCR and correctly carries out next step vector construction.
Conclusion: the end green plant bug nuclear hormone receptor E75 5' and 3' respectively obtains about 400bp (Figure 1A) and 800bp (figure
1B) the segment of size.Recovered, sequencing and splicing, obtain the full length sequence of green plant bug green plant bug nuclear hormone receptor E75
(Fig. 2).
The full length sequence is as follows:
CCTTCATCCCTTGTGTACATCAATACACCACTATAAACCACACACCACCTTCACCACAAACCACCAGAA
AAACCGGTGATCACCGCCAACCCCGAAACTGCCTTCAACCGCGAAAACTCGATTTTCACTTCAGTTTCGCCGTCGAC
GAAAATTCACCAGTTTTCCGCCGTTGAACGTTTCATTTTGTTGCCGCTGAACGATTTTGTGCTTTTTTGGATTTTAC
ATCGGATTGCCGTGCCGCGAATGTGATGTCTCCGGATGGAGGTGATTTCCGGGTACCGCAGGCGCCGCCGCTGGTGT
CGAGCTCCACTTGCCCGCCGCCCCCGGCGCCGCAGATGCTCCCGCCCCCGCCGCCACCGCCGCCCCCACAGCACAAG
CACCGGACACAGCACGAGGAACCGTCAAGCTCCATGCCTGATCTCGAATTCGACGGGACGACCGTCCTCTGCAGGGT
TTGTGGTGACAAGGCCTCTGGATTCCACTATGGCGTGCACTCCTGCGAAGGATGCAAGGGTTTCTTCCGGCGGAGCA
TCCAGCAGAAGATCCAGTACCGCCCGTGCACCAAGAACCAGCAGTGCTCCATCCTGCGAATCAACAGGAACCGCTGC
CAGTACTGCAGACTCAAAAAATGCATCGCAGTTGGAATGTCCCGAGACGCGGTGAGATTCGGTCGCGTTCCCAAAAG
GGAAAAGGCGAGGATTTTGGCTGCCATGCAGCAAAGTACCAACTCGCGGTGCCAAGAAAAGGCGCTGGCGGCAGAGC
TCGAAGATGATCAGAGGCTGCTGCGCACAGTCATCAGAGCGCATCTGGACACGTGCGATTTTACTCGCGACAAAGTC
GAGCCTATGATCATCAGAGCCCGAGAACACCCTTCCTTCACCGCCTCACCTCCTACCTTGGCTTGCCCGCTCAATCC
CAATCCTCAACCACTGACGGGTCAGCAAGAACTCCTTCAGGACTTTTCCAAGAGATTCTCCCCCGCCATCCGCGGCG
TCGTCGAATTCGCCAAGCGAATCCCCGGATTCAGCCTCCTCTCCCAAGACGACCAGGTCACCCTCCTCAAAGCCGGC
GTGTTCGAGGTTCTACTCGTCCGCCTCGCTTGCATGTTCGACACGCAAAACAACAGCATGATTTGCCTCAATGGTCA
AGTCCTCAAGAGGGAGTCCATCCACAGCGGATCCAATGCCAGGTTTCTCATGGACTCCATGTTCGACTTCGCCGAGC
GCCTCAACTCCCTGAGGCTCACTGACGCAGAAATCGGCCTCTTCAGCTCGATAGTCGTCATTGCACCTGATCGGCCT
GGACTGAGAAACACAGAGCTCATTGAAATGATGCAGAATAAACTGAGAGACGGCCTCCACCTCATGATTGCCCAAAA
CCACCACAACCAACCGGGTTTGGCGCAAGAACTGATGAAGAAGATACCCGACCTGAGAACGTTGAACACGCTGCATA
GCGAGAAATTGCTTGCATTCAAAATGACTGAGCAGCAGCATCTGCAAGAGCAAAACCACCAGGCGAGCTTGGGAATG
TGGAGCAGTATGGTTGGTGAAGACGAGAGCAAAAGCCCCAGTGGCACGTCCTGGTCATCCAGCTCCGATGTCGCGAT
GGAAGAAGTTAAAAGTCCGCTGGGATCAGTCTCGAGCACGGAGTCCATGTGCTCGGGCGAAGTTGAGTACCACAGCA
ACCACGCCGCTTCTGCACCTCTTCTGGCGGCCACACTAGCTGGAGGCTGCCCCATGCGACACCGCCAGAACATGTGT
GATGACAGTAAAGACATCAAACCCATCATCATCCACAGACATGAAAGGACTTTTAGGAAGTTGGATTCTCCCAGTGA
TTCGGGGATTGAGTCTGGAACGGAAAAACATGAAAAGTTGACGTGTAGTGCTCCAACGTCTGTTTGCTCCAGTCCGA
GGTCTTCAGTTGAGGATAAGGATGATAGTCACCATATCGAAGATATGCCCGTTTTGAAACGAGTGCTCCAAGCGCCG
CCATTGTACGACACAAATTCACTGATGGATGAAGCGTACAAACCTCACAAGAAATTCAGAGCGTTGAGGAAAGAATG
CGGAGACTCGGAAAGCGACAACACAACAACGACGACATCCAGCGCCTGCTCGTCTCCACTCCTGTCGTCGTCCTTGT
CGTCGACGCACTCCACCCTCGCCAAGTCCCTCATGGAGAGCCCGAGGATGACGCCCGAGCAGATGAAGCGAACGGAC
ATCATCCACAGCTACATCATGCGAGGGGAAGGTTGCTGGCAGCAGCAGTCCGTCATCACGACGTCCTCGGCGTCTCG
GGGCAACGCCTACGTCATCGTCAACAACACTCCCAGTCCTGGACCATCCCTCTACGTCCAATCCAACTCTCCCTGCC
CCTCCTCCACCTCCTTCGTCGAGCTCCAAGTCGGCACTCAACCCCTCAATTTGTCCAAGAAAACGCCGCCTCCTTCC
CCCAGGCCGAAAGTCCTTTCACTCGAAGCCTAACCTACAAACTAACATAATAAACCCTAAAAATATTATGTTTAAGT
ACAAACTCAACAACTGAGGCAATGTGGTACAAAATAAACTTGCTCTTGTGATCTCTCCCTCCTCTGTAAATATATCC
ACCTTTATCTATTTGTGTATATAGAACCTTTCTTCTTCTGTAGTAACTTTTTTTCGTGTATTTTATCGTTAAAAACG
AACTATTCTAATGGTGCCACCCTTTTTGAACCCTATATTATAATTGAAAATACCAAATAATTTATTATAAATCCGAT
TTTTTAATTAAAATTTGAACAAATAAGTGTGGCGAAGAAAAACGCAGAAGTTTCATTTTTTGGTAAACTTGTTACTA
AGTGTTTTAAAAGTCGTAGGACCTTAGCAGTGTTGTGGTGTATCCTATCTATTCTTGAGAAAAAATATTTTCATCTT
ACGAATGCGAGTCATGGCCTTTTGTCAGACCTCAATCCAATTTCTCGTGTTCCAGTTATTTTTTTCGCATCTACGTT
TCTCAAAAAAACTTATATATCTATATTTGTTCTAGATATATTTGAAGAAAAAATTATGAATGTGATAATTCGATATA
GTGGGGCTTGCATTTTAAAAGAAAACATTTTTGTGTGTGTACATTTTAGTCTCATATAAATCAATAATGTAAAAAAA
AAAAAAAAAAAAAAAAAAAAA
Gene ORF frame includes the nucleotide of 2241bp, encodes 746 amino acid.
The amino acid sequence of coding is as follows:
MSPDGGDFRVPQAPPLVSSSTCPPPPAPQMLPPPPPPPPPQHKHRTQHEEPSSSMPDLEFDGTTVLCR
VCGDKASGFHYGVHSCEGCKGFFRRSIQQKIQYRPCTKNQQCSILRINRNRCQYCRLKKCIAVGMSRDAVRFGRVP
KREKARILAAMQQSTNSRCQEKALAAELEDDQRLLRTVIRAHLDTCDFTRDKVEPMIIRAREHPSFTASPPTLACP
LNPNPQPLTGQQELLQDFSKRFSPAIRGVVEFAKRIPGFSLLSQDDQVTLLKAGVFEVLLVRLACMFDTQNNSMIC
LNGQVLKRESIHSGSNARFLMDSMFDFAERLNSLRLTDAEIGLFSSIVVIAPDRPGLRNTELIEMMQNKLRDGLHL
MIAQNHHNQPGLAQELMKKIPDLRTLNTLHSEKLLAFKMTEQQHLQEQNHQASLGMWSSMVGEDESKSPSGTSWSS
SSDVAMEEVKSPLGSVSSTESMCSGEVEYHSNHAASAPLLAATLAGGCPMRHRQNMCDDSKDIKPIIIHRHERTFR
KLDSPSDSGIESGTEKHEKLTCSAPTSVCSSPRSSVEDKDDSHHIEDMPVLKRVLQAPPLYDTNSLMDEAYKPHKK
FRALRKECGDSESDNTTTTTSSACSSPLLSSSLSSTHSTLAKSLMESPRMTPEQMKRTDIIHSYIMRGEGCWQQQS
VITTSSASRGNAYVIVNNTPSPGPSLYVQSNSPCPSSTSFVELQVGTQPLNLSKKTPPPSPRPKVLSLEA*
Conserved sequence:
CCGTCGACGAAAATTCACCAGTTTTCCGCCGTTGAACGTTCCATTTTGTTGCCGCTGAACGATTTTGTG
TTTTTTTGGATTTTACATCGGATTGCCGTGCCGCGAATGTGATGCAAGTTTCGGGAGGTGATTTCCGGGTACCGCAG
GCGCCGCCGCTGGTGTCGAGCTCCACTTGCCCGCCGCCCCCGGCGCCGCAGATGCTCCCGCCCCCGCCGCCACCGCC
GCCCCCACAGCACAAGCACCGGACACAGCACGAGGAACCGTCAAGCTCCATGCCTGATCTCGAATTCGACGGGACGA
CCGTCCTCTGCAGGGTTTGTGGTGACAAGGCCTCTGGATTCCACTATGGCGTGCACTCCTGCGAAGGATGCAAGGGT
TTCTTCCGGCGGAGCATCCAGCAGAAGATCCAGTACCGCCCGTGCACCAAGAACCAGCAGTGCTCCATCCTGCGAAT
CAACAGGAACCGCTGCCAGTACTGCAGACTCAAAAAATGCATCGCAGTTGGAATGTCCCGAGACGCGGTGAGATTCG
GTCGCGTTCCCAAAAGGGAAAAGGCGAGGATTTTGGCTGCCATGCAGCAAAGTACCAACTCGCGGTGCCAAGAAAAG
GCGCTGGCGGCAGAGCTCGAAGATGATCAGAGGCTGCTGCGCACAGTCATCAGAGCGCATCTGGACACGTGCGATTT
TACTCGCGACAAAGTCGAGCCTATGATCATCAGAGCCCGAGAACACCCTTCCTTCACCGCCTCACCTCCTACCTTGG
CTTGCCCGCTCAATCCCAATCCTCAACCACTGACGGGTCAGCAAGAACTCCTTCAGGACTTTTCCAAGAGATTCTCC
CCCGCCATCCGCGGCGTCGTCGAATTCGCCAAGCGAATCCCCGGATTCAGCCTCCTCTCCCAAGACGACCAGGTCAC
CCTCCTCAAAGCCGGCGTGTTCGAGGTTCTACTCGTCCGCCTCGCTTGCATGTTCGACACGCAAAACAACAGCATGA
TTTGCCTCAATGGTCAAGTCCTCAAGAGGGAGTCCATCCACAGCGGATCCAATGCCAGGTTTCTCATGGACTCCATG
TTCGACTTCGCCGAGCGCCTCAACTCCCTGAGGCTCACTGACGCAGAAATCGGCCTCTTCAGCTCGATAGTCGTCAT
TGCACCTGATCGGCCTGGACTGAGAAACACAGAGCTCATTGAAATGATGCAGAATAAACTGAGAGACGGCCTCCACC
TCATGATTGCCCAAAACCACCACAACCAACCGGGTTTGGCGCAAGAACTGATGAAGAAGATACCCGACCTGAGAACG
TTGAACACGCTGCATAGCGAGAAATTGCTTGCATTCAAAATGACTGAGCAGCAGCATCTGCAAGAGCAAAACCACCA
GGCGAGCTTGGGAATGTGGAGCAGTATGGTTGGTGAAGACGAGAGCAAAAGCCCCAGTGGCACGTCCTGGTCATCCA
GCTCCGATGTCGCGATGGAAGAAGTTAAAAGTCCGCTGGGATCAGTCTCGAGCACGGAGTCCATGTGCTCGGGCGAA
GTTGAGTACCACAGCAACCACGCCGCTTCTGCACCTCTTCTGGCGGCCACACTAGCTGGAGGCTGCCCCATGCGACA
CCGCCAGAACATGTGTGATGACAGTAAAGACATCAAACCCATCATCATCCACAGACATGAAAGGACTTTTAGGAAGT
TGGATTCTCCCAGTGATTCGGGGATTGAGTCTGGAACGGAAAAACATGAAAAGTTGACGTGTAGTGCTCCAACGTCT
GTTTGCTCCAGTCCGAGGTCTTCAGTTGAGGATAAGGATGATAGTCACCATATCGAAGATATGCCCGTTTTGAAACG
AGTGCTCCAAGCGCCGCCATTGTACGACACAAATTCACTGATGGATGAAGCGTACAAACCTCACAAGAAATTCAGAG
CGTTGAGGAAAGAATGCGGAGACTCGGAAAGCGACAACACAACAACGACGACATCCAGCGCCTGCTCGTCTCCACTC
CTGTCGTCGTCCTTGTCGTCGACGCACTCCACCCTCGCCAAGTCCCTCATGGAGAGCCCGAGGATGACGCCCGAGCA
GATGAAGCGAACGGACATCATCCACAGCTACATCATGCGAGGGGAAGGTTGCTGGCAGCAGCAGTCCGTCATCACGA
CGTCCTCGGCGTCTCGGGGCAACGCCTACGTCATCGTCAACAACACTCCCAGTCCTGGACCATCCCTCTACGTCCAA
TCCAACTCTCCCTGCCCCTCCTCCACCTCCTTCGTCGAGCTCCAAGTCGGCACTCAACCCCTCAATTTGTCCAAGAA
AACGCCGCCTCCTTCCCCCAGGCCGAAAGTCCTTTCACTCGAAGCCTAACCTACAAAC
The building of embodiment 2:AlE75 prokaryotic expression vector
Prokaryotic expression vector construction method containing AlE75 gene carrier T are as follows: by the correct segment green plant bug of above-mentioned sequencing
Green plant bug nuclear hormone receptor E75 is connected into the target gene of pMD18-T carrier and pCzn1 carrier uses restriction enzyme
Ndel and Xbal double digestion carries out large fragment connection (Fig. 2).Digestion system are as follows: 2 kinds of restriction enzymes (Sigma company) are each
0.25 μ l, 10 × Buffer buffer, 1.0 μ l, the 3 μ l of genetic fragment with suitable restriction enzyme site, uses ddH2O complements to 10 μ l,
37 DEG C of water-bath 3h.Linked system are as follows: digestion 5.0 μ l of carrier after the recovery, 10.0 μ l, T4DNA ligase (Sigma of genetic fragment
Company) 1.0 μ l, 10 × Buffer buffer, 2.0 μ l, uses ddH2O complements to 20 μ l, 16 DEG C of 12~16h of reaction.Connection product turns
PCR screening positive clone is used after changing Escherichia coli TOP10.It will identify that correct recombinant plasmid pCzn1-AlE75 is transformed into greatly
It is applied to after enterobacteria Arctic Express competent cell on the plate containing kanamycins (50 μ g/mL), 37 DEG C, 220r/
Min overnight incubation；Monoclonal is selected on the plate of next day in post-conversion to LB culture medium (containing 50 μ g/ml kanamycins, 10mL)
In, 37 DEG C, 250r/min overnight incubation；Bacterium solution is inoculated into LB liquid medium (containing 50 μ g/mL cards in 1% ratio by next day
That mycin) in, 2~3h is cultivated in 37 DEG C of shaking tables to be added when the OD600 of bacterium solution reaches between 0.6~0.8 to remaining culture
Enter IPTG to final concentration of 0.5mM, reduces cultivation temperature to 11 DEG C, 220rpm shaking overnight, the expression of induced fusion albumen.From
The a small amount of thallus of the heart, takes supernatant to precipitate respectively, and 2 isometric × SDS is added after adding appropriate TE solution (pH 8.0), concussion to suspend
Buffer, 100 DEG C of 5min, 12%SDS-PAGE electrophoresis detection recombinate the expression (Fig. 3) of AlE75.
Conclusion: through 11 DEG C, after 0.5mM IPTG induction AlE75 gene in pCzn1 carrier can high efficient expression, mainly with packet
Contain body form to exist.
The purifying of the denaturation of embodiment 3:AlE75 fusion protein, renaturation and albumen
The denaturation and renaturation of inclusion body protein are carried out in the steps below: bacterial sediment is resuspended in 20ml lysis buffer
(sigma company, 20mM Tris-HCl containing 1mM PMSF and bacteria protease inhibitor
Cocktail, pH 8.0), ultrasonication (power 400W, work 4sec, interval 8sec, total 20min)；By the thin of ultrasonication
4 DEG C of 10000g of cellular lysate liquid are centrifuged 20min, collect precipitating；Use inclusion body cleaning solution (20mM Tris, 1mM EDTA, 2M urine
Element, 1M NaCl, 1%Triton X-100, pH8.0) washing inclusion body 3 times；With dissolution buffer (20mM Tris, 5mM
DTT, 8M urea pH8.0), inclusion body is dissolved by a certain percentage, and 4 degree stand overnight；Room temperature, 15000rpm are centrifuged 15min；It will be upper
Solution is stated to be added dropwise in 20mM Tris-HCl 100mM NaCl Buffer pH8.0 (sigma company) buffer, it is gradually terraced at double
Degree dilution is slowly stirred, and protein solution loading bag filter is dialysed in 20mM Tris-HCl, 100mM NaCl, pH8.0 solution
Overnight.The albumen that renaturation is obtained is dissolved in PBS buffer solution (pH 7.4) after being concentrated by ultrafiltration and obtains forgiving liquid solution.
The purifying of AlE75 albumen is carried out in the steps below: being utilized low pressure chromatography system, is forgiven liquid solution with 0.5ml/min
The Ni-IDA-Sepharose CL-6B affinity column (sigma that flow velocity loading is pre-equilibrated to Ni-IDA Binding-Buffer
Company)；With Ni-IDA Binding-Buffer with the flushing of 0.5ml/min flow velocity, until efflux OD280 value reaches baseline；With
Ni-IDA Washing-Buffer (20mM Tris-HCl, 20mM imidazoles, 0.15M NaCl, pH8.0) is with the punching of 1ml/min flow velocity
It washes, until efflux OD280 value reaches baseline；With Ni-IDA Elution-Buffer (20mM Tris-HCl, 250mM imidazoles,
0.15M NaCl, pH8.0) with 1ml/min flow velocity elution destination protein, collect efflux；The protein solution of above-mentioned collection is added
In bag filter, using 20mM Tris-HCl, 0.10M NaCl, pH8.0 carry out dialysed overnight；12%SDS-PAGE analysis is carried out,
With protein quantification kit measurement protein concentration (Fig. 4).
Conclusion: after albuminous degeneration and renaturation, AlE75 albumen can be successfully obtained, size is about 75KD, the protein concentration
For 0.4mg/mL.
The above is only a preferred embodiment of the present invention, it should be pointed out that: for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>Jiangsu Province Agriculture Science Institute
<120>a kind of green plant bug nuclear hormone receptor E75, its coded sequence, carrier and bacterial strain
<130> SG20170509001
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 2241
<212> DNA
<213>green plant bug nuclear hormone receptor E75
<220>
<221> CDS
<222> (1)..(2241)
<400> 1
atg tct ccg gat gga ggt gat ttc cgg gta ccg cag gcg ccg ccg ctg 48
Met Ser Pro Asp Gly Gly Asp Phe Arg Val Pro Gln Ala Pro Pro Leu
1 5 10 15
gtg tcg agc tcc act tgc ccg ccg ccc ccg gcg ccg cag atg ctc ccg 96
Val Ser Ser Ser Thr Cys Pro Pro Pro Pro Ala Pro Gln Met Leu Pro
20 25 30
ccc ccg ccg cca ccg ccg ccc cca cag cac aag cac cgg aca cag cac 144
Pro Pro Pro Pro Pro Pro Pro Pro Gln His Lys His Arg Thr Gln His
35 40 45
gag gaa ccg tca agc tcc atg cct gat ctc gaa ttc gac ggg acg acc 192
Glu Glu Pro Ser Ser Ser Met Pro Asp Leu Glu Phe Asp Gly Thr Thr
50 55 60
gtc ctc tgc agg gtt tgt ggt gac aag gcc tct gga ttc cac tat ggc 240
Val Leu Cys Arg Val Cys Gly Asp Lys Ala Ser Gly Phe His Tyr Gly
65 70 75 80
gtg cac tcc tgc gaa gga tgc aag ggt ttc ttc cgg cgg agc atc cag 288
Val His Ser Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Ile Gln
85 90 95
cag aag atc cag tac cgc ccg tgc acc aag aac cag cag tgc tcc atc 336
Gln Lys Ile Gln Tyr Arg Pro Cys Thr Lys Asn Gln Gln Cys Ser Ile
100 105 110
ctg cga atc aac agg aac cgc tgc cag tac tgc aga ctc aaa aaa tgc 384
Leu Arg Ile Asn Arg Asn Arg Cys Gln Tyr Cys Arg Leu Lys Lys Cys
115 120 125
atc gca gtt gga atg tcc cga gac gcg gtg aga ttc ggt cgc gtt ccc 432
Ile Ala Val Gly Met Ser Arg Asp Ala Val Arg Phe Gly Arg Val Pro
130 135 140
aaa agg gaa aag gcg agg att ttg gct gcc atg cag caa agt acc aac 480
Lys Arg Glu Lys Ala Arg Ile Leu Ala Ala Met Gln Gln Ser Thr Asn
145 150 155 160
tcg cgg tgc caa gaa aag gcg ctg gcg gca gag ctc gaa gat gat cag 528
Ser Arg Cys Gln Glu Lys Ala Leu Ala Ala Glu Leu Glu Asp Asp Gln
165 170 175
agg ctg ctg cgc aca gtc atc aga gcg cat ctg gac acg tgc gat ttt 576
Arg Leu Leu Arg Thr Val Ile Arg Ala His Leu Asp Thr Cys Asp Phe
180 185 190
act cgc gac aaa gtc gag cct atg atc atc aga gcc cga gaa cac cct 624
Thr Arg Asp Lys Val Glu Pro Met Ile Ile Arg Ala Arg Glu His Pro
195 200 205
tcc ttc acc gcc tca cct cct acc ttg gct tgc ccg ctc aat ccc aat 672
Ser Phe Thr Ala Ser Pro Pro Thr Leu Ala Cys Pro Leu Asn Pro Asn
210 215 220
cct caa cca ctg acg ggt cag caa gaa ctc ctt cag gac ttt tcc aag 720
Pro Gln Pro Leu Thr Gly Gln Gln Glu Leu Leu Gln Asp Phe Ser Lys
225 230 235 240
aga ttc tcc ccc gcc atc cgc ggc gtc gtc gaa ttc gcc aag cga atc 768
Arg Phe Ser Pro Ala Ile Arg Gly Val Val Glu Phe Ala Lys Arg Ile
245 250 255
ccc gga ttc agc ctc ctc tcc caa gac gac cag gtc acc ctc ctc aaa 816
Pro Gly Phe Ser Leu Leu Ser Gln Asp Asp Gln Val Thr Leu Leu Lys
260 265 270
gcc ggc gtg ttc gag gtt cta ctc gtc cgc ctc gct tgc atg ttc gac 864
Ala Gly Val Phe Glu Val Leu Leu Val Arg Leu Ala Cys Met Phe Asp
275 280 285
acg caa aac aac agc atg att tgc ctc aat ggt caa gtc ctc aag agg 912
Thr Gln Asn Asn Ser Met Ile Cys Leu Asn Gly Gln Val Leu Lys Arg
290 295 300
gag tcc atc cac agc gga tcc aat gcc agg ttt ctc atg gac tcc atg 960
Glu Ser Ile His Ser Gly Ser Asn Ala Arg Phe Leu Met Asp Ser Met
305 310 315 320
ttc gac ttc gcc gag cgc ctc aac tcc ctg agg ctc act gac gca gaa 1008
Phe Asp Phe Ala Glu Arg Leu Asn Ser Leu Arg Leu Thr Asp Ala Glu
325 330 335
atc ggc ctc ttc agc tcg ata gtc gtc att gca cct gat cgg cct gga 1056
Ile Gly Leu Phe Ser Ser Ile Val Val Ile Ala Pro Asp Arg Pro Gly
340 345 350
ctg aga aac aca gag ctc att gaa atg atg cag aat aaa ctg aga gac 1104
Leu Arg Asn Thr Glu Leu Ile Glu Met Met Gln Asn Lys Leu Arg Asp
355 360 365
ggc ctc cac ctc atg att gcc caa aac cac cac aac caa ccg ggt ttg 1152
Gly Leu His Leu Met Ile Ala Gln Asn His His Asn Gln Pro Gly Leu
370 375 380
gcg caa gaa ctg atg aag aag ata ccc gac ctg aga acg ttg aac acg 1200
Ala Gln Glu Leu Met Lys Lys Ile Pro Asp Leu Arg Thr Leu Asn Thr
385 390 395 400
ctg cat agc gag aaa ttg ctt gca ttc aaa atg act gag cag cag cat 1248
Leu His Ser Glu Lys Leu Leu Ala Phe Lys Met Thr Glu Gln Gln His
405 410 415
ctg caa gag caa aac cac cag gcg agc ttg gga atg tgg agc agt atg 1296
Leu Gln Glu Gln Asn His Gln Ala Ser Leu Gly Met Trp Ser Ser Met
420 425 430
gtt ggt gaa gac gag agc aaa agc ccc agt ggc acg tcc tgg tca tcc 1344
Val Gly Glu Asp Glu Ser Lys Ser Pro Ser Gly Thr Ser Trp Ser Ser
435 440 445
agc tcc gat gtc gcg atg gaa gaa gtt aaa agt ccg ctg gga tca gtc 1392
Ser Ser Asp Val Ala Met Glu Glu Val Lys Ser Pro Leu Gly Ser Val
450 455 460
tcg agc acg gag tcc atg tgc tcg ggc gaa gtt gag tac cac agc aac 1440
Ser Ser Thr Glu Ser Met Cys Ser Gly Glu Val Glu Tyr His Ser Asn
465 470 475 480
cac gcc gct tct gca cct ctt ctg gcg gcc aca cta gct gga ggc tgc 1488
His Ala Ala Ser Ala Pro Leu Leu Ala Ala Thr Leu Ala Gly Gly Cys
485 490 495
ccc atg cga cac cgc cag aac atg tgt gat gac agt aaa gac atc aaa 1536
Pro Met Arg His Arg Gln Asn Met Cys Asp Asp Ser Lys Asp Ile Lys
500 505 510
ccc atc atc atc cac aga cat gaa agg act ttt agg aag ttg gat tct 1584
Pro Ile Ile Ile His Arg His Glu Arg Thr Phe Arg Lys Leu Asp Ser
515 520 525
ccc agt gat tcg ggg att gag tct gga acg gaa aaa cat gaa aag ttg 1632
Pro Ser Asp Ser Gly Ile Glu Ser Gly Thr Glu Lys His Glu Lys Leu
530 535 540
acg tgt agt gct cca acg tct gtt tgc tcc agt ccg agg tct tca gtt 1680
Thr Cys Ser Ala Pro Thr Ser Val Cys Ser Ser Pro Arg Ser Ser Val
545 550 555 560
gag gat aag gat gat agt cac cat atc gaa gat atg ccc gtt ttg aaa 1728
Glu Asp Lys Asp Asp Ser His His Ile Glu Asp Met Pro Val Leu Lys
565 570 575
cga gtg ctc caa gcg ccg cca ttg tac gac aca aat tca ctg atg gat 1776
Arg Val Leu Gln Ala Pro Pro Leu Tyr Asp Thr Asn Ser Leu Met Asp
580 585 590
gaa gcg tac aaa cct cac aag aaa ttc aga gcg ttg agg aaa gaa tgc 1824
Glu Ala Tyr Lys Pro His Lys Lys Phe Arg Ala Leu Arg Lys Glu Cys
595 600 605
gga gac tcg gaa agc gac aac aca aca acg acg aca tcc agc gcc tgc 1872
Gly Asp Ser Glu Ser Asp Asn Thr Thr Thr Thr Thr Ser Ser Ala Cys
610 615 620
tcg tct cca ctc ctg tcg tcg tcc ttg tcg tcg acg cac tcc acc ctc 1920
Ser Ser Pro Leu Leu Ser Ser Ser Leu Ser Ser Thr His Ser Thr Leu
625 630 635 640
gcc aag tcc ctc atg gag agc ccg agg atg acg ccc gag cag atg aag 1968
Ala Lys Ser Leu Met Glu Ser Pro Arg Met Thr Pro Glu Gln Met Lys
645 650 655
cga acg gac atc atc cac agc tac atc atg cga ggg gaa ggt tgc tgg 2016
Arg Thr Asp Ile Ile His Ser Tyr Ile Met Arg Gly Glu Gly Cys Trp
660 665 670
cag cag cag tcc gtc atc acg acg tcc tcg gcg tct cgg ggc aac gcc 2064
Gln Gln Gln Ser Val Ile Thr Thr Ser Ser Ala Ser Arg Gly Asn Ala
675 680 685
tac gtc atc gtc aac aac act ccc agt cct gga cca tcc ctc tac gtc 2112
Tyr Val Ile Val Asn Asn Thr Pro Ser Pro Gly Pro Ser Leu Tyr Val
690 695 700
caa tcc aac tct ccc tgc ccc tcc tcc acc tcc ttc gtc gag ctc caa 2160
Gln Ser Asn Ser Pro Cys Pro Ser Ser Thr Ser Phe Val Glu Leu Gln
705 710 715 720
gtc ggc act caa ccc ctc aat ttg tcc aag aaa acg ccg cct cct tcc 2208
Val Gly Thr Gln Pro Leu Asn Leu Ser Lys Lys Thr Pro Pro Pro Ser
725 730 735
ccc agg ccg aaa gtc ctt tca ctc gaa gcc taa 2241
Pro Arg Pro Lys Val Leu Ser Leu Glu Ala
740 745
<210> 2
<211> 746
<212> PRT
<213>green plant bug nuclear hormone receptor E75
<400> 2
Met Ser Pro Asp Gly Gly Asp Phe Arg Val Pro Gln Ala Pro Pro Leu
1 5 10 15
Val Ser Ser Ser Thr Cys Pro Pro Pro Pro Ala Pro Gln Met Leu Pro
20 25 30
Pro Pro Pro Pro Pro Pro Pro Pro Gln His Lys His Arg Thr Gln His
35 40 45
Glu Glu Pro Ser Ser Ser Met Pro Asp Leu Glu Phe Asp Gly Thr Thr
50 55 60
Val Leu Cys Arg Val Cys Gly Asp Lys Ala Ser Gly Phe His Tyr Gly
65 70 75 80
Val His Ser Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Ile Gln
85 90 95
Gln Lys Ile Gln Tyr Arg Pro Cys Thr Lys Asn Gln Gln Cys Ser Ile
100 105 110
Leu Arg Ile Asn Arg Asn Arg Cys Gln Tyr Cys Arg Leu Lys Lys Cys
115 120 125
Ile Ala Val Gly Met Ser Arg Asp Ala Val Arg Phe Gly Arg Val Pro
130 135 140
Lys Arg Glu Lys Ala Arg Ile Leu Ala Ala Met Gln Gln Ser Thr Asn
145 150 155 160
Ser Arg Cys Gln Glu Lys Ala Leu Ala Ala Glu Leu Glu Asp Asp Gln
165 170 175
Arg Leu Leu Arg Thr Val Ile Arg Ala His Leu Asp Thr Cys Asp Phe
180 185 190
Thr Arg Asp Lys Val Glu Pro Met Ile Ile Arg Ala Arg Glu His Pro
195 200 205
Ser Phe Thr Ala Ser Pro Pro Thr Leu Ala Cys Pro Leu Asn Pro Asn
210 215 220
Pro Gln Pro Leu Thr Gly Gln Gln Glu Leu Leu Gln Asp Phe Ser Lys
225 230 235 240
Arg Phe Ser Pro Ala Ile Arg Gly Val Val Glu Phe Ala Lys Arg Ile
245 250 255
Pro Gly Phe Ser Leu Leu Ser Gln Asp Asp Gln Val Thr Leu Leu Lys
260 265 270
Ala Gly Val Phe Glu Val Leu Leu Val Arg Leu Ala Cys Met Phe Asp
275 280 285
Thr Gln Asn Asn Ser Met Ile Cys Leu Asn Gly Gln Val Leu Lys Arg
290 295 300
Glu Ser Ile His Ser Gly Ser Asn Ala Arg Phe Leu Met Asp Ser Met
305 310 315 320
Phe Asp Phe Ala Glu Arg Leu Asn Ser Leu Arg Leu Thr Asp Ala Glu
325 330 335
Ile Gly Leu Phe Ser Ser Ile Val Val Ile Ala Pro Asp Arg Pro Gly
340 345 350
Leu Arg Asn Thr Glu Leu Ile Glu Met Met Gln Asn Lys Leu Arg Asp
355 360 365
Gly Leu His Leu Met Ile Ala Gln Asn His His Asn Gln Pro Gly Leu
370 375 380
Ala Gln Glu Leu Met Lys Lys Ile Pro Asp Leu Arg Thr Leu Asn Thr
385 390 395 400
Leu His Ser Glu Lys Leu Leu Ala Phe Lys Met Thr Glu Gln Gln His
405 410 415
Leu Gln Glu Gln Asn His Gln Ala Ser Leu Gly Met Trp Ser Ser Met
420 425 430
Val Gly Glu Asp Glu Ser Lys Ser Pro Ser Gly Thr Ser Trp Ser Ser
435 440 445
Ser Ser Asp Val Ala Met Glu Glu Val Lys Ser Pro Leu Gly Ser Val
450 455 460
Ser Ser Thr Glu Ser Met Cys Ser Gly Glu Val Glu Tyr His Ser Asn
465 470 475 480
His Ala Ala Ser Ala Pro Leu Leu Ala Ala Thr Leu Ala Gly Gly Cys
485 490 495
Pro Met Arg His Arg Gln Asn Met Cys Asp Asp Ser Lys Asp Ile Lys
500 505 510
Pro Ile Ile Ile His Arg His Glu Arg Thr Phe Arg Lys Leu Asp Ser
515 520 525
Pro Ser Asp Ser Gly Ile Glu Ser Gly Thr Glu Lys His Glu Lys Leu
530 535 540
Thr Cys Ser Ala Pro Thr Ser Val Cys Ser Ser Pro Arg Ser Ser Val
545 550 555 560
Glu Asp Lys Asp Asp Ser His His Ile Glu Asp Met Pro Val Leu Lys
565 570 575
Arg Val Leu Gln Ala Pro Pro Leu Tyr Asp Thr Asn Ser Leu Met Asp
580 585 590
Glu Ala Tyr Lys Pro His Lys Lys Phe Arg Ala Leu Arg Lys Glu Cys
595 600 605
Gly Asp Ser Glu Ser Asp Asn Thr Thr Thr Thr Thr Ser Ser Ala Cys
610 615 620
Ser Ser Pro Leu Leu Ser Ser Ser Leu Ser Ser Thr His Ser Thr Leu
625 630 635 640
Ala Lys Ser Leu Met Glu Ser Pro Arg Met Thr Pro Glu Gln Met Lys
645 650 655
Arg Thr Asp Ile Ile His Ser Tyr Ile Met Arg Gly Glu Gly Cys Trp
660 665 670
Gln Gln Gln Ser Val Ile Thr Thr Ser Ser Ala Ser Arg Gly Asn Ala
675 680 685
Tyr Val Ile Val Asn Asn Thr Pro Ser Pro Gly Pro Ser Leu Tyr Val
690 695 700
Gln Ser Asn Ser Pro Cys Pro Ser Ser Thr Ser Phe Val Glu Leu Gln
705 710 715 720
Val Gly Thr Gln Pro Leu Asn Leu Ser Lys Lys Thr Pro Pro Pro Ser
725 730 735
Pro Arg Pro Lys Val Leu Ser Leu Glu Ala
740 745
<210> 3
<211> 2360
<212> DNA
<213>conserved sequence of AlE75 gene
<400> 3
ccgtcgacga aaattcacca gttttccgcc gttgaacgtt ccattttgtt gccgctgaac 60
gattttgtgt ttttttggat tttacatcgg attgccgtgc cgcgaatgtg atgcaagttt 120
cgggaggtga tttccgggta ccgcaggcgc cgccgctggt gtcgagctcc acttgcccgc 180
cgcccccggc gccgcagatg ctcccgcccc cgccgccacc gccgccccca cagcacaagc 240
accggacaca gcacgaggaa ccgtcaagct ccatgcctga tctcgaattc gacgggacga 300
ccgtcctctg cagggtttgt ggtgacaagg cctctggatt ccactatggc gtgcactcct 360
gcgaaggatg caagggtttc ttccggcgga gcatccagca gaagatccag taccgcccgt 420
gcaccaagaa ccagcagtgc tccatcctgc gaatcaacag gaaccgctgc cagtactgca 480
gactcaaaaa atgcatcgca gttggaatgt cccgagacgc ggtgagattc ggtcgcgttc 540
ccaaaaggga aaaggcgagg attttggctg ccatgcagca aagtaccaac tcgcggtgcc 600
aagaaaaggc gctggcggca gagctcgaag atgatcagag gctgctgcgc acagtcatca 660
gagcgcatct ggacacgtgc gattttactc gcgacaaagt cgagcctatg atcatcagag 720
cccgagaaca cccttccttc accgcctcac ctcctacctt ggcttgcccg ctcaatccca 780
atcctcaacc actgacgggt cagcaagaac tccttcagga cttttccaag agattctccc 840
ccgccatccg cggcgtcgtc gaattcgcca agcgaatccc cggattcagc ctcctctccc 900
aagacgacca ggtcaccctc ctcaaagccg gcgtgttcga ggttctactc gtccgcctcg 960
cttgcatgtt cgacacgcaa aacaacagca tgatttgcct caatggtcaa gtcctcaaga 1020
gggagtccat ccacagcgga tccaatgcca ggtttctcat ggactccatg ttcgacttcg 1080
ccgagcgcct caactccctg aggctcactg acgcagaaat cggcctcttc agctcgatag 1140
tcgtcattgc acctgatcgg cctggactga gaaacacaga gctcattgaa atgatgcaga 1200
ataaactgag agacggcctc cacctcatga ttgcccaaaa ccaccacaac caaccgggtt 1260
tggcgcaaga actgatgaag aagatacccg acctgagaac gttgaacacg ctgcatagcg 1320
agaaattgct tgcattcaaa atgactgagc agcagcatct gcaagagcaa aaccaccagg 1380
cgagcttggg aatgtggagc agtatggttg gtgaagacga gagcaaaagc cccagtggca 1440
cgtcctggtc atccagctcc gatgtcgcga tggaagaagt taaaagtccg ctgggatcag 1500
tctcgagcac ggagtccatg tgctcgggcg aagttgagta ccacagcaac cacgccgctt 1560
ctgcacctct tctggcggcc acactagctg gaggctgccc catgcgacac cgccagaaca 1620
tgtgtgatga cagtaaagac atcaaaccca tcatcatcca cagacatgaa aggactttta 1680
ggaagttgga ttctcccagt gattcgggga ttgagtctgg aacggaaaaa catgaaaagt 1740
tgacgtgtag tgctccaacg tctgtttgct ccagtccgag gtcttcagtt gaggataagg 1800
atgatagtca ccatatcgaa gatatgcccg ttttgaaacg agtgctccaa gcgccgccat 1860
tgtacgacac aaattcactg atggatgaag cgtacaaacc tcacaagaaa ttcagagcgt 1920
tgaggaaaga atgcggagac tcggaaagcg acaacacaac aacgacgaca tccagcgcct 1980
gctcgtctcc actcctgtcg tcgtccttgt cgtcgacgca ctccaccctc gccaagtccc 2040
tcatggagag cccgaggatg acgcccgagc agatgaagcg aacggacatc atccacagct 2100
acatcatgcg aggggaaggt tgctggcagc agcagtccgt catcacgacg tcctcggcgt 2160
ctcggggcaa cgcctacgtc atcgtcaaca acactcccag tcctggacca tccctctacg 2220
tccaatccaa ctctccctgc ccctcctcca cctccttcgt cgagctccaa gtcggcactc 2280
aacccctcaa tttgtccaag aaaacgccgc ctccttcccc caggccgaaa gtcctttcac 2340
tcgaagccta acctacaaac 2360
<210> 4
<211> 22
<212> DNA
<213> AlE75-F
<400> 4
cattatggwg tytayagytg tg 22
<210> 5
<211> 22
<212> DNA
<213> AlE75-R
<400> 5
aghagttcat tccavcctgc tc 22
<210> 6
<211> 16
<212> DNA
<213> 5′- E75A -F
<400> 6
gcttgacggt tcctcg 16
<210> 7
<211> 18
<212> DNA
<213> 5′- E75A -R
<400> 7
gggcaagtgg agctcgac 18
<210> 8
<211> 25
<212> DNA
<213> 3′- E75A -F
<400> 8
atcgtcaaca acactcccag tcctg 25
<210> 9
<211> 25
<212> DNA
<213> 3′- E75A -R
<400> 9
caatccaact ctccctgccc ctcct 25
Claims (10)
1. a kind of for encoding the DNA sequence dna of green plant bug nuclear hormone receptor E75, base sequence such as SEQ ID NO:1 institute
Show.
2. a kind of green plant bug nuclear hormone receptor E75, which is characterized in that its amino acid sequence is as shown in SEQ ID NO:2.
3. being carried containing the recombinant expression described in claim 1 for encoding the DNA sequence dna of green plant bug nuclear hormone receptor E75
Body, transgenic cell system or transgenosis recombinant bacterium.
4. recombinant expression carrier according to claim 3, which is characterized in that DNA sequence dna described in claim 1 to be inserted into
To the recombinant expression carrier for expressing green plant bug nuclear hormone receptor E75 obtained in coli expression carrier.
5. recombinant expression carrier according to claim 4, which is characterized in that the coli expression carrier is pCzn1.
6. transgenosis recombinant bacterium according to claim 3, which is characterized in that the recombinant bacterium is by claim 4 or 5 institutes
The recombinant expression carrier stated imports in Escherichia coli, and screening obtains transgenosis recombinant bacterium.
7. transgenosis recombinant bacterium according to claim 6, which is characterized in that the Escherichia coli are Arctic
Express。
8. DNA sequence dna described in claim 1, recombinant expression carrier as claimed in claim 3, transgenic cell system turn base
Because of application of the recombinant bacterium in production green plant bug nuclear hormone receptor E75.
9. a kind of preparation method of green plant bug nuclear hormone receptor E75 is transgenosis weight described in culture claim 3 or 6
Group bacterium obtains green plant bug nuclear hormone receptor E75.
10. a kind of preparation method of green plant bug nuclear hormone receptor E75, which comprises the following steps:
1), for encoding the amplification of green plant bug nuclear hormone receptor E75 gene, the E75 gene order such as claim 1 institute
Shown in the SEQ ID NO:1 stated；
2), the Prokaryotic expression vector construction of green plant bug nuclear hormone receptor E75 gene；By the prokaryotic expression carrier of building big
It is converted in enterobacteria, is induced using IPTG and obtain green plant bug nuclear hormone receptor E75；
3), the denaturation and renaturation of green plant bug nuclear hormone receptor E75；
4), the purifying of green plant bug nuclear hormone receptor E75；
PCR screening positive clone is used after the step 2 conversion Escherichia coli TOP10.
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|---|---|---|---|---|
| CN103789319A (en) * | 2014-01-15 | 2014-05-14 | 江苏省农业科学院 | Ultraspiracle protein of green plant bug, coding sequence, carrier and strain thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103789319A (en) * | 2014-01-15 | 2014-05-14 | 江苏省农业科学院 | Ultraspiracle protein of green plant bug, coding sequence, carrier and strain thereof |
Non-Patent Citations (2)
| Title |
|---|
| Ecdysone receptor isoform-B mediates soluble trehalase expression to regulate growth and development in the mirid bug, Apolygus lucorum (Meyer-Dur);Y.-A. Tan et al.;《Insect Molecular Biology》;20150903;第24卷(第6期);第611-623页 * |
| The nuclear hormone receptor E75A regulates vitellogenin gene (Al-Vg) expression in the mirid bug Apolygus lucorum;Y.-A. Tan et al.;《Insect Molecular Biology》;20171129;第27卷(第2期);第188-197页 * |
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