CN1514003A - Construction method using detoxiase gene as stable expression system in silkworm - Google Patents
Construction method using detoxiase gene as stable expression system in silkworm Download PDFInfo
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Abstract
A process for configuring the stable expression system of detoxicating gene in silkworm includes designing and synthesizing the oligodeoxyribonucleotide primer, linking its cDNA or DNA sequence to the promoter and terminator of silkworm silkprotein L chain to configure fusion gene, inserting them between two opposite repetitive terminal sequences of transposable factor piggyBac to obtain recombinant transposon, configuring transposase gene expression carrier, using said transposon and expression carrier to transfect silkworm ova, and screening and culturing the young silkworm to obtain the transgenic silkworm able to stably express the external detoxicating enzyme lipase BI.
Description
Technical field
The present invention relates to the expression of detoxifying gene in transgenic bombyx mori, more specifically relate to the construction process of a kind of detoxifying gene stably express system in silkworm.
Background technology
Pesticide intoxication and agricultural chemicals more and more receive global concern to the destruction of ecotope.The whole world in 1997 only pesticide intoxication just has 500,000, and example surplus nineteen ninety-five China's pesticide intoxication incident 100,000 wherein mainly is the hypertoxic type pesticide intoxication.Except acute poisoning, pesticide residue and also more and more big to the pollution of soil, water body, air etc. produce delayed neurotoxicity, disturbed ecological balance.For pesticide intoxication and pesticidal contamination, mainly be to adopt coromegine and Pyridine a-Aldoxime Methiodide class medicine at present, these medicines have certain side effect.
Utilization is separated toxenzyme the pesticide residue in pesticide intoxication, crop and the environment is decomposed, and is a new approach.In recent years, quite active for the research of separating toxenzyme in microorganism and the resistant insects, developed into the molecular biology level, though Chinese scholars has been carried out multiple detoxifying gene order-checking, clone and transgenic research, especially more careful to the research of lipase B1, and carried out successful expression at intestinal bacteria, blue-green algae, gram-positive microorganism, Flavobacterium etc., but it is glycosylation modified that the lipase B1 that utilizes these microorganisms and algae to express can not carry out, and brings suitable difficulty for separation, Purification after expressing., be present numerous investigator and investor's focus of attention, but yet there are no as the polypeptide of the synthetic economically valuable of bio-reactor, albumen etc. with silkworm with the report of silkworm as foreign protein genes expression of receptor detoxifying gene.
Summary of the invention
The object of the present invention is to provide a kind of detoxifying gene (lipase B1) construction process of stably express in silkworm.It is easy to implement the method, easy and simple to handle, transformation efficiency is high, has obtained recombinant silkworm lipase B1 albumen and transgenic bombyx mori.
The present invention expresses lipase B1 expression of gene system in silkworm construction process may further comprise the steps:
(1) clone of design of primers and a plurality of genes
Synthetic 14 oligodeoxynucleotides of design see the following form as primer:
| ??A3-0 | ??????5’tgg?atc?cgc?ggc?gcg?tta?cca?tat?atg?3’ | ||
| ??A3-1 | ??????5’gcg?ata?tca?cgc?cct?gat?gg3’ | ||
| ??A3-2 | ??????5’gcg?gat?ccc?atc?ttg?aat?tag?tct?gc3’ | ||
| ??A3-3 | ???23 | ??????5’agtccatgggccttccgacgatc3’ | |
| ??L-1 | ???32 | ??????Ctggatccataacagaccactaaaatgaagcc | |
| ??L-2 | ???26 | ??????Cgaagcttgtcgcgtcattaccgttg | |
| ??L-3 | ???30 | ??????Cgtctagactcgaggtggtgcctatcccac | |
| ??L-4 | ???29 | ??????Gcggatccaatcggtatatactatacagg | |
| ??L-5 | ???20 | ??????Cgaagcttagttgctaatgc | |
| ??L-6 | ???22 | ??????Ctggtaccatgtacccactgtc | |
| ??Tra-1 | ??????5’gcg?gat?cct?ctt?tag?acg?atg3’ | ||
| ??Tra-2 | ??????5’tac?tgc?agg?tca?tca?cag?3’ | ||
| ??Gfp-1 | ???24 | ??????5′-gtggatccgcggccgctttacttg-3′ | |
| ??Gfp-2 | ??????Acggatccatggtgagcaagggc |
Take out posterior division of silkgland from 5 ages the children silkworm and extract total DNA.With this total DNA is template, carry out pcr amplification reaction with above-mentioned primer respectively, again with corresponding restriction enzyme digestion, be connected with the plasmid Bluescript M13 (pBS) that cuts processing through same enzyme, the screening recon, obtained the clone of each pcr amplified fragment, clone's dna fragmentation is respectively: the 1. different lengths dna sequence dna of tenuigenin actin gene promotor, what have does not contain signal peptide sequence, be fused into helper plasmid with the transposase coding region of transposon piggyBac, what have contains signal peptide sequence and part coding region, can merge with reporter gene-multitube jellyfish green fluorescence protein gene gfp; 2. domestic silk core albumen light chain (Light chain) gene promoter; 3. domestic silk core albumen light chain (Light chain) gene terminator sequence; 4. transposase coding region partial sequence; 5. green fluorescence protein gene coding region partial sequence.
(2) cDNA of detoxifying gene or its aminoacid sequence height repeating part corresponding DNA sequences and domestic silkworm silk albumen L chain gene cDNA are merged, and place the dna fragmentation that merges after the L chain gene promotor and before the L chain gene terminator, structure obtains complete fusion gene, complete fusion gene is linked to each other with a kind of reporter gene, and between two reverse repetition end sequences that insert transposable element piggyBac together, the transposable element that obtains recombinating;
(3) make up the transposase gene expression vector with transposase gene;
(4) transform silkworm egg simultaneously with obtaining the transposase gene expression vector in the reorganization transposable element that obtains in the step (2) and the step (3);
(5) the young silkworm that forms of the silkworm egg that transforms by the quilt of cultivating and screening is obtained by step (4) obtains the transgenic bombyx mori that stable expression of exogenous is separated toxenzyme lipase B1.
The unitary correct structure of lipase B1 genetic expression all has decisive role for its formation at the intravital expression of silkworm, secretion, regulation and control and zymoprotein.Therefore the present invention is the be in downstream of fibroin L chain gene of lipase B1 gene fusion, the synthetic and secretion that utilizes synthesizing of domestic silkworm silk albumen L chain to guide to separate toxenzyme.
The present invention utilizes the L chain gene promotor of silkworm self to start transcribing of L chain-lipase B1 fusion gene: 5 ' end of the dna fragmentation of lipase B1 gene cDNA or its aminoacid sequence height repeating part correspondence is right after 3 ' end at fibroin L chain gene cDNA, form the fusion gene of correctly reading over, and this fusion gene is placed under the tight control of silkworm self L chain gene promotor and terminator, be built into complete " silk L chain-lipase B1 " recombinant gene expression unit.Therefore this fusion gene is subjected to the accuracy controlling of silkworm self at silkworm intravital expression L chain gene promotor and terminator; And the secretion of fusion gene expression product and the formation of zymoprotein have been guaranteed in the existence of silk L catenin.Silk L chain gene promotor and terminator can obtain through pcr amplification by domestic silkworm gene group DNA, and L chain gene cDNA then can obtain through RT-PCR (reverse transcription PCR) amplification by total RNA or mRNA in the extraction silkworm posterior division of silkgland in five ages.
The present invention places the downstream of the promotor of bombyx mori cell matter actin gene A3 with the GFP reporter gene, is convenient to transgenic bombyx mori is screened and identifies.
For " silk L chain-lipase B1 " fusion gene is transferred in the domestic silkworm gene group effectively, the present invention adopts the gene transformation system that makes up according to transposon piggyBac.The about 2kb size of transposon piggyBac is from lepidopterous insects cabbage looper Trichoplusia ni.Studies show that, this transposon can be in multiple lepidopteran and dipteral insect swivel base.Its mechanism that swivel base takes place is: piggyBac transposon two ends respectively have a tumor-necrosis factor glycoproteins, and the orientation of two tumor-necrosis factor glycoproteinss is opposite, are called reverse terminal repeat.Comprise between two reverse terminal repeats that two are read frame, one of them reads frame coding transposase.When swivel base took place, transposase promoted the swivel base of the dna fragmentation between the two reverse terminal repeats by the reverse terminal repeat of identification.The present invention utilizes the DNA recombinant technology, complete reorganization " silk L chain-lipase B1 " gene is linked to each other by a manual splice with complete GFP reporter gene, and substitute sequence between the reverse terminal repeat of piggyBac transposon with the correct DAN fragment that obtains that connects, thereby the piggyBac transposon is transformed into gene transfer vector in the future.This gene transfer vector can stably shift goal gene and be incorporated in the domestic silkworm gene group.
Transposon generation swivel base except that the reverse terminal repeat of needs, the participation of also essential transposase.Transposase is discerned reverse terminal repeat, and realizes two reverse terminal repeats and inner dna fragmentation generation swivel base thereof.For realizing that the stable gene that shifts is incorporated in the domestic silkworm gene group, transposase can be separated between two reverse terminal repeats of piggyBac transposon, be cloned in another carrier, and add the A3 promotor, constitute the helper plasmid carrier with this at its front end.When carrying out the transgenic bombyx mori test, the transposase gene generation transient expression on the helper plasmid, assurance swivel base successful reaction is carried out.
This two plasmid transgenosis system by piggyBac transposon reconstruction will need the goal gene of swivel base to separate with transposase, not only can realize the high frequency integration of goal gene in the silkworm genome, and guarantee the genetic stability of integrator gene in the transgenic bombyx mori.
Above-mentioned lipase B1 gene that obtains and silk L chain integrative gene expression vector are transformed silkworm egg simultaneously with the helper plasmid that contains transposase.Silkworm egg after the injection forms young silkworm through conventional incubation and cultivation.
Silkworm seed after the injection is raised through conventional, to children silkworm in 5 ages, detects the expression level of reporter gene GFP in the silkworm under wavelength 390 nano enzyme ultraviolet rays, mark GFP positive individuals up to long.After silkworm is put on an arrow and cocoons, GFP positive individuals silk cocoon physico-chemical property is detected, mark contains the individuality of the enzyme component that detoxifies.To containing the individual cross-breeding of the enzyme component that detoxifies,, obtain the transgenic bombyx mori of stable expression of exogenous detoxifying gene by the silkworm of cultivating and screening is transformed.This transgenic bombyx mori that seed selection obtains can synthesize and secrete the detoxifcation zymoprotein when cocooing, contain the nucleotide sequence of lipase B1 gene at its genome, and efficiently expressed in pupal cell.
The reverse terminal repeat of the piggyBac transposon that adopts among the present invention has special dna sequence dna, can be discerned by transposase, and under the effect of transposase, drive the dna fragmentation generation swivel base that is included in reverse terminal repeat inside.Can adopt the synthetic dna sequence dna identical, be convenient to DNA reorganization operation with the reverse terminal repeat of piggyBac transposon.Another artificial synthesized sequence is that complete fibroin L chain cDNA-lipase B1 gene cDNA is expressed the catenation sequence between unit and the GFP reporter gene expression unit: the manual splice in the multiple clone site district of one section 30bp length.
The present invention links to each other lipase B1 gene cDNA with silkworm L chain gene cDNA, form recombination.Guarantee to understand synthetic smoothly, the secretion of toxenzyme on the one hand, on the other hand, the product of this recombination is the fusion rotein of lipase B1 and domestic silkworm silk albumen L chain, and separating toxenzyme becomes the proteic organic component of this novel silkworm.
The present invention adopts two pUC pUCs to carry out goal gene and shifts: will be transformed into gene transfer vector from the piggyBac transposon of cabbage looper, and can be in the genome of silkworm the recombination high-frequency integration of silk L chain gene and lipase B1 genomic constitution; And transposase gene is deleted from gene transfer vector, be built into the assistant carrier of transient expression.Therefore the recombination lipoidase B1 gene that is incorporated in the domestic silkworm gene group can stably remain in the genome.The characteristic that the transgenic bombyx mori of the recombination lipoidase B1 gene that isozygotys that obtains by cross-breeding and screening, its secretion reorganization are separated toxenzyme will stably entail filial generation with transgenic bombyx mori THE REPLICATION OF CHROMOSOME, separation.
In sum, this invention obtain by genophore plasmid and the two domestic silkworm gene transfer system that constitutes jointly of helper plasmid, can guarantee multiple copied, the high frequency integration of external source goal gene in the silkworm genome, further improve the expression of exogenous gene level.Therefore, the integrative vector of development by the mode of swivel base, can be realized the integration of foreign gene at silkworm genome high frequency, and transformation frequency can reach 7%.
The present invention compared with prior art has the following advantages and effect: easy to implement the method, easy and simple to handle, and the transformation efficiency height, lipase B1 content is 52% in the transgenic bombyx mori pupa after testing, has opened up new approach for obtaining to separate toxenzyme, has significant social and economic benefit.
Description of drawings
Fig. 1 transgenic bombyx mori rapid molecular is identified and the Southern hybridization analysis
Wherein: Fig. 2-1:PCR positive products that increases; 7: negative control Fig. 2-2:PCR positive products that increases; 7: negative control
The structure of Fig. 2 transposon expression plasmid pZAGFP
Embodiment
" fibroin L chain-lipase B1 " fusion gene is expressed unitary structure
At first extract the mRNA in the silkworm posterior division of silkgland, sequence according to known domestic silkworm silk albumen L chain gene, (the upstream and downstream primer sequence is respectively design upstream and downstream primer: 5 '-tggatccgcggcgcgttaccatatatg-3 ', 5 '-gcgatatcacgccctgatgg-3 '), obtain domestic silkworm silk albumen L chain cDNA through the RT-PCR amplification, be approximately 0.8Kb, comprising signal peptide sequence.
From the total DNA of silkworm, according to known array design primer (upstream primer sequence: 5 '-tggatccgcggcgcgttaccatatatg-3 ', downstream primer sequence: 5 '-gcgatatcacgccctgatgg-3 '), amplify the dna fragmentation of the about 1.2Kb of promoter region of L chain 5 ' end and upstream thereof, and the terminator fragment of L chain 3 ' end 0.5Kb.
According to (Proc.Natl.Acad.Sci.USA.1990,87:2574-2578 such as Mouches.Mouches C et al.) Bao Dao lipase B1cDAN sequence, extract the mRNA of anti-temephos Culex quinquefasciatus (TEM-R), design primer (upstream primer sequence: 5 '-cgcatatgatgagtttggaaagc-3 ', the downstream primer sequence: 5 '-ctcccagacattacgatacgc-3 '), 665bp fragment lipase B1 cDNA 5 ' the end B1 (a) after the initial secret numeral ATG of lipase B1cDNA is obtained in the RT-PCR amplification.Its B1 (b) with lipase B13 ' end cDNA 1.33kb is built into total length lipase B1 gene.
Above-mentioned fragment is connected into the silk L chain-lipase B1 gene of fusion by correct reading frame.At first the pcr amplification sequence of lipase B1 gene cDNA is inserted among the 7th exon of 3 ' stub area of L chain cDNA by correct reading frame, be built into L-EB1 and merge fragment, merge segmental 5 ' end and 3 ' at this respectively then and hold and to add L chain promoter sequence and terminator sequence, form the complete lipase B1 and the fusion gene L-EB1 of L chain.
The structure of integrated genophore
Extract domestic silkworm gene group DNA, obtain the A3 promoter fragment through pcr amplification according to filamentous actin gene (A3) sequence of bombyx mori cell matter flesh.The A3 promoter fragment is connected with GFP genes encoding region sequence with suitable reading frame.With the SV40polyA signal sequence, be inserted into the GFP gene downstream of fusion, form complete GFP genetic expression unit.Add in complete GFP gene fragment upstream the multiple clone site district (MCS) of a segment length 30bp manual splice (nucleotides sequence is classified as:
5’-ACCGCGGTCTAGAGGATCCCGGGCTGCAGT-3’)。The fusion gene L-EB1 expression unit of lipase B1 gene and L chain and complete GFP genetic expression unit are linked together by the multiple clone site district, obtain recombinant dna fragment.Add the reverse terminal repeat (nucleotide sequence 5 '-CGCCGCGGCC CTAGAAAGATAGTCTGCGTA AAATTGACGC ATGCTGCAGT G-3 ') of piggyBac transposon respectively in the outside of recombinant dna fragment gene, and with this fragment cloning in carrier pBS, be built into the gene delivery plasmid of silkworm transgenosis system.
Helper plasmid makes up
Adopt round pcr to clone the transposase coding region sequence in the piggyBac transposon.Front, coding region at transposase gene adds the A3 promotor, and is cloned in the plasmid pUC18, is the helper plasmid of gene transfer system.The two has constituted the domestic silkworm gene transfer system jointly L-EB1 genophore plasmid and helper plasmid.This system realizes the integration of foreign gene at silkworm genome high frequency by the mode of swivel base, and transformation frequency can reach 7%.This system can guarantee multiple copied, the high frequency integration of external source goal gene in the silkworm genome, further improves the expression of exogenous gene level.
Change the acquisition of lipase B1 gene silkworm
The gene transformation of silkworm.The gene transfer system of above-mentioned structure, inject the silkworm silkworm seed with microinjection instrument, 25 ℃ of cultivations, hatching forms newly-hatched silkworm, feeds with mulberry leaf.The screening of transgenic bombyx mori and evaluation.Silkworm enters second day of 5 length of times, promptly can be used for the evaluation of transgenic bombyx mori.With 350nm-390nm fluorescent inspection instrument, by the detection to the GFP reporter gene, preliminary evaluation goes out transgenic bombyx mori.In the research, injected in 1500 silkworm seeds, survived 825 young silkworms, 510 can form the moth that lays eggs, and G0 is the 7% demonstration fluorescin positive in generation.Transgenic bombyx mori is carried out succeeding transfer culture, and its GFP fluorescent characteristic shows as Mendelian inheritance.The positive silkworm of the fluorescin that obtains is backcrossed and adelphogamy, with the silkworm seed (G that gives birth to
1Generation) be divided into two parts, a primer up and down of fluorescence protein gene that adopts carries out pcr amplification, if show negatively, discards another part silkworm seed, if positive, then keeps another part, and after 5 ages second day, detect with luminoscope again.Positive G to fluoroscopic examination
1In generation, is partly individual, extract total DNA, use primer at the GFP gene to carry out the PCR rapid molecular and identify, and use specific probe at lipase B1 gene to carry out Southern hybridization analysis (Fig. 1 is for lipase B1 gene specific fragment being the result of the Southern hybridization carried out of probe) total DNA of positive individuals.Positive individuals is further carried out sisters' friendship, and until G3 generation, the G3 offspring positive individuals of acquisition is the transgenic bombyx mori of foreign gene stable integration with the purifying transgenic bombyx mori.Extract the protein of transgenic bombyx mori posterior division of silkgland, the protein in the sampling observation silkworm chrysalis carries out series evaluations such as SDS-PAGE analysis, and the result shows lipase B1 protein content height, accounts for 52% of gross protein.
Sequence table
Silkworm kytoplasm actin gene promotor and part coding region sequence:
Regulating and controlling sequence SRE, ActE1, TATA and initiator codon are identified respectively, first row: A3.1 sequencing fragment result:
Second row: Europe 200 * 300 cultivated silkworm breed variety BmA3 gene corresponding sequence; The third line: BmA4 gene corresponding sequence
SRE???????????????????????????????????????????????????????ActEl
A3.1：?????CCGCOTTACCATATATGGTGC-AAAA-CTGAGT-----------CAGCUCGCGATTGGTGGAAAAACAAACTGGAGCCGATACTGTGTAAATTGTGATAAC--G
||||||||||||||||||||??||||?||||||???????????|||||||||||||||||||||||||||||||||||||||||||||||||||||||||??|
Europ?A3：GCGCGTTACCATATATCOTGACAAAAAUTGAGT-----------CAOCCCCCOATTOOTGGAAAAACAAACTGGAGCCGATACTGTGTAAATTOTOATAAC--G
BmA4：????Gtaa--TACCATATATGGacacaaaa-cttcGTgtattgtaccctAGCGCGCGATTGGAGGAgAgtctgcggcggcggGgcaggGgcgcccc---GATAACcaG
TATA-box?????????????????+l
9l?GCTCT
TTTATATAGTTTATCCTCACGAGTCGGTTCTCATTTACT---AAGGTGTGCTCGAACAGTGCGCATTCGCATCTACGTACCTACTTGT
|||||?||||||||||?|||||||||||||||||||||||||||||???||||||||||||||||||||||||||||||||||||||?????|||
GCTCT
TTTATATAGTTTATCCTCACGAGTCGGTTCTCATTTACT---AAGGTGTGCTCGAACAGTGCGCATTCGCATCTACGTAC----TTGT
CCTCA
TTTATATAGTccgcCaagcgcacTcaccaacattccacgaagtgaGctTGggtcgttgcgttgtAcagcaATaacgaagCtgtg.....
18l?CACTTATTTAATAATACTATGTAAGTTTTAATTTTAAAATTGCGAAAGAAAAAAAAACATATTTATTTATTTGTAAAATTTGAATTTCGA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CACTTATTTAATAATACTATGTAAGTTTTAATTTTAAAATTGCGAAAGAAAAAAAAACATATTTATTTATTTGTAAAATTTGAATTTCGA
27l?AGGTTCTCCGTCCCTTTACCTTTAAGTATTACATATGTTTGAGTGTTTTTTTTTT--AATAATACGCGAATGATGATAACGTGTTACGTTAC
|||||||||||||||||||||||||||||||||||||||||||||||||||||||??|||||||||||||||||?|??????||||||||||
AGGTTCTCCGTCCCTTTACCTTTAAGTATTACATATGTTTGAGTGTTTTTTTTTTTTAATAATACGCTAATGATAACG---TGTTACGTTAC
331?ACATATGTTTGAGTGTTTTTTTTTT--MTAATACGCGAATGATGATAACGTGTTACGTTACATAATcGTTGcATAACTAGTGAAGTGAAAT
|||||||||||||||||||||||||??||||||||||||||||?|??????||||||||||||||||||||||||||||||||||||||||
ACATATGTTTGAGTGTTTTTTTTTTTTAATAATACGCTAATGATAACG---TGTTACGTTACATAATtGTTGCATAACrAGTGAAGTGAAA
39l?TTTTTATAAAAAAAAACAgTTTTCGGAATT-AGTGTAaTGCcGtTG-----------CTACTAAATAAGAAATAAGTTTATTGAACGCACATTTCAAAATGT
||||||||||||||||||?|||||||||||?||||||?|||?|?||???????????||||||||||||||||||||||???||||||||||||||||||||
TTTTTTATAAAAAAAAACAtTTTTCGGAATTTAGTGTAcTGcaGaTGTTAATAAACACTACTAAATAAGAAATAAGTTTATTGACGCACATTTCAAAGTGT
481?CCACTCGCATCGATCAATTCGGAAACAGAAATTGGGAACAGTGAATTATGAATCTTAgAtAGITTTTCTTTAACGTCACTAAATTGATGa
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||?|||||||||||||||||||||||||||||||
CCACTCGCATCGATCAATTCGGAAACAGAAATTGGGAACAGTGAATTATGAATCTTAtAcAGITTTTCTTTAACGTCACTAAATAGATGg
54l?ACGCAAATAAATTTGTCGTTTACT----ATAATTTATGGAATGAGAATGTAGTTTGAATTGTTTTTTTCTTTTTCTTGCAGACTAATTCAAGATG
||||||||||||||||||||||||???||||||||||||||||||||||||||||||||||?|||||||||||||||||||||||||||||||||
ACGCAAATAAATTTGTCGTTTACTTAGTATAATGTATGGAATGAGAATGTAGTTTGAATTGTTTTTTTTCTTTTCTTGCAGACTAATTCAAGATG
66l
TGCGACGAgGAAGTTGCCGCGTTGGTAGTAGACAATGGCTCCGGTATGTGCAAGGCCGGTTTCGCAGGAGATGATGCTCCTCCCGCCGTa
||||||||?|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TGCGACGAaGAAGTTGCCGCGTTGGTAGTAGACAATGGCTCCGGTATGTGCAAGGCCGGTTTCGCAGGAGATGATGCTCCTCGCGCCGTg
75l?TTCCCCTCGATCGTCGGAAGGCGCCGCCATCAGGGCGTGATG
||||||||||||||||||||||||||||||||||||||||||
TTCCCCTCGATCGTCGGAAGGCCCCGCCATCAGGGCGTGATC
The nucleotide sequence of domestic silkworm silk albumen L chain cDNA:
ACCACTAAA
A?TGAAGCTATA???????TTTTTGGTAT????TACTCGTCGC????TACAAGCGCC???????????????????50
TACGCTGCAC?????CATCGGTGAC?????CATCAATCAA????TACAGTGATA????ATGAAATTCC?????ACGCGACATT????110
GATGATGGAA?????AAGCTAGTTC?????CGTAATCTCA????CGTGCATGGG????ACTACGTCGA?????TGACACTGAC????170
AAAAGCATCG?????CCATCCTCAA?????CGTTCAAGAG????ATCTTGAAGG????ACATGGCCAG?????CCAGGGCGAT????230
TATGCAAGTC?????AAGCATCAGC?????GGTGGCCCAA????ACCGCCGGAA????TTATCGCCCA?????TCTATCTGCC????290
G?GT
AGGTCCGGAA?????ACTTCGCCGG?????CTTCAGACAA????TCTCTCGGTC????CCTTCTTCGG?????ACACGTGGGA????410
CAAAACTTGA?????ATCTTATCAA?????TCAACTCGTC????ATCAACCCTG????GTCAACTCCG?????ATACTCTGTC????470
GGACCAGCCC?????TGGGTTGTGC?????CGGAGGTGGA????AGAATCTATG????ACTTCGAAGC?????CGCTTGGGAT????530
AGT
ATA
CCT
GGCGTTGGCA?????ACGGTAATGC????GCGAC?????????????????????????????????????????????????????735
The nucleotide sequence of L-chain promoter fragment:
1????GGCTATATTC?AGACTCGCCA?AGTTACGTCA?GTCGTATTGT?AATGAGCGAT?TTAGTGGGCA
C
61???ACTTCATTCT?GTTAATTTTG?TGTCACGGTG?CGCGCGCATC?GTAAAATTTC?ACTCTCATAG
C
121??ATTTTTCATA?ACGTGCCTAA?AGAAGTATAA?CTTCAATGAT?TTAAATT?AA?AAAAAAACAT
A??????????T
181??GCATAGAATA?ATTATATGAA?TTATTTAAAA?TGTCATTTAC?CGACATTGAC?ATAACAGACG
241??ACGTTAACAC?TACAAAACAT?T
TTAATTCCA?CATTGTTACA?TATTCAAC
AG?TTAAATTTGC
7????????????????????????????6
301?
GTTAATTCTC?GATGCGAACA?AATATAAGAA?CAA
TCGGATC?AATTAGATCG?CTTTGTTTCG
5
361??AGCAACACTT?AGTTTAACTA?GAGGCGTACA?CC
TCAAGAAA?TCATCTTCAT?TAGAAACTAA
A??????????????????????????????????????????????4
421??ACCTTAAAAT?CG
CATTAATA?AAGCATAGTC?AATTTTAACT?GAAATGCAAA?ATCTTTTGAA
A??3??????????????????????????????????????G
481??CGTTAGATGC?TGTCAGCGTT?CGTTGGTACA?GT
TGTTTGAT?ATTTATT
TTA?ATTGTCTTTT
2
541?TATATATAAA?TAGTGGAA
CA?TTAATCACGG?AATCCT
1?????????????+1
The segmental nucleotide sequence of L chain 3 ' end terminator:
taaataa?gaactgtaaa?taatgtatat?atataattat?ataaaagata?tatataacca
tatacaaaca?tatatatcat?tataagacaa?tctacctata?taaaaacaga?ctaaaattaa
taattatgta?tactttaatt?gtgtttagga?cattttatgc?aaattgtgtt?tgcgttagga
ttttttttgg?aagtttttta?gattatttat?gaatatataa?ataaatatac?gttaatataa
tatatattat?ataaatcaac?gacacggctt?ttcattttgg?tgatgatcaa?tcttattgtt
cttctaattg?atttttttgt?acaataaaga?tgtatccagt?tttccagata?aagaatttag
tttgttattt?ctggccccat?taaaataagt?acggtattcg?acaataccac?atagtatata
cccaaagacg?gtggattgga?cagtgggtac?atggatttcg?gtactgttgt?catgct
Claims (6)
1, the construction process of a kind of detoxifying gene stably express system in silkworm, it may further comprise the steps:
A, the synthetic oligomeric deoxynucleotidyl primer of design, from young silkworm, take out posterior division of silkgland and extract total DNA, with total DNA is template, carry out pcr amplification reaction with primer respectively, digest with restriction enzyme again, the plasmid Bluescript M13 that cuts processing with enzyme is connected, the screening recon, obtain pcr amplified fragment, clone's dna fragmentation is respectively bombyx mori cell matter actin gene promotor and coding region sequence, the nucleotide sequence of domestic silk core albumen L chain cDNA, the pulsating nucleotide sequence of L chain gene promotor, L chain gene 3 ' terminator segment nucleotide sequence;
B, with after the Cdna of detoxifying gene or its aminoacid sequence corresponding DNA sequences and the domestic silkworm silk albumen L chain gene promotor and before the L chain gene terminator, gene fusion construct, to melt gene links to each other with reporter gene, and two reverse repetitions of inserting transposable element piggyBac together obtain the transposon of recon between the end sequence;
C, make up the transposase gene expression vector with transposase gene;
D, the transposase gene expression vector that obtains among the reorganization transposable element that obtains among the step B and the step C is transformed silkworm egg simultaneously;
The young silkworm that E, the silkworm egg that is transformed by the quilt that obtains among the step D by cultivation and screening form obtains the transgenic bombyx mori that stable expression of exogenous is separated toxenzyme lipase B1.
2, the construction process of a kind of detoxifying gene according to claim 1 stably express system in silkworm is characterized in that the primer of oligodeoxynucleotide is:
???A3-0 ????5’tgg?atc?cgc?ggc?gcg?tta?cca?tat?atg?3’
???A3-1 ????5’gcg?ata?tca?cgc?cct?gat?gg3’
???A3-2 ????5’gcg?gat?ccc?atc?ttg?aat?tag?tct?gc3’
???A3-3 ???23 ????5’agtccatgggccttccgacgatc3’
???L-1 ???32 ????Ctggatccataacagaccactaaaatgaagcc
???L-2 ???26 ????Cgaagcttgtcgcgtcattaccgttg
???L-3 ???30 ????Cgtctagactcgaggtggtgcctatcccac
???L-4 ???29 ????Gcggatccaatcggtatatactatacagg
???L-5 ???20 ????Cgaagcttagttgctaatgc
???L-6 ???22 ????Ctggtaccatgtacccactgtc
???Tra-1 ????5’gcg?gat?cct?ctt?tag?acg?atg3’
???Tra-2 ????5’tac?tgc?agg?tca?tca?cag?3’
???Gfp-1 ???24 ????5′-gtggatccgcggccgctttacttg-3′
???Gfp-2 ????Acggatccatggtgagcaagggc
3, the construction process of a kind of detoxifying gene according to claim 1 stably express system in silkworm is characterized in that bombyx mori cell matter actin gene promotor and encoding sequence are:
SRE??????????????????????????????????????????????????????????ActEl
A3.1：????GCGCGTTACCATATATGGTGC-AAAA-CTGAGT-----------CAGCCCGCGATTGGTGGAAAAACAAACTGGAGCCGATACTGTGTAAATTGTGATAAC-G
|||||||||||||||||||||?||||?||||||???????????|||||||||||||||||||||||||||||||||||||||||||||||||||||||||?|
EuropA3：GCGCGTTACCATATATGGTGACAAAAACTGAGT-----------CAGCCCGCGATTGGTGGAAAAACAAACTGGAGCCGATACTGTGTAAATTGTGATAAC--G
BmA4：????Gtaa-TACCATATATGGacacaaaa-cttcGTgtattgtaccctAGCGCGCGATTGGAGGAgAgtctgcggcggcggGgcaggGgcgcccc---GATAACcaG
TATA-box????????????????????+1
91?GCTCT
TTTATATAGTTTATCCTCACGAGTCGGTTCTCATTTACT---AAGGTGTGCTCGAACAGTGCGCATTCGCATCTACGTACCTACTTGT
|||||?||||||||||?||||||||||?||||||||||||||||||???||||||||||||||||||||||||||||||||||||||?????|||
GCTCT
TTTATATAGTTTATCCTCACGAGTCGGTTCTCATTTACT---AAGGTGTGCTCGAACAGTGCGCATTCGCATCTACGTAC----TTGT
CCTCA
TTTATATAGTccgcCaagcgcacTcaccaacattccacgaagtgaGctTGggtcgttgcgttgtAcagcaATaacgaagCtgtg......
181?CACTTATTTAATAATACTATGTAAGTTTTAATTTTAAAATTGCGAAAGAAAAAAAAACATATTTATTTATTTGTAAAATTTGAATTTCGA
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CACTTATTTAATAATACTATGTAAGTTTTAATTTTAAAATTGCGAAAGAAAAAAAAACATATTTATTTATTTGTAAAATTTGAATTTCGA
271?AGGTTCTCCGTCCCTTTACCTTTAAGTATTACATATGTTTGAGTGTTTTTTTTTT--AATAATACGCGAATGATGATAACGTGTTACGTTAC
|||||||||||||||||||||||||||||||||||||||||||||||||||||||??||||||||||?||||||?|?????|||||||||||
AGGTTCTCCGTCCCTTTACCTTTAAGTATTACATATGTTTGAGTGTTTTTTTTTTTTAATAATACGCTAATGATAACG---TGTTACGTTAC
331?ACATATGTTTGAGTGTTTTTTTTTT-AATAATACGCGAATGATGATAACGTGTTACGTTACATAATcGTTGCATAACTAGTGAAGTGAAAT
|||||||||||||||||||||||||?||||||||||?||||||?|?????|||||||||||||||||||||||||||||||||||||||||
ACATATGTTTGAGTGTTTTTTTTTTTTAATAATACGCTAATGATAACG---TGTTACGTTACATAATtGTTGCATAACTAGTGAAGTGAAA
391?TTTTTATAAAAAAAAACAgTTTTCGGAATT-AGTGTAaTGCcGtTG----------CTACTAAATAAGAAATAAGTTTATTGAACGCACATTTCAAAATGT
||||||||||||||||||?|||||||||||?||||||?|||?|?||??????????|||||||||||||||||||||||||||||||||||||||||||||
TTTTTATAAAAAAAAACAtTTTTCGGAATTTAGTGTAcTGCaGaTGTTAATAAACACTACTAAATAAGAAATAAGTTTATTGGACGCACATTTCAAAGTGT
481?CCACTCGCATCGATCAATTCGGAAACAGAAATTGGGAACAGTGAATTATGAATCTTAgAtAGTTTTTCTTTAACGTCACTAAATTGATGa
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||??||||||||||||||||||||||||||||||
CCACTCGCATCGATCAATTCGGAAACAGAAATTGGGAACAGTGAATTATGAATCTTAtAcAGTTTTTCTTTAACGTCACTAAATAGATGg
541?ACGCAAATAAATTTGTCGTTTACT----ATAATTTATGGAATGAGAATGTAGTTTGAATTGTTTTTTTCTTTTTCTTGCAGACTAATTCAAGATG
||||||||||||||||||||||||????|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ACGCAAATAAATTTGTCGTTTACTTAGTATAATGTATGGAATGAGAATGTAGTTTGAATTGTTTTTTTCTTTTCTTGCAGACTAATTCAAGATG
661
TGCGACGAgGAAGTTGCCGCGTTGGTAGTAGACAATGGCTCCGGTATGTGCAAGGCCGGTTTCGCAGGAGATGATGCTCCTCGCGCCGTa
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TGCGACGAaGAAGTTGCCGCGTTGGTAGTAGACAATGGCTCCGGTATGTGCAAGGCCGGTTTCGCAGGAGATGATGCTCCTCGCGCCGTg
751?TTCCCCTCGATCGTCGGAAGGCCCCGCCATCAGGGCGTGATG
||||||||||||||||||||||||||||||||||||||||||
TTCCCCTCGATCGTCGGAAGGCCCCGCCATCAGGGCGTGATG
4, the construction process of a kind of detoxifying gene according to claim 1 stably express system in silkworm is characterized in that the nucleotide sequence of domestic silkworm silk albumen L chain cDNA is:
ACCACTAAA
A?TGAAGCTATA???????TTTTTGGTAT????TACTCGTCGC?????TACAAGCGCC??????????????????50
TACGCTGCAC?????CATCGGTGAC?????CATCAATCAA????TACAGTGATA?????ATGAAATTCC????ACGCGACATT????110
GATGATGGAA?????AAGCTAGTTC?????CGTAATCTCA????CGTGCATGGG?????ACTACGTCGA????TGACACTGAC????170
AAAAGCATCG?????CCATCCTCAA?????CGTTCAAGAG????ATCTTGAAGG?????ACATGGCCAG????CCAGGGCGAT????230
TATGCAAGTC?????AAGCATCAGC?????GGTGGCCCAA????ACCGCCGGAA?????TTATCGCCCA????TCTATCTGCC????290
G?GT
AGGTCCGGAA?????ACTTCGCCGG?????CTTCAGACAA????TCTCTCGGTC????CCTTCTTCGG?????ACACGTGGGA????410
CAAAACTTGA?????ATCTTATCAA?????TCAACTCGTC????ATCAACCCTG????GTCAACTCCG?????ATACTCTGTC????470
GGACCAGCCC?????TGGGTTGTGC?????CGGAGGTGGA????AGAATCTATG????ACTTCGAAGC?????CGCTTGGGAT????530
AGT
ATA
CCT
GGCGTTGGCA?????ACGGTAATGC????GCGAC?????????????????????????????????????????????????????735
5, the construction process of a kind of detoxifying gene according to claim 1 stably express system in silkworm is characterized in that the pulsating nucleotide sequence of domestic silkworm silk albumen L chain promotor is:
1????GGCTATATTC?AGACTCGCCA?AGTTACGTCA?GTCGTATTGT?AATGAGCGAT?TTAGTGGGCA
C
61???ACTTCATTCT?GTTAATTTTG?TGTCACGGTG?CGCGCGCATC?GTAAAATTTC?ACTCTCATAG
C
121??ATTTTTCATA?ACGTGCCTAA?AGAAGTATAA?CTTCAATGAT?TTAAATT?AA?AAAAAAACAT
A??????????T
181??GCATAGAATA?ATTATATGAA?TTATTTAAAA?TGTCATTTAC?CGACATTGAC?ATAACAGACG
241??ACGTTAACAC?TACAAAACAT?
TTTAATTCCA?CATTGTTACA?TATTCAAC
AG?TTAAATTTGC
7????????????????????????????6
301??
GTTAATTCTC?GATGCGAACA?AATATAAGAA?CAA
TGGGATC?AATTAGATCG?CTTTGTTTCG
5
361??AGCAACACTT?AGTTTAACTA?GAGGCGTACA?CC
TCAAGAAA?TCATCTTCAT?TAGAAACTAA
A??????????????????????????????????????????????4
421??ACCTTAAAAT?CG
CATTAATA?AAGCATAGTC?AATTTTAACT?GAAATGCAAA?ATCTTTTGAA
A??3??????????????????????????????????????G
481??CGTTAGATGC?TGTCAGCGTT?CGTTGGTACA?GT
TGTTTGAT?ATTTATT
TTA?ATTGTCTTTT
2
541??TATATATAAA?TAGTGGAA
CA?TTAATCACGG?AATCCT
1?????????????+1
6, the construction process of a kind of detoxifying gene according to claim 1 stably express system in silkworm is characterized in that the nucleotide sequence of domestic silkworm silk albumen L chain 3 ' end terminator is:
taaataa??gaactgtaaa?taatgtatat??atataattat??ataaaagata
tatataacca?tatacaaaca?tatatatcat?tataagacaa?tctacctata
taaaaacaga?ctaaaattaa?taattatgta?tactttaatt?gtgtttagga
cattttatgc?aaattgtgtt?tgcgttagga?ttttttttgg?aagtttttta
gattatttat?gaatatataa?ataaatatac?gttaatataa?tatatattat
ataaatcaac?gacacggctt?ttcattttgg?tgatgatcaa?tcttattgtt
cttctaattg?atttttttgt?acaataaaga?tgtatccagt?tttccagata
aagaatttag??tttgttattt?ctggccccat?taaaataagt?acggtattcg
acaataccac??atagtatata?cccaaagacg?gtggattgga?cagtgggtac
atggatttcg??gtactgttgt?catgct
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006122442A1 (en) * | 2005-05-14 | 2006-11-23 | Fudan University | Piggybac as a tool for genetic manipulation and analysis in vertebrates |
| CN101177692B (en) * | 2007-12-05 | 2010-10-13 | 浙江大学 | Method for developing transgenic domestic silkworm by using A3 promotor defect piggyBac transposon |
| CN102187845A (en) * | 2010-03-05 | 2011-09-21 | 中国科学院上海生命科学研究院 | Transgenic method for improving silk yield |
| CN103232977A (en) * | 2013-05-16 | 2013-08-07 | 西南大学 | Application of phiC31 recombinase system and piggyBac transposon and fixed point transgenetic system of silkworm and preparation method of fixed point transgenetic system |
-
2003
- 2003-03-27 CN CN 03118855 patent/CN1234871C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006122442A1 (en) * | 2005-05-14 | 2006-11-23 | Fudan University | Piggybac as a tool for genetic manipulation and analysis in vertebrates |
| CN101177692B (en) * | 2007-12-05 | 2010-10-13 | 浙江大学 | Method for developing transgenic domestic silkworm by using A3 promotor defect piggyBac transposon |
| CN102187845A (en) * | 2010-03-05 | 2011-09-21 | 中国科学院上海生命科学研究院 | Transgenic method for improving silk yield |
| CN102187845B (en) * | 2010-03-05 | 2013-06-05 | 中国科学院上海生命科学研究院 | Transgenic method for improving silk yield |
| CN103232977A (en) * | 2013-05-16 | 2013-08-07 | 西南大学 | Application of phiC31 recombinase system and piggyBac transposon and fixed point transgenetic system of silkworm and preparation method of fixed point transgenetic system |
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|---|---|
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