CN1483823A - Engineering systhesized gene cry LC of pests-killing crytal protein of Bacillus thuringiensis Berliner - Google Patents
Engineering systhesized gene cry LC of pests-killing crytal protein of Bacillus thuringiensis Berliner Download PDFInfo
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Abstract
The present invention is characterized by that it utilizes a series method to design and synthesize a bacillus thuringiensis Berlinear insecticidal protein gene CrylC. As compared with sequence of 1890 nucleotides of 5' end of original CrylCa5 gene the amino acid composition of said gene coded protein is not changed, but the richly-contained AT sequence, existed invented repeats and undefined eukaryotic gene intron sequence in the original gene are eliminated, C+G content is 44.64%, and the homology with original sequence is 84.0%, on the 5' end of coding sequence the guide sequence (SEQ ID No:3) is added and on the 3' end the tailing identification signal sequence (SEQ ID No.4) is added.
Description
Technical field:
The invention belongs to gene engineering technology field, belong to a kind of innovation bacillus thuringiensis (Bt) insecticidal crystal protein (ICP) gene.Be specifically related to: part changes the codon of original gene and forms, the dna sequence dna that keeps the N end toxicity district of coded insect-killing crystallin in the protogene, part is removed the dna sequence dna of coding C end in the protogene, add the homing sequence of raising gene expression efficiency and add the tailing recognition sequence at 5 ' end, transform the synthetic killing gene Cry1C that transgenic plant efficiently express that is used at 3 ' end
*
Background technology:
Insect pest is an important factor that causes the agriculture production loss.According to statistics, the direct economic loss that causes to agriculture production in insect pest every year is up to 13%.
Chemical insecticide once to pest control, stablize agriculture production and made significant contribution.Along with people to the understanding of chemical insecticide environmental hazard, the reinforcement day by day of environmental consciousness, environmentally safe biotic pesticide have become the focus of research.In biotic pesticide, what research at present was the clearest, application is the most successful is a class Bt preparation.The effective constituent of Bt preparation is Bt insecticidal crystal protein (ICP).Bt insecticidal crystal protein (ICP) is that bacillus thuringiensis produces in the gemma forming process.But find also in the production that the Bt preparation exists problem of unstable, easily run off in the field on the one hand that drug effect is short by rain drop erosion; On the other hand, because the active ingredient of Bt sterilant is a protein, under the irradiation of sunlight middle-ultraviolet lamp, easily is decomposed and lost efficacy.The success of plant transgene provides a brand-new route for the application of Bt insecticidal crystal protein.
Bt insecticidal crystal protein (ICP) is to be produced by the Bt genes encoding.There is homology in various degree in the aminoacid sequence of many ICP.1989, (53:241-255) about 42 genes that will find at that time according to the homology of the insecticidal spectrum of ICP and aminoacid sequence were divided into five big classes, 15 subclass for H fte H and Whiteley HR, Microbio.Rev. for H fte and Whiteley.The gene family (Cry) that wherein preceding four classes are crystallin, the 5th class is called as cytolysis protein gene (Cyt).The CryI genes encoding is to the virose CryI albumen of lepidopterous insects, the CryII genes encoding is to lepidopteran and the virose CryII albumen of dipteral insect, the CryIII genes encoding is to the virose CryIII albumen of coleopteron, and the CryIV genes encoding is to the virose CryIV albumen of dipteral insect.Because the continuous isolation identification of new killing gene, two-fold-classification method according to H fte and Whiteley, 1992, (Feitelson et al. such as Feitelson, Bio/Technology, 10:271-275,1992) original classification is replenished, the Bt gene is divided into 7 big classes, 29 subclass.Remove 5 original big classes, increased CryV and CryVI two big classes newly.Along with the continuous increase of new Bt gene dosage, it is found that original sorting technique exists amino acid identity and the conflicting problem of desinsection specificity.Therefore, do not have spondylopathy Neo-Confucianism in nineteen ninety-five and can set up the Bt unnamed gene council that forms by people such as Crickmore specially in the annual meeting, proposed with the insecticidal proteins amino acid sequence homology is the classification naming system of sole criterion, the Bt gene is divided into 17 big classes, 3 subclass (Crickmore et al, 1995), augmenting in 1996 is 21 big classes, 44 subclass; To on August 2nd, 2002, the Bt gene reached 42 big classes, 110 subclass, amounts to more than 200 Bt gene order.
Typical ICP is made up of two portions, the structure fragment of the active fragments of N end and C end, and the ICP that has structure fragment is called as parent toxin.It produces activated toxicity peptide through after the digestion of proteolytic enzyme.The someone points out recently, and the active fragments of holding at N is divided into toxicity district and cell land again.After the Bt insecticidal crystal protein is by the target insect's food-taking, produce the active toxin molecule through dissolving and two steps of enzymolysis, the active toxin that discharges can pass the direct and gastrointestinal epithelial cells effect in trophocyte space, insect midgut road.The cell land is with after the receptor-specific of insect midgut tract epithelial cell combines, and the toxicity district directly acts on cytolemma, makes membrane perforation, destroys the osmotic equilibrium of cell, causes the cracking of cell at last.
1987, (Vaeck et al. such as Vaeck, Nature, 328:33-37,1987), (Barton etal., Plant Physiol.85:1103-1109 such as Barton, 1987), (fischoff et al. such as Fischoff, Bio/Technology, 5:807-813,1987) obtained the trans Bt gene plant.But the insect-resistance of these early stage trans Bt gene plant that they obtain is all very weak, is difficult to detect transcribing of mRNA, and the protein expression amount is very low.Cause the low reason of Bt gene expression amount in plant to have many: for example, 1, be rich in the AT sequence, the mRNA instability of in plant, expressing in the wild Bt gene; Intron cleavage site, the transcription termination signal sequence that 2, may have eukaryotic gene in the wild Bt gene cause the abnormal processing of the imperfect or transcript of transcript; 3, microorganism and plant have very big-difference on the frequency of utilization to codon in translation, and translation efficiency is reduced; 4,5 ' of eukaryotic gene-UTR sequence and prokaryotic gene are very different, and 3 ' end of eukaryotic gene needs tailing recognition signal sequence.Therefore, make the Bt gene in transgenic plant, efficiently express, must effectively transform wild Bt gene.
Nineteen ninety, (Adang et al such as Adang, EP0359472,1990) by the A+T content of adjustment wild gene and the frequency of utilization of codon, itself and dicotyledon gene are consistent, having removed influences the AATGAA that gene is expressed in plant, synthesized a new Btt gene: the homology of new gene and protogene is 85%, and A+T content drops to the level (55%) of normal plants gene.Utilize the gene-transformed plant of transforming, the proteic expression amount of Bt is improved.
1991, (Perlak et al such as Perlak, PNAS USA, 7164 88:3324-3328 1991) under the situation that does not change the crystallin aminoacid sequence, the Cry1Ab gene has been carried out partly transforming or transforming fully by synthetic, selected the codon of plant-preference for use, part has been removed the element of interference base because of expressing in the former sequence in plant, as the ATTTA sequence, PM and FM gene have been obtained; As a result, the target protein expression amount acquisition of changeing PM and FM gene plant improves.
1992, people such as Guo's three heaps (Guo's three heaps etc., Chinese invention patent application number: 95119563.8) by the double chain synthesising DNA method, synthetic the GFM killing gene that merges of the Cry1Ab of total length 1824bp and Cry1Ac, the expression amount of Bt toxalbumin in plant increases substantially as a result, and complete synthesis gene has improved about 100 times than the expression amount of protogene.Afterwards, many scientific workers have carried out a large amount of transformation research to the Bt killing gene, and utilize the Bt killing gene of transforming to do a large amount of Plant Transformation work, lay the foundation for cultivating the Bt zoophobous.According to incompletely statistics, Bt gene (the being used for transgenic plant) patent 40 of existing so far part improvement or synthetic is multinomial.
The about 130kD of coded insect-killing crystallin of typical C ryI gene, and at present reforming composite most of CryI genes remove for part coded insect-killing crystallin C end dna sequence dna prescind gene.Three examples more than enumerating are the genetic modification of prescinding encoding sequence.To the transformation Cry1 Gene Sequence Analysis delivered as can be seen, keep the needs that the proteic dna sequence dna of original gene 5 ' the end about 70kB of coding just can satisfy plant transgene.
Summary of the invention:
The objective of the invention is to artificial reconstructed synthetic new Cry1C
*Gene (DNA) sequence.
The present invention realizes by following scheme:
A kind of Cry1C that is named as
*Bacillus thuringiensis (Bt) dna sequence dna, it has the sequence shown in the nucleotide coding sequence table SEQ ID NO:1.Also has the 5 ' sequence of holding the 3 ' tailing of holding shown in noncoding homing sequence and the sequence table SEQ ID NO:3 to discern shown in sequence table SEQ ID NO:2.
The C+G content of described coding nucleotide sequence SEQ ID NO:1 is 44.62%, with the homology of original DNA sequence be 84.0%.
The codon of described dna sequence dna is formed as shown in Figure 1.
Described dna sequence dna comprises the expression of this dna sequence dna in vegetable cell, further the application on the plant transgene breeding for pest resistance.
Concrete steps comprise:
(1) is source with Cry1Ca5, removes 128 amino acid whose 384 Nucleotide of 3 ' end coding, obtain 630 amino acid whose sequences that contain 1890 Nucleotide of 5 ' end coding.
(2) preferences of codon being used according to plant is keeping amino acid to form under the constant situation, replaces the codon of the sequence of 1890 Nucleotide with the plant gene high frequency subdivision that accesses to your password, and tentatively obtains the dna sequence dna of a transformation.
(3) get rid of exist in the dna sequence dna typically cause this unsettled AT of being rich in sequence of plant gene transcription and restriction endonuclease sites commonly used, the method by permutation cipher corrects then.
(4) carry out the Blast2 analysis with the normal chain and the corresponding minus strand of improved encoding sequence, get rid of in the gene by the method for permutation cipher and have big inverted repeats.
(5) determine Cry1C
*The encoding sequence of gene shown in sequence table SEQ ID NO:1.
(6) the Cry1C of determining
*5 ' end of the encoding sequence of gene adds the sequence shown in sequence table SEQ ID NO:2, in the sequence of 3 ' end interpolation shown in sequence table SEQ ID NO:3.
(7) add the restriction enzyme enzyme recognition site sequence that further clone needs at the sequence two ends.Final definite Cry1C
*The sequence of gene shown in sequence table SEQ ID NO:4.
(8) sequence of chemosynthesis shown in sequence table SEQ ID NO:4.
Below nucleotides sequence tabulation is described:
1, sequence table SEQ ID NO:1 is the Cry1C of design
*Encoding sequence
2, sequence table SEQ ID NO:2 is Cry1C
*5 ' end homing sequence:
3, sequence table SEQ ID NO:3 is Cry1C
*3 ' end tailing recognition signal and transcription termination sequence:
4, sequence table SEQ ID NO:4 is synthetic Cry1C
*Gene order
Accompanying drawing and explanation thereof:
Accompanying drawing 1: the comparative analysis that is the plant gene codon usage frequency that the present invention relates to
Accompanying drawing 2: the codon feature of the sequence of 1890 Nucleotide of 5 ' end of reforming composite Cry1C* of the present invention and former Cry1Ca5 relatively
Concrete embodiment:
From Genbank, get 984 plant gene coded sequences and 20 plant ribosome protein gene coding sequences of highly expressing, add up codon usage frequency respectively and handle as Fig. 1.Therefrom as can be seen, the 3rd of the codon of plant gene the is waved base preference use G or C.
Embodiment 2:Cry1C
*The codon feature of the sequence of 1890 Nucleotide of 5 ' end of encoding sequence and former Cry1Ca5 relatively
From Genbank, search the Cry1Ca sequence, obtain 6 altogether, be respectively Cry1Ca1, Cry1Ca2, Cry1Ca3, Cry1Ca4, Cry1Ca5 and Cry1Ca6.Therefrom select the shortest Cry1Ca5 of sequence, part is removed its 3 ' end encoding sequence, keeps the sequence of 630 amino acid whose 1890 Nucleotide of 5 ' end coding.According to table 1 statistics, adopt the access to your password corresponding codon of sequence of 1890 Nucleotide of subdivision displacement of plant gene high frequency, ATTTA, AATGAA etc. that part is removed wherein are rich in AT sequence and indefinite intron sequences, and get rid of big inverted repeats and the restriction enzyme enzyme recognition site sequence commonly used that exists in the gene; Design target synthetic Cry1C
*The encoding sequence of gene is shown in sequence table SEQ ID NO:1.Target synthetic Cry1C
*The codon feature of gene as shown in Figure 2.
Sequence and synthetic Cry1C with 1890 Nucleotide of former Cry1Ca5
*Encoding sequence carries out Blast2 to be analyzed, and the homology of two sequences is 84.0%.The statistics of based composition is: the C+G% of original gene is 36.55%, and the C+G% of new synthetic gene is 44.62%.The Blast2 of amino acid sequence coded analyzes and shows that both amino acid sequence coded are in full accord.
By structural analysis to 5 ' end homing sequence of plant gene, implementation sequence 2, this sequence and is added in Cry1C shown in sequence table sequence table SEQ ID NO:2
*5 ' end of gene coded sequence.The sequence of design is shown in sequence table SEQ ID NO:3 in addition, and this sequence is added on Cry1C
*3 ' end of gene coded sequence.
The further interpolation of clone's restriction enzyme enzyme recognition site sequence of embodiment 5
According to the needs that gene is further cloned, at 5 ' end interpolation BamHI restriction endonuclease recognition site sequence ggatcc of implementation sequence, 3 ' end adds SacI, BamHI and HindIII restriction endonuclease recognition site sequence gagctcggatccaagctt.
By above step, design reforming composite Cry1C
*Gene order is shown in sequence table sequence table SEQ ID NO:4.By this gene of chemosynthesis, be loaded on the plasmid vector pUC18 then.
With synthetic Cry1C
*Gene constructed to escherichia coli plasmid expression vector pGEX-KG, transformed into escherichia coli DHIOB; Inoculate single bacterium colony to 20mL LB culture medium culturing 4 hours, add IPTG abduction delivering reagent, continue to cultivate 2-3 hour; Centrifugal then collection thalline, adding 20mL sterilized water is resuspended; Liquid nitrogen freezes molten 6 times repeatedly, the centrifugal thalline that goes, supernatant liquor feeding lepidopterous insects cabbage caterpillar and striped rice borer; The toxicity qualification result sees Table 3 and table 4:
Table 3 synthetic gene expression product is identified the toxicity of cabbage caterpillar
| One, 24 hours statistics of feeding | Handle (three repetitions) | Examination borer population (only) | Average mortality (%) | Corrected mortality (%) |
| Clear water | ????18 | ????5.56 | ||
| Blank carrier | ????18 | ????11.11 | ????5.88 | |
| ????Cry1C * | ????18 | ????22.22 | ????17.64 | |
| Two, 48 hours statistics of feeding | Handle (three repetitions) | Examination borer population (only) | Average mortality (%) | Corrected mortality (%) |
| Clear water | ????18 | ????5.56 | ||
| Blank carrier | ????18 | ????22.22 | ????17.64 | |
| ????Cry1C * | ????18 | ????66.67 | ????64.71 |
Table 4 synthetic gene expression product is identified the toxicity of striped rice borer
| One, 48 hours statistics of feeding | Handle (three repetitions) | Examination borer population (only) | Average mortality (%) | Corrected mortality (%) |
| Clear water | ????30 | ????0 | ????0 | |
| Blank carrier | ????30 | ????10.0 | ????10.0 | |
| ????Cry1C * | ????30 | ????36.67 | ????36.67 | |
| Two, 72 hours statistics of feeding | Handle (three repetitions) | Examination borer population (only) | Average mortality (%) | Corrected mortality (%) |
| Clear water | ????30 | ????0 | ????0 | |
| Blank carrier | ????30 | ????16.67 | ????16.67 | |
| ????Cry1C * | ????30 | ????80.0 | ????78.57 |
Thuringiensis gene SEQ:ID:NO:4.WorkFileOrganization Applicant----------------------
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EmailAddress: zhanghb@mail.hzau.edu.cn <110> OrganizationName: Huazhong Agricultural University Application Project ------------------- <120> Title: transformation of synthetic Bacillus thuringiensis insecticidal crystal protein gene of Bacillus Cry1C * <130> AppFileReference: <140> CurrentAppNumber: <141> CurrentFilingDate :2002-09-20Sequence -------- <213> OrganismName: Bacillus thuringiensis (Bacillus thuringiensis) <400 > PreSequenceString: 1atggaggaga acaatcagaa ccagtgtatc ccttacaatt gtctttctaa tcctgaagaa 60gttcttttgg atggagaaag gatctcaact ggtaactcat caattgacat ctctctctca 120cttgttcagt tcttggtttc taactttgtg ccaggaggag gattccttgt tggacttatc 180gacttcgttt ggggaatcgt tggaccttct caatgggatg catttctcgt tcagatcgaa 240cagctcatca acgaaagaat cgctgagttc gctaggaatg ctgctattgc taaccttgaa 300ggacttggaa acaacttcaa catctacgtg gaggcattca aggaatggga agaagatcct 360aacaacccag caaccaggac cagagtgatc gataggttcc gtatccttga tggacttctt 420gaaagggaca ttcctagctt taggatctct ggatttgaag ttccacttct ctctgtttac 480gctcaagctg ctaatctcca tcttgctatc cttagagatt ctgtgatctt cggagaaaga 540tggggattga caaccatcaa cgtgaacgag aactacaaca gactcatcag gcacatcgat 600gagtacgctg atcactgtgc taacacttac aaccgtggac tcaacaacct tcctaagtct 660acctatcaag attggatcac atacaaccga cttaggagag accttacatt gactgttctt 720gatatcgctg ctttctttcc aaactatgac aataggagat atccaattca gccagttggt 780caacttacaa gggaagttta cactgaccca ctcatcaact tcaacccaca gcttcagtct 840gttgctcagc ttcctacctt caacgttatg gagagcagcg caatcagaaa tcctcacctc 900ttcgacatct tgaacaacct tacaatcttt accgattggt ttagtgttgg acgtaacttc 960tactggggag gacatcgagt gatctctagc ctcatcggag gtggtaacat cacatctcct 1020atctacggaa gagaggctaa ccaggagcct ccaagatcat tcactttcaa cggacctgtg 1080ttcaggactc tttcaaatcc tactcttcga cttcttcagc aaccttggcc agctccacca 1140ttcaaccttc gtggtgttga aggagttgag ttctctacac ctacaaacag cttcacctat 1200cgtggaagag gtactgttga ttctcttact gaacttccac ctgaggacaa cagtgtgcca 1260cctcgtgaag gatacagtca tcgtctttgt catgcaacct tcgttcaaag atctggaaca 1320cctttcctta caactggtgt tgtgttctct tggactcatc gtagtgcaac tcttaccaac 1380acaattgatc cagagaggat caaccagatc cctcttgtga aaggattcag agtttgggga 1440ggaacctctg tgattacagg accaggattc acaggaggtg atatccttcg aagaaacacc 1500tttggtgact tcgtttctct tcaagtgaac atcaactcac caatcaccca aagataccgt 1560...
Bacillus thuringiensis gene SEQ:ID:NO:4.WorkFilecttagatttc gttacgcttc tagtagggat gcacgagtta tcgttcttac aggagctgca 1620tctacaggag tgggaggtca agttagtgtg aacatgcctc ttcagaaaac tatggagatc 1680ggagagaacc tcacatctag aacattcaga tacaccgact tcagtaatcc tttctcattc 1740agagctaatc cagacatcat cggtatcagt gaacaacctc tcttcggtgc aggttctatc 1800agtagcggtg aactttacat cgacaagatc gagatcatcc ttgcagatgc aacatttgaa 1860gcagaatctg accttgaaag agcacaaaag tagtgaccaa cgtatttata tcagaaaata 1920gatgagtcga aattaaaagc ttatacccgt taatga 1956＜212〉Type:DNA＜211〉Length:1956
SequenceName：SEQ?ID?NO：1
SequenceDescription：Feature-------Sequence：SEQ?ID?NO：1：<221>FeatureKey：CDS<222>LocationFrom：1<222>LocationTo：1956
Other?Information：
CDSJoin:NoSequence--------＜213〉OrganismName: Tribactur (Bacillus thuringiensis)＜400〉PreSequenceString:2agactcactc tgagcgtcgt cacacgcagc ttgtgcggga tatcatttgc ctgtaaccgg 60tttccttaaa gcgaaaaccc ccccacccaa aggtaaggct 100＜212〉Type:DNA＜211〉Length:100
SequenceName：SEQ?ID?NO：2
SequenceDescription：Feature--------Sequence：SEQ?ID?NO：2：<221>FeatureKey：5′UTR<222>LocationFrom：1<222>LocationTo：100
Other?Information：
CDSJoin:NoSequence---------＜213〉OrganismName: thuringiensis (Bacillus thuringiensis)＜400〉PreSequenceString:3tgacgaattc ccgatctagt aacatagatg acaccgcgcg cgataattta tcctagtttg 60cgcgctatat tttgttttct atcgcgtatt aaatgtataa ttgcgggact ctaatcataa 120aaacccatct cataaataac gtcatgcacc tgaatagatc ttggacaagc gttaggccta 180tctgtgcatt acatgttaat tattacatgc ttaacgtaat tcaacagaaa ttatatgata 240
Thuringiensis gene SEQ ID NO:4.WorkFileatcatcgcaa gaccggcaac aggattcaat cttaagaaac tttattgcca aatgtttgaa 300cgatcgggga aattc 315＜212〉Type:DNA＜211〉Length:315
SequenceName：SEQ?ID?NO：3
SequenceDescription：Feature-------Sequence：SEQ?ID?NO：3：<221>FeatureKey：3′UTR<222>LocationFrom：1<222>LocationTo：315
Other?Information：
CDSJoin: NoSequence ---------- <213> OrganismName: Bacillus thuringiensis (Bacillus thuringiensis) <400> PreSequenceString: 4cggatccaga ctcactctga gcgtcgtcac acgcagcttg tgcgggatat catttgcctg 60taaccggttt ccttaaagcg aaaacccccc cacccaaagg taaggctatg gaggagaaca 120atcagaacca gtgtatccct tacaattgtc tttctaatcc tgaagaagtt cttttggatg 180gagaaaggat ctcaactggt aactcatcaa ttgacatctc tctctcactt gttcagttct 240tggtttctaa ctttgtgcca ggaggaggat tccttgttgg acttatcgac ttcgtttggg 300gaatcgttgg accttctcaa tgggatgcat ttctcgttca gatcgaacag ctcatcaacg 360aaagaatcgc tgagttcgct aggaatgctg ctattgctaa ccttgaagga cttggaaaca 420acttcaacat ctacgtggag gcattcaagg aatgggaaga agatcctaac aacccagcaa 480ccaggaccag agtgatcgat aggttccgta tccttgatgg acttcttgaa agggacattc 540ctagctttag gatctctgga tttgaagttc cacttctctc tgtttacgct caagctgcta 600atctccatct tgctatcctt agagattctg tgatcttcgg agaaagatgg ggattgacaa 660ccatcaacgt gaacgagaac tacaacagac tcatcaggca catcgatgag tacgctgatc 720actgtgctaa cacttacaac cgtggactca acaaccttcc taagtctacc tatcaagatt 780ggatcacata caaccgactt aggagagacc ttacattgac tgttcttgat atcgctgctt 840tctttccaaa ctatgacaat aggagatatc caattcagcc agttggtcaa cttacaaggg 900aagtttacac tgacccactc atcaacttca acccacagct tcagtctgtt gctcagcttc 960ctaccttcaa cgttatggag agcagcgcaa tcagaaatcc tcacctcttc gacatcttga 1020acaaccttac aatctttacc gattggttta gtgttggacg taacttctac tggggaggac 1080atcgagtgat ctctagcctc atcggaggtg gtaacatcac atctcctatc tacggaagag 1140aggctaacca ggagcctcca agatcattca ctttcaacgg acctgtgttc aggactcttt 1200caaatcctac tcttcgactt cttcagcaac cttggccagc tccaccattc aaccttcgtg 1260gtgttgaagg agttgagttc tctacaccta caaacagctt cacctatcgt ggaagaggta 1320ctgttgattc tcttactgaa cttccacctg aggacaacag tgtgccacct cgtgaaggat 1380acagtcatcg tctttgtcat gcaaccttcg ttcaaagatc tggaacacct ttccttacaa 1440ctggtgttgt gttctcttgg actcatcgta gtgcaactct taccaacaca attgatccag 1500agaggatcaa ccagatccct cttgtgaaag gattcagagt ttggggagga acctctgtga 1560ttacaggacc aggattcaca ggaggtgata tccttcgaag aaacaccttt ggtgacttcg 1620tttctcttca agtgaacatc aactcaccaa tcacccaaag ataccgtctt agatttcgtt 1680acgcttctag tagggatgca cgagttatcg ttcttacagg agctgcatct acaggagtgg 1740...
CDSJoin: NoSequence ---------- <213> OrganismName: Bacillus thuringiensis (Bacillus thuringiensis) <400> PreSequenceString: 4cggatccaga ctcactctga gcgtcgtcac acgcagcttg tgcgggatat catttgcctg 60taaccggttt ccttaaagcg aaaacccccc cacccaaagg taaggctatg gaggagaaca 120atcagaacca gtgtatccct tacaattgtc tttctaatcc tgaagaagtt cttttggatg 180gagaaaggat ctcaactggt aactcatcaa ttgacatctc tctctcactt gttcagttct 240tggtttctaa ctttgtgcca ggaggaggat tccttgttgg acttatcgac ttcgtttggg 300gaatcgttgg accttctcaa tgggatgcat ttctcgttca gatcgaacag ctcatcaacg 360aaagaatcgc tgagttcgct aggaatgctg ctattgctaa ccttgaagga cttggaaaca 420acttcaacat ctacgtggag gcattcaagg aatgggaaga agatcctaac aacccagcaa 480ccaggaccag agtgatcgat aggttccgta tccttgatgg acttcttgaa agggacattc 540ctagctttag gatctctgga tttgaagttc cacttctctc tgtttacgct caagctgcta 600atctccatct tgctatcctt agagattctg tgatcttcgg agaaagatgg ggattgacaa 660ccatcaacgt gaacgagaac tacaacagac tcatcaggca catcgatgag tacgctgatc 720actgtgctaa cacttacaac cgtggactca acaaccttcc taagtctacc tatcaagatt 780ggatcacata caaccgactt aggagagacc ttacattgac tgttcttgat atcgctgctt 840tctttccaaa ctatgacaat aggagatatc caattcagcc agttggtcaa cttacaaggg 900aagtttacac tgacccactc atcaacttca acccacagct tcagtctgtt gctcagcttc 960ctaccttcaa cgttatggag agcagcgcaa tcagaaatcc tcacctcttc gacatcttga 1020acaaccttac aatctttacc gattggttta gtgttggacg taacttctac tggggaggac 1080atcgagtgat ctctagcctc atcggaggtg gtaacatcac atctcctatc tacggaagag 1140aggctaacca ggagcctcca agatcattca ctttcaacgg acctgtgttc aggactcttt 1200caaatcctac tcttcgactt cttcagcaac cttggccagc tccaccattc aaccttcgtg 1260gtgttgaagg agttgagttc tctacaccta caaacagctt cacctatcgt ggaagaggta 1320ctgttgattc tcttactgaa cttccacctg aggacaacag tgtgccacct cgtgaaggat 1380acagtcatcg tctttgtcat gcaaccttcg ttcaaagatc tggaacacct ttccttacaa 1440ctggtgttgt gttctcttgg actcatcgta gtgcaactct taccaacaca attgatccag 1500agaggatcaa ccagatccct cttgtgaaag gattcagagt ttggggagga acctctgtga 1560ttacaggacc aggattcaca ggaggtgata tccttcgaag aaacaccttt ggtgacttcg 1620tttctcttca agtgaacatc aactcaccaa tcacccaaag ataccgtctt agatttcgtt 1680acgcttctag tagggatgca cgagttatcg ttcttacagg agctgcatct acaggagtgg 1740...
SequenceName：SEQ?ID?NO：4
SequenceDescription：Feature-------Sequence：SEQ?ID?NO：4：<221>FeatureKey：gene<222>LocationFrom：1<222>LocationTo：2407
Other?Information：
CDSJoin：No
Claims (5)
1, a reforming composite bacillus thuringiensis (Bt) gene C ry1C
*, it has the sequence shown in the Nucleotide nuclear code sequence tabulation SEQ ID NO:1, also has 5 ' the noncoding homing sequence of end shown in sequence table SEQ ID NO:2 and the tailing recognition sequence of 3 ' end shown in the sequence table SEQ ID NO:3
2, according to claim 1, reforming composite Cry1C
*Gene is characterized in that: the C+G content of encoding sequence is 44.64%, with the homology of original gene be 84.0%; Codon is formed as shown in Figure 2.
3, according to claim 1, comprising the method that obtains said gene.
4, according to claim 1, comprising middle transition carrier that contains said gene that makes up and the expression vector that is used for plant transgene.
5, according to claim 1, comprising the expression of this gene in vegetable cell; The further application on the plant transgene breeding for pest resistance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02139081 CN1219883C (en) | 2002-09-20 | 2002-09-20 | Engineering systhesized gene cry LC of pests-killing crytal protein of Bacillus thuringiensis Berliner |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02139081 CN1219883C (en) | 2002-09-20 | 2002-09-20 | Engineering systhesized gene cry LC of pests-killing crytal protein of Bacillus thuringiensis Berliner |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1483823A true CN1483823A (en) | 2004-03-24 |
| CN1219883C CN1219883C (en) | 2005-09-21 |
Family
ID=34147361
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02139081 Expired - Fee Related CN1219883C (en) | 2002-09-20 | 2002-09-20 | Engineering systhesized gene cry LC of pests-killing crytal protein of Bacillus thuringiensis Berliner |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1219883C (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060458A (en) * | 2013-01-17 | 2013-04-24 | 中国检验检疫科学研究院 | Primer, probe, kit and method for detecting transgenic rice strain T1c-19 |
| CN103074353A (en) * | 2011-10-26 | 2013-05-01 | 华中农业大学 | Synthetic Bt bivalent fusion protein with high virulence against lepidoptera, and coding gene thereof |
| CN103421840A (en) * | 2013-08-01 | 2013-12-04 | 华中农业大学 | Method for improving resistance of rape to Lepidoptera pests by transgenic technology |
| CN103421838A (en) * | 2013-04-19 | 2013-12-04 | 华中农业大学 | Cultivating method for transgenic lepidoptera pest resistant rice |
| CN103667340A (en) * | 2013-12-05 | 2014-03-26 | 华中农业大学 | Construction method for anti-insect transgene corn with stable heredity |
| CN104292314A (en) * | 2013-07-19 | 2015-01-21 | 中国科学院亚热带农业生态研究所 | Codon optimized Cry1Ca<#> gene, recombinant vector and method for improving crop resistance |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NZ591992A (en) | 2005-10-14 | 2012-11-30 | Resmed Ltd | Breathing mask with cushion attached to frame via lip of cushion engaging within recess between frame outer and inner walls, and guided in via angled protrusion of frame inner wall |
| US8517023B2 (en) | 2007-01-30 | 2013-08-27 | Resmed Limited | Mask system with interchangeable headgear connectors |
| EP2837398B1 (en) | 2008-03-04 | 2018-08-22 | ResMed Limited | Mask system |
-
2002
- 2002-09-20 CN CN 02139081 patent/CN1219883C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103074353A (en) * | 2011-10-26 | 2013-05-01 | 华中农业大学 | Synthetic Bt bivalent fusion protein with high virulence against lepidoptera, and coding gene thereof |
| CN103060458A (en) * | 2013-01-17 | 2013-04-24 | 中国检验检疫科学研究院 | Primer, probe, kit and method for detecting transgenic rice strain T1c-19 |
| CN103421838A (en) * | 2013-04-19 | 2013-12-04 | 华中农业大学 | Cultivating method for transgenic lepidoptera pest resistant rice |
| CN104292314A (en) * | 2013-07-19 | 2015-01-21 | 中国科学院亚热带农业生态研究所 | Codon optimized Cry1Ca<#> gene, recombinant vector and method for improving crop resistance |
| CN103421840A (en) * | 2013-08-01 | 2013-12-04 | 华中农业大学 | Method for improving resistance of rape to Lepidoptera pests by transgenic technology |
| CN103667340A (en) * | 2013-12-05 | 2014-03-26 | 华中农业大学 | Construction method for anti-insect transgene corn with stable heredity |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1219883C (en) | 2005-09-21 |
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