CN102296070A - Bombyx mori midgut specific high-level expression promoter P2 and use thereof - Google Patents
Bombyx mori midgut specific high-level expression promoter P2 and use thereof Download PDFInfo
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Abstract
The invention relates to gene engineering, in particular to a bombyx mori gene promoter, namely bombyx mori midgut specific high-level expression promoter P2 represented by SEQ ID No.1 or SEQ ID No.2, and the use thereof in specific expression of foreign proteins in bombyx mori midgut. The invention also relates to the recombinant vector of the bombyx mori midgut specific high-level expression promoter P2 and the preparation method thereof. The promoter is capable of specifically promote the high-level expression of a downstream gene in bombyx mori midgut, and provides a powerful tool for the study and utilization of a bombyx mori midgut specific gene, particularly midgut specific high-level expression immune resistance associated gene; and meanwhile, the promoter can realize the specific high-level expression of the foreign gene in the bombyx mori midgut, and thus, has a bright application prospect.
Description
Technical Field
The invention relates to gene engineering, in particular to a bombyx mori gene promoter. .
Background
The silkworm is an insect with important economic value, and has important roles in theoretical research, production and application as a model organism of lepidoptera. The midgut is an important immune tissue organ of the silkworm, a plurality of pathogens can infect the silkworm by eating the silkworm, and after the silkworm eats the pathogens, the midgut is the first infected tissue organ and is also the first defense line of the silkworm. At present, the research on the midgut of silkworms is less, and the main reason is that available research means are limited. With the successive completion of the silkworm genome frame map and the fine map drawing work, the research and the knowledge of human beings on silkworms have entered the posterior genome era, and with the breakthrough and the improvement of the silkworm transgenic technology, transgenes have become powerful tools for the research of genes and the utilization of genes of silkworms, but the transgenic technology also needs the cooperation of other research tools to better exert the efficacy. Many silkworm midgut specific expression genes are expressed only in the midgut and are expressed in a very high amount in the midgut, and according to the results of information analysis, the midgut specific high expression genes are found to have very important functions, including a plurality of genes related to silkworm immune resistance, and a silkworm midgut specific high expression promoter is required for researching and utilizing the silkworm midgut specific high expression genes through transgenosis, but no related report of the silkworm midgut specific high expression promoter exists at present.
Disclosure of Invention
It is an object of the present invention to provide a promoter which can be expressed in a specific high amount in the midgut of Bombyx mori.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the silkworm midgut specific promoter P2 and the silkworm midgut specific promoter P2 contain the nucleotide sequence shown as SEQ ID NO: 1.
Further, the bombyx mori midgut specific promoter P2 is shown as SEQ ID NO: 2.
The invention also aims to provide a recombinant vector which contains a silkworm midgut specific promoter P2 and can ensure the specificity expression of the silkworm midgut.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the recombinant vector containing the silkworm midgut specific promoter P2.
Further, the recombinant vector is a recombinant expression vector P2-EGFP-SV40-1180, wherein P2 represents a promoter P2, the upstream and the downstream of the promoter P2 respectively contain SalI and BamHI enzyme digestion sites, EGFP-SV40-1180 is specifically a pSfa 1180fa vector containing an EGFP T clone and an SV40 terminator, the upstream and the downstream of the EGFP T clone respectively contain BamHI and Not I enzyme digestion sites, and the upstream and the downstream of the SV40 terminator respectively contain Not I and HindIII enzyme digestion sites.
Further, the recombinant vector is a recombinant expression vector pBac [ P2-EGFP-SV40-3 xP 3-DsRed af ], the recombinant expression vector is formed by connecting P2-EGFP-SV40 and pBac [3 xP 3-DsRed af ], the P2-EGFP-SV40 is obtained by using a restriction enzyme AscI to cut P2-EGFP-SV40-1180, and the pBac [3 xP 3-DsRed af ] is a 5' end dephosphorylated linear fragment cut by the AscI.
The invention further aims to provide application of the promoter P2, and the application provides a new idea for expression of the silkworm midgut exogenous protein.
The silkworm midgut specific promoter P2 is applied to the application of the silkworm midgut specific expression exogenous protein.
The technical scheme is carried out through the following technical route: screening a gene BGIBMGA014298 specifically expressed in high quantity in the midgut of the silkworm, and taking a genome sequence of the gene ATG (1080 bp ahead) for analysis to obtain a gene shown as SEQ ID NO:1 and SEQ ID NO:2, the sequence contains silkworm midgut specific promoter P2; an expression vector pBac [ P2-EGFP-SV40-3 xP 3-DsRed af ] is prepared, the expression vector is injected into silkworm eggs by a microscope, the silkworm eggs are hatched and raised to moth killing, only the midgut has strong green fluorescence by a fluorescence microscope, and the EGFP with high expression quantity is detected only by the midgut through RT-PCR and fluorescent quantitative PCR detection.
The invention has the beneficial effects that: through the combination of experimental means such as information analysis, RT-PCR, fluorescent quantitative PCR, silkworm egg embryo microinjection, microscope fluorescence observation and the like, the specific high-level expression promoter of the middle intestine of the silkworm is identified by utilizing transgenic cloning for the first time. The promoter can be specifically expressed in silkworm midgut at a high level, and provides a powerful tool for researching and utilizing specific genes of the silkworm midgut, particularly immune resistance related genes specifically expressed in a high level in the midgut; meanwhile, the recombinant expression vector can specifically express exogenous genes in the midgut of the silkworm in high quantity, and has good application prospect.
Drawings
Fig. 1 is a photograph under a microscope of a transgenic silkworm and a negative control silkworm subjected to microinjection, in which a is a photograph under a bright field of the transgenic silkworm, B is a photograph under a bright field of the negative control silkworm, C is a photograph under a bright field of the transgenic silkworm, and D is a photograph under GFP excitation light of the transgenic silkworm.
Fig. 2 is a microscopic photograph of the dissected transgenic silkworm and the dissected negative control silkworm after microinjection, in which a is a photograph under a dissected transgenic silkworm bright field, B is a photograph under a dissected negative control silkworm bright field, C is a photograph under GFP excitation light of the dissected transgenic silkworm, and D is a photograph under GFP excitation light of the dissected negative control silkworm.
Fig. 3 is a photograph under microscope of midgut of a transgenic silkworm and a midgut of a negative control silkworm subjected to microinjection, in which a is a photograph under a microscope bright field of the negative control silkworm, B is a photograph under GFP excitation light of the negative control silkworm, C is a photograph under a microscope bright field of the midgut of the transgenic silkworm, and D is a photograph under GFP excitation light of the midgut of the transgenic silkworm.
FIG. 4 is a diagram of RT-PCR, in which FIG. A is a transgenic silkworm, FIG. B is a negative control silkworm, wherein 1 represents blood, 2 represents fat body, and 3 represents midgut.
In the above figures, the transgenic silkworms refer to silkworms that were successfully subjected to microinjection of pBac [ P2-EGFP-SV40-3 XP 3-DsRed af ], and the negative control silkworms refer to silkworms that were not subjected to any treatment.
Detailed Description
Example 1: construction of P2-mediated EGFP expression vector P2-PR
(1) The method comprises the steps of screening genes specifically expressed in the middle and intestine of silkworms by using silkworm chip data (http:// silkwork. swu. edu. cn/silkdb /) in a silkworm genome database, selecting the first 5 genes with the highest expression quantity, downloading CDS sequences and ETS sequences of the 5 genes in the silkworm genome database, and respectively designing specific detection primers by using software Primer Premier 5.0. And (2) carrying out RT-PCR detection by using cDNA templates of various tissues of 5-day old and 3-day old, wherein the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 4 min, denaturation at 94 ℃ for 40 sec, annealing at 55 ℃ for 40 sec, extension at 72 ℃ for 20 sec for 30 cycles, and final extension at 72 ℃ for 10 min. The PCR product is detected by agarose gel electrophoresis, and the BGIBMGA014298 gene (the gene has the full length of 672bp and comprises 7 exons and 6 introns) is indeed specifically expressed in high quantity in the midgut of the silkworm. Detection primers BGIBMGA 014298F: 5 'GATTTGAACCACCGCAGTAT 3', BGIBMGA014298 (SEQ ID NO: 3) R: 5'CGTTGACGGCGACAGAAG3' (SEQ ID NO: 4);
(2) in the silkworm genome database, the translation initiation site ATG of the gene is positioned at the 1081 th base of scaffold1148, therefore, a genome sequence 1080bp before ATG of the gene is selected for analysis, and the gene is found through website prediction (http: html), there are typical promoter structural regions in this sequence, therefore, a specific primer was designed (P2F: 5 'ACGCGTCGACGTCCCCCATCCAGCAGTC 3' (SEQ ID NO: 5) P2R: 5 'cgGGATCCTTTTTTGCTGAAAGAAACGTACA 3' (SEQ ID NO: 6) the 1080bp sequence P2 was cloned, taking a large genome as a template, performing PCR amplification by using a P2 primer to obtain a target strip, recovering a PCR product, connecting the recovered PCR product with a pMD19-T Simple vector, transforming DH5 alpha competent cells, obtaining positive clones, and sending the positive clones to Shanghai Sangon for sequencing, wherein a sequencing result shows that an amplified sequence is consistent with an expected result;
the sequence of the bombyx mori midgut specific expression promoter P2 obtained by the method is shown as SEQ ID NO: 2.
gtcccccatccagcagtcctggtcggtggggacgaagtacggctccgcagcgacagcgacgtcgattgaccactccgccatggtgtggacgaggagatcctgtgccctggcggagtggttcaaattactttgaaggaagcgatggacacaacccattacggggctacatccatcgctccctcgtctgttccgccctgcggctcgacaacagccgcggcgggaactgactcgccggctgcccttgttgcagccggcttctttttctttccgcccctcctcgtgtttgtagaggagggggcggacttgcacgccgggcccccggcccggtggtcagccttccttttagccgcgtcgcacaagacgcagtgcggcgcggctgcggtgcaggaggctgccttgtgccccggttggccgcagcggaagcacaggttgctgcggtccccgatagctgttgtcctatccaattccatttttttttcaagtaagccaaatttggagtatgacaacgtattaagactaaataatttcaggcatcttgaagtaatgccatacatgtattaaagaacagttcaaaaacatttcatgaatcttgacataataaatatatctttaatgtcttaaatgaactcatcacgaaaatgaacgaaaactctgcaataaggatgataatatttacttctttgtttcagtaatattttccaaatttatcaccaatctatcgatttcgtgattatcacgtctcaatttaattttgttttcactttgaaatgtaataatatacacatttcaatcagataatcttgatcaagattgttttaatgtacgtcagctaatagagaatacgttatcagcttaagcgtaggaaacagTATAAAtaccgaatgaaaattcaataaatcGtacacatttatttggtgaggtaagagcatttgtgtttctcagggaaacactgaaaattacataaattttaacttctgttctctctatcaaaacaatttcaaatcataccagtttaaatcgcaggtgccaagtaacatttatacttcattattgtacgtttctttcagcaaaaaatcgacaATG
Wherein, Grepresents a transcription initiation site; ATGindicating the translation initiation site, TATAAA represents the TATA box. Then the silkworm midgut specific promoter P2 contains a sequence shown in SEQ ID No:1, and the nucleotide sequence shown in the figure:
gtcccccatccagcagtcctggtcggtggggacgaagtacggctccgcagcgacagcgacgtcgattgaccactccgccatggtgtggacgaggagatcctgtgccctggcggagtggttcaaattactttgaaggaagcgatggacacaacccattacggggctacatccatcgctccctcgtctgttccgccctgcggctcgacaacagccgcggcgggaactgactcgccggctgcccttgttgcagccggcttctttttctttccgcccctcctcgtgtttgtagaggagggggcggacttgcacgccgggcccccggcccggtggtcagccttccttttagccgcgtcgcacaagacgcagtgcggcgcggctgcggtgcaggaggctgccttgtgccccggttggccgcagcggaagcacaggttgctgcggtccccgatagctgttgtcctatccaattccatttttttttcaagtaagccaaatttggagtatgacaacgtattaagactaaataatttcaggcatcttgaagtaatgccatacatgtattaaagaacagttcaaaaacatttcatgaatcttgacataataaatatatctttaatgtcttaaatgaactcatcacgaaaatgaacgaaaactctgcaataaggatgataatatttacttctttgtttcagtaatattttccaaatttatcaccaatctatcgatttcgtgattatcacgtctcaatttaattttgttttcactttgaaatgtaataatatacacatttcaatcagataatcttgatcaagattgttttaatgtacgtcagctaatagagaatacgttatcagcttaagcgtaggaaacagtataaataccgaatgaaaattcaataaatc
example 2 recombinant vector containing Bombyx mori midgut specific promoter P2
The Bombyx mori midgut specific promoter P2T clone described in example 1 and shown in SEQ ID NO:2 was cloned into a vector containing SalI and BamHI cleavage sites at the upstream and downstream, and P2-mediated EGFP expression vector P2-EGFP-SV40-1180 was constructed using the constructed vector 1180-EGFP-SV40 (the EGFP T clone containing BamHI and Not I cleavage sites at the upstream and downstream, and SV40 termination signal sequences containing Not I and HindIII cleavage sites at the upstream and downstream, respectively, were added to pSLfa1180fa vector).
Then, restriction enzyme AscI is used for respectively digesting P2-EGFP-SV40-1180 and pBac [3 xP 3-DsRed af ], and because pSLfa1180fa vector skeleton contains an AscI site at both ends of a multiple cloning site, the cut EGFP expression skeleton structure can be connected with pBac [3 xP 3-DsRed af ] linear fragment which is dephosphorylated at the 5' end after digestion by AscI to form the final P2 mediated EGFP transgenic expression vector pBac [ P2-EGFP-SV40-3 xP 3-DsRed af ].
Example 3 promoter functional verification
The function verification of the silkworm midgut specific expression promoter P2 was performed by using the recombinant expression vector pBac [ P2-EGFP-SV40-3 XP 3-DsRed af ] obtained in example 2 as a microinjection vector.
Pickling silkworm eggs of large silkworm breeds to remove diapause, placing the silkworm eggs in a dark environment with 15 ℃ and 80% humidity for incubation, collecting the newly-hatched silkworms, placing the newly-hatched silkworms in a standard environment for feeding (the temperature is 25 ℃ and the humidity is 80%), mating male and female silkworms for 4 hours after moth dissolving, and taking the eggs laid after dismantling as non-diapause silkworms for the next microinjection;
the laid silkworm eggs are arranged neatly on a clean glass slide, P2-PR and an auxiliary plasmid A3H are injected into 205 large silkworm eggs by an Eppendorf microinjection instrument 2 hours after the silkworm eggs are delivered, the silkworm eggs are sealed by non-toxic glue and then placed in an environment with the temperature of 25 ℃ and the relative humidity of 80% for hatching, 139G 0 hatched silkworms are collected and fed to moths by mulberry leaves (detailed in figure 1), 29 moth rings G1 silkworm eggs are obtained by selfing or backcrossing of the G0 silkworm moths, 5 positive moth rings are observed and screened by an Olympus electric macroscopic fluorescence microscope, and the conversion efficiency is 17.20%.
And feeding the obtained positive moth pens into respective single moth pens, subculturing and expanding to obtain 5P 2 transgenic systems, and randomly selecting 2 transgenic systems (P2-1 and P2-2) for fluorescence observation. The green fluorescence of the middle intestine was observed even through the blood and epidermis of P2 under the fluorescence microscope (results are shown in fig. 2); the body wall is cut off from the tail angle of the silkworm larva along the back center line towards the head by using scissors, the body cavity is opened, and the body walls at two sides are fixed in a wax tray by obliquely inserting a pin, which is shown in figure 3: in the P2 transgenic system, the intestine had intense green fluorescence, and other tissues had no green fluorescence.
Taking tissue materials of P2-1 such as midgut, fat body and blood, extracting RNA, and reverse transcribing into cDNA. Performing RT-PCR detection by using specific primers of EGFP, wherein the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 4 min, denaturation at 94 ℃ for 40 sec, annealing at 57 ℃ for 40 sec, extension at 72 ℃ for 10 sec for 30 cycles, and final extension at 72 ℃ for 10 min. The PCR product is detected by agarose gel electrophoresis, as shown in FIG. 4, the expression of EGFP is detected only in the midgut and the expression level is very high, which indicates that P2 is really a bombyx mori midgut specific high-level expression promoter.
Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that, while the invention has been described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
<110> university of southwest
<120> bombyx mori midgut specific high-level expression promoter P2 and application thereof
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<213>Silkworm (silkworm)Bombyx moriL.)
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<223> truncation of specific promoter P2 for specific high-level expression of silkworm midgut
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gtcccccatc cagcagtcct ggtcggtggg gacgaagtac ggctccgcag cgacagcgac 60
gtcgattgac cactccgcca tggtgtggac gaggagatcc tgtgccctgg cggagtggtt 120
caaattactt tgaaggaagc gatggacaca acccattacg gggctacatc catcgctccc 180
tcgtctgttc cgccctgcgg ctcgacaaca gccgcggcgg gaactgactc gccggctgcc 240
cttgttgcag ccggcttctt tttctttccg cccctcctcg tgtttgtaga ggagggggcg 300
gacttgcacg ccgggccccc ggcccggtgg tcagccttcc ttttagccgc gtcgcacaag 360
acgcagtgcg gcgcggctgc ggtgcaggag gctgccttgt gccccggttg gccgcagcgg 420
aagcacaggt tgctgcggtc cccgatagct gttgtcctat ccaattccat ttttttttca 480
agtaagccaa atttggagta tgacaacgta ttaagactaa ataatttcag gcatcttgaa 540
gtaatgccat acatgtatta aagaacagtt caaaaacatt tcatgaatct tgacataata 600
aatatatctt taatgtctta aatgaactca tcacgaaaat gaacgaaaac tctgcaataa 660
ggatgataat atttacttct ttgtttcagt aatattttcc aaatttatca ccaatctatc 720
gatttcgtga ttatcacgtc tcaatttaat tttgttttca ctttgaaatg taataatata 780
cacatttcaa tcagataatc ttgatcaaga ttgttttaat gtacgtcagc taatagagaa 840
tacgttatca gcttaagcgt aggaaacagt ataaataccg aatgaaaatt caataaatc 899
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<213>Silkworm (silkworm)Bombyx moriL.)
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<223> Bombyx mori midgut specific high-level expression promoter P2
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gtcccccatc cagcagtcct ggtcggtggg gacgaagtac ggctccgcag cgacagcgac 60
gtcgattgac cactccgcca tggtgtggac gaggagatcc tgtgccctgg cggagtggtt 120
caaattactt tgaaggaagc gatggacaca acccattacg gggctacatc catcgctccc 180
tcgtctgttc cgccctgcgg ctcgacaaca gccgcggcgg gaactgactc gccggctgcc 240
cttgttgcag ccggcttctt tttctttccg cccctcctcg tgtttgtaga ggagggggcg 300
gacttgcacg ccgggccccc ggcccggtgg tcagccttcc ttttagccgc gtcgcacaag 360
acgcagtgcg gcgcggctgc ggtgcaggag gctgccttgt gccccggttg gccgcagcgg 420
aagcacaggt tgctgcggtc cccgatagct gttgtcctat ccaattccat ttttttttca 480
agtaagccaa atttggagta tgacaacgta ttaagactaa ataatttcag gcatcttgaa 540
gtaatgccat acatgtatta aagaacagtt caaaaacatt tcatgaatct tgacataata 600
aatatatctt taatgtctta aatgaactca tcacgaaaat gaacgaaaac tctgcaataa 660
ggatgataat atttacttct ttgtttcagt aatattttcc aaatttatca ccaatctatc 720
gatttcgtga ttatcacgtc tcaatttaat tttgttttca ctttgaaatg taataatata 780
cacatttcaa tcagataatc ttgatcaaga ttgttttaat gtacgtcagc taatagagaa 840
tacgttatca gcttaagcgt aggaaacagt ataaataccg aatgaaaatt caataaatcg 900
tacacattta tttggtgagg taagagcatt tgtgtttctc agggaaacac tgaaaattac 960
ataaatttta acttctgttc tctctatcaa aacaatttca aatcatacca gtttaaatcg 1020
caggtgccaa gtaacattta tacttcatta ttgtacgttt ctttcagcaa aaaatcgaca 1080
atg 1083
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<213> Artificial sequence
<220>
<223> primer BGIBMGA 014298F
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gatttgaacc accgcagtat 20
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<213> Artificial sequence
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<223> primer BGIBMGA 014298R
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cgttgacggc gacagaag 18
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<213> Artificial sequence
<220>
<223> primer P2F
<400> 5
acgcgtcgac gtcccccatc cagcagtc 28
<210> 6
<211> 31
<212> DNA
<213> Artificial sequence
<220>
<223> primer P2R
<400> 6
cgggatcctt ttttgctgaa agaaacgtac a 31
Claims (6)
1. The silkworm midgut specific promoter P2 is characterized in that the silkworm midgut specific promoter P2 contains a nucleotide sequence shown as SEQ ID NO: 1.
2. The silkworm midgut-specific promoter P2 of claim 1, wherein the silkworm midgut-specific promoter P2 is as set forth in SEQ ID NO: 2.
3. A recombinant vector comprising the bombyx mori midgut specific promoter P2 according to claim 1 or 2.
4. The recombinant vector of the silkworm midgut specific promoter P2 of claim 3, wherein the recombinant vector is a recombinant expression vector P2-EGFP-SV40-1180, wherein P2 represents a promoter P2, the upstream and downstream of the promoter P2 respectively contain SalI and BamHI enzyme cleavage sites, EGFP-SV40-1180 specifically is a pSLfa1180fa vector containing an EGFP TA clone and an SV40 terminator, the upstream and downstream of the EGFP T clone respectively contain BamHI and Not I enzyme cleavage sites, and the upstream and downstream of the SV40 terminator respectively contain Not I and HindIII enzyme cleavage sites.
5. The recombinant vector of the bombyx mori midgut specific promoter P2 according to claim 3, wherein the recombinant vector is a recombinant expression vector pBac [ P2-EGFP-SV40-3 xP 3-DsRed af ], the recombinant expression vector is formed by connecting EGFP and pBac [3 xP 3-DsRed af ], the EGFP is obtained by using a restriction enzyme AscI to cut P2-EGFP-SV40-1180, and the pBac [3 xP 3-DsRed af ] is a 5' dephosphorylated linear fragment cut by AscI.
6. The use of the bombyx mori midgut specific promoter P2 of claim 1 or 2 for specifically expressing a foreign protein in the bombyx mori midgut.
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| CN102925484A (en) * | 2012-11-21 | 2013-02-13 | 西南大学 | Application and recombinant vector of bombyx mori nuclear polyhedrosis virus polygene inverted repeat sequence and bombyx mori lipase-1 gene |
| CN103757023A (en) * | 2014-01-23 | 2014-04-30 | 西南大学 | Ecdysone-replying silkworm vitellogenin promoter as well as preparation method and application thereof |
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| 《BMC Biotechnology》 20060616 Abhimanyu Sarkar Insulated piggyBac vectors for insect transgenesis 1-9 5 第6卷, 第27期 * |
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| CN102925484A (en) * | 2012-11-21 | 2013-02-13 | 西南大学 | Application and recombinant vector of bombyx mori nuclear polyhedrosis virus polygene inverted repeat sequence and bombyx mori lipase-1 gene |
| CN102925484B (en) * | 2012-11-21 | 2014-09-10 | 西南大学 | Application and recombinant vector of bombyx mori nuclear polyhedrosis virus polygene inverted repeat sequence and bombyx mori lipase-1 gene |
| CN103757023A (en) * | 2014-01-23 | 2014-04-30 | 西南大学 | Ecdysone-replying silkworm vitellogenin promoter as well as preparation method and application thereof |
| CN103757023B (en) * | 2014-01-23 | 2015-09-23 | 西南大学 | Silkworm egg xanthan protein promoter of response moulting hormone and its preparation method and application |
| CN105886511A (en) * | 2016-06-24 | 2016-08-24 | 南阳师范学院 | Bombyx mori BmP56 gene promoter as well as recombinant expression vector and application thereof |
| CN105886511B (en) * | 2016-06-24 | 2018-11-06 | 南阳师范学院 | Silkworm BmP56 gene promoters and its recombinant expression carrier and application |
| CN107151668A (en) * | 2017-06-30 | 2017-09-12 | 西南大学 | A kind of authentication method of the promoter with silkworm haemocyte activity specific |
| CN112852825A (en) * | 2021-01-27 | 2021-05-28 | 南阳师范学院 | Antheraea pernyi midgut specific gene ApLITAF and promoter and application thereof |
| CN112852825B (en) * | 2021-01-27 | 2023-05-26 | 南阳师范学院 | Tussah midgut specific gene ApLITAF and promoter and application thereof |
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