CN102604953B - BmCP274 promoter of bombyx mori cuticular protein, recombinant expression vector and application of the promoter - Google Patents
BmCP274 promoter of bombyx mori cuticular protein, recombinant expression vector and application of the promoter Download PDFInfo
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- CN102604953B CN102604953B CN 201210085855 CN201210085855A CN102604953B CN 102604953 B CN102604953 B CN 102604953B CN 201210085855 CN201210085855 CN 201210085855 CN 201210085855 A CN201210085855 A CN 201210085855A CN 102604953 B CN102604953 B CN 102604953B
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Abstract
The invention discloses a BmCP274 promoter of bombyx mori cuticular protein. The promoter consists of nucleotide sequences shown in SEQ ID No.1, can drive foreign proteins to specifically express at a front silk gland of the bombyx mori and can be used as a specific promoter at the front silk gland of the silkworm. The invention further discloses a recombinant expression vector containing the promoter, which provides a powerful tool for a next-step study on regulating ion channel protein expression at the front silk gland of the silkworm to affect the behavior of the bombyx mori and improve the performance of the silk fiber.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of tissue-specific promoter and expression vector thereof and application.
Background technology
The silky fibre of silkworm output under natural condition is a kind of filamentary material that has widespread use in fields such as textile industry, biomedicines.Spider has similar spinning body to silkworm, but the silky fibre of its output has stronger hardness and outstanding toughness than silk.Silky fibre formation in vivo is an extremely complicated process, does not all have so far to obtain to illustrate fully.Existing synthon are all the processes of the natural product silk of manual simulation insect, synthesize external, but its mechanical property also far can not compare with spider silk fiber.If can make silkworm efficiently, the class spider silk fiber of a large amount of high-qualitys of output specifically, have very important significance for obtaining of high-quality filamentary material.The silkworm anterior division of silkgland is the place that nematic liquid crystal forms, and ionic strength has vital role to the formation of silky fibre.Utilize for example some ion path albumen of silkworm anterior division of silkgland of silkworm anterior division of silkgland specific promoter overexpression foreign protein, silky fibre character is changed, obtain better silk fiber.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of silkworm epidermal protein promotor and recombinant expression vector and application, this promotor can make foreign protein specific expressed at the silkworm anterior division of silkgland, utilize this promotor to regulate and control some ion path protein expression at the silkworm anterior division of silkgland, silky fibre character is changed, obtain better silk fiber.
For achieving the above object, the invention provides following technical scheme:
1, silkworm epidermal protein BmCP274 promotor is comprised of nucleotide sequence shown in SEQ ID No.1.
2, the recombinant expression vector that contains described silkworm epidermal protein BmCP274 promotor.
further, described recombinant expression vector is pBac[BmCP274-expressing gene-SV40, 3xP3EGFP], will be by silkworm epidermal protein BmCP274 promotor, the expression cassette [BmCP274-expressing gene-SV40] that expressing gene sequence and SV40 termination signal form inserts in the multiple clone site of shuttle vectors pSLfa1180fa, obtain recombinant vectors pSL[BmCP274-expressing gene-SV40], use again Asc I from recombinant vectors pSL[BmCP274-expressing gene-SV40] downcut [BmCP274-expressing gene-SV40] fragment, with the carrier pBac[3xP3-EGFPafm that cuts through Asc I enzyme equally] be connected, and get final product.
Further, described expressing gene is red fluorescent protein DeRed gene.
3, described silkworm epidermal protein BmCP274 promotor is as the application of silkworm anterior division of silkgland specificity promoter.
beneficial effect of the present invention is: the present invention utilizes the proteomics means to identify an epidermal protein BmCP274 special at the silkworm anterior division of silkgland and a large amount is expressed, its promoter sequence is cloned, and take the DsRed gene as representing gene constructed by the DsRed expression vector of BmCP274 promoter regulation, this expression vector microinjection is entered to have obtained the transgenic bombyx mori positive individuals in Silkworm, Bombyx mori, research to this transgenic bombyx mori positive individuals is found, BmCP274 promoters driven DsRed is at silkworm anterior division of silkgland specifically expressing, therefore, the BmCP274 promotor can be used as silkworm anterior division of silkgland specificity promoter and uses.Utilize the BmCP274 promotor to regulate and control some ion path protein expression at the silkworm anterior division of silkgland, silky fibre character is changed, obtain better silk fiber, ion path protein expression by regulation and control silkworm anterior division of silkgland affects house silkworms spin silk behavior and improvement silky fibre performance lays the foundation thereby the present invention is for next step research, and the recombinant expression vector of the BmCP274 of containing promotor provided by the invention provides strong instrument for above-mentioned research.
Description of drawings
Fig. 1 is the photo of BmCP274 promotor transgenic bombyx mori different times under white light and fluorescence irradiation, wherein a and b are respectively the ovum under white light and fluorescence irradiation, c and d are respectively the adult under white light and fluorescence irradiation, and e and f are respectively the Cocoon shell under white light and fluorescence irradiation.
Fig. 2 is the sericterium internal anatomy of BmCP274 promotor transgenic bombyx mori, and wherein a and b are respectively the transgenic bombyx mori sericterium under white light and fluorescence irradiation, and the non-transgenic silkworm that c and d are respectively under white light and fluorescence irradiation is made greatly sericterium.
Fig. 3 is that the Southern blot of BmCP274 promotor transgenic bombyx mori analyzes, wherein 1 is Tran 2K plus DNA Marker, 2 is to make greatly silkworm moth DNA through the non-transgenic silkworm that Bgl II enzyme is cut, 3 for the transgenic bombyx mori G1 that cuts through Bgl II enzyme for silkworm moth DNA, 4 is enhanced green fluorescence protein (EGFP) gene (positive control).
Fig. 4 is the forwardly Western blot analysis of sericterium specifically expressing DsRed of BmCP274 promotor transgenic bombyx mori, and wherein 1 is transgenic bombyx mori anterior division of silkgland albumen, and 2 make greatly anterior division of silkgland albumen for the non-transgenic silkworm.
Embodiment
In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing.The experimental technique of unreceipted actual conditions in embodiment, usually according to normal condition, the condition described in molecular cloning experiment guide (third edition, J. Pehanorm Brooker etc. work) for example, or the condition of advising according to manufacturer.
The cultivated silkworm breed variety that uses in embodiment is for making greatly, provided by Southwest China university domestic silkworm gene resources bank.
One, the clone of silkworm epidermal protein BmCP274 promotor
According to sequence nscaf1681 in domestic silkworm gene group database, design 1 pair of upstream and downstream primer.The primer of design entrusts Sangon Biotech (Shanghai) Co., Ltd. to synthesize.Primer sequence is as follows:
274-F:5'-tt
GtcgacGtagcctttacataatatgccg-3'(SEQ ID No.2, underscore are partly Sal I restriction enzyme site);
274-R:5'-ga
AgatctCtttcctggtgatcgatgg-3'(SEQ ID No.3, underscore are partly Bgl II restriction enzyme site).
Take the 3rd day five ages make greatly genomic dna as template, adopt primer 2 74-F and 274-R amplification BmCP274 promoter fragment (SEQ ID No.1).The PCR reaction conditions is: 94 ℃ of denaturations 4 minutes; Then 94 ℃ of sex change are 40 seconds, 51 ℃ of annealing 40 seconds, and 72 ℃ were extended 1.5 minutes, totally 24 circulations; Last 72 ℃ were extended 10 minutes.The PCR product carries out agarose gel electrophoresis to be separated, and cuts glue and reclaims purifying purpose fragment, then be cloned into carrier pMD19-T Simple(TaKaRa), obtain recombinant vectors pMD19-BmCP274.
Two, the structure of the DsRed expression vector of BmCP274 promoter regulation
With recombinant vectors pMD19-BmCP274 Sal I and Bgl II double digestion, reclaim the BmCP274 promoter fragment, again with the same carrier pSL[MCS-DsRed-SV40 of being connected with Bgl II double digestion through Sal I] be connected, obtain recombinant vectors pSL[BmCP274-DsRed-SV40].Described carrier pSL[MCS-DsRed-SV40] be reference literature method (Horn and Wimmer, 2000), utilize pSLfa1180fa clone shuttle vectors, adopt two step cloning to build and get, concrete construction process is seen Chinese patent application 20110423280.3.
With recombinant vectors pSL[BmCP274-DsRed-SV40] cut with Asc I enzyme, reclaim the BmCP274-DsRed-SV40 fragment, again with the carrier pBac[3xP3-EGFPafm that cuts through Asc I enzyme equally] be connected, obtain recombinant vectors pBac[BmCP274-DsRed-SV40,3xP3EGFP].Described carrier pBac[3xP3-EGFPafm] be that reference literature method (Horn and Wimmer, 2000) builds and gets.
Three, the acquisition of the DsRed expression vector transgenic bombyx mori of BmCP274 promoter regulation
with recombinant vectors pBac[BmCP274-DsRed-SV40, 3xP3EGFP] mix with the transgenosis assistant carrier pHA3PIG equimolar ratio of coding piggyBac transposase, made greatly body early embryo (the last laying eggs 2 ~ 5 hours by what microinjection entered termination of diapause, G0 generation) in, the nontoxic glue sealing of silkworm seed after injection, 25 ℃ are hastened the hatching of silkworms to hatching, the larva of hatching adopts artificial diet to raise, carry out the selfing production of hybrid seeds to adult, the silkworm seed of the 7th day ovum phase that obtains (G1 generation) utilizes wavelength under the stereoscopic fluorescent microscope of macroscopic view (Olympus MVX10) be the exciting light detection green fluorescence of 460 ~ 490nm, filter out the transgenic positive individual (Fig. 1 a-d) that excites green fluorescence at eyes or neural specific, namely obtain BmCP274 promotor transgenic bombyx mori.
Choose at random BmCP274 promotor transgenic bombyx mori G1 for silkworm moth, extract genomic dna and carry out Southern blot detection, result as shown in Figure 3, the insertion copy number of EGFP is 1, DsRed and EGFP are in same transposon zone, it inserts copy number is also 1, shows that the swivel base event has occured in the genome of BmCP274 promotor transgenic bombyx mori 1 time the piggyBac transposable element that carries DsRed.
Four, the DsRed expression vector of BmCP274 promoter regulation is at the specifically expressing of transgenic bombyx mori anterior division of silkgland
1, period and the tissue specificity of Fluirescence observation DsRed in transgenic bombyx mori
Choose at random BmCP274 promotor transgenic bombyx mori, begin to carry out continuous fluoroscopic examination in Late Embryogenesis, found that, just can observe anterior division of silkgland and send red fluorescence when newly-hatched silkworm, along with the growth of transgenic bombyx mori, red fluorescence is more and more stronger afterwards.
With five age first day BmCP274 promotor transgenic bombyx mori dissect rear discovery, red fluorescence is to send and asexuality difference from anterior division of silkgland, and do not observe red fluorescence in other tissue such as middle intestines, sexual gland etc., do not observe red fluorescence (Fig. 1 e, f) in the cocoon shell of transgenic bombyx mori yet.The sericterium of BmCP274 promotor transgenic bombyx mori and non-transgenic silkworm is dissected rear discovery, red fluorescence (Fig. 2) only just can be detected in the anterior division of silkgland of BmCP274 promotor transgenic bombyx mori.
2, RT-PCR detects period and the tissue specificity of DsRed gene in transgenic bombyx mori
Take from respectively the transgenic bombyx mori that plays silkworm second day in period to pupa time five ages individual, and five ages the 3rd day transgenic bombyx mori each tissue sample, grind fast and extract total RNA in liquid nitrogen, reverse transcription becomes cDNA, employing DsRed gene specific primer DsRed-F:5'-atggtgcgctcctccaagaacg-3'(SEQ ID No.4); DsRed-R:5'-ctacaggaacaggtggtggc-gg-3'(SEQ ID No.5) ］ carry out RT-PCR and detect, take Actin3 as contrast.The PCR reaction conditions is: 94 ℃ of denaturations 4 minutes; Then 94 ℃ of sex change are 15 seconds, 65 ℃ of annealing 30 seconds, and 72 ℃ were extended 1 minute, totally 24 circulations; Last 72 ℃ were extended 10 minutes.The PCR product adopts 1% agarose gel electrophoresis to detect.Result shows, aspect the period specificity, DsRed gene silkworm from five ages had a large amount to express in 12 hours period to being placed on small straw bundles to spin cocoons, and after certainly being placed on small straw bundles to spin cocoons 12 hours, expression amount sharply descends; Aspect tissue specificity, the DsRed gene is only expressed at the silkworm anterior division of silkgland.
3, Western blot detects the expression of DsRed in the transgenic bombyx mori anterior division of silkgland
Get respectively the BmCP274 promotor transgenic bombyx mori in the 3rd day five ages and the anterior division of silkgland of non-transgenic silkworm, grind fast in liquid nitrogen and be dissolved in the 8mol/L urea buffer solution, placed 1 hour for 4 ℃, the centrifuging and taking supernatant is measured total protein concentration, then is mixed with 5 * sample loading buffer, 100 ℃ of sex change 10 minutes, carry out 10% SDS-polyacrylamide glue gel electrophoresis, after electrophoresis is complete, take Anti-DsRed antibody as primary antibodie, Tubulin antibody analyzes as internal reference antibody carries out Western blot.Result as shown in Figure 4, DsRed expresses in the anterior division of silkgland of BmCP274 promotor transgenic bombyx mori, and does not express in non-transgenic silkworm anterior division of silkgland.
Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and not depart from the spirit and scope of the present invention that appended claims limits.
＜110〉Southwestern University
＜120〉silkworm epidermal protein BmCP274 promotor and expression vector thereof and application
<160> 5
<210> 1
<211> 1564
<212> DNA
＜213〉silkworm (Bombyx mori Linnaeus)
<220>
＜223〉silkworm epidermal protein BmCP274 promoter sequence
<400> 1
ctaatttatt ttttttatat ttcccaaagt tttggtagcc tttacataat atgccggtcc 60
ggcgtctgtg ggacggcggg atggatgtca tatgctctac gtcaaccctg tcccgccgtc 120
tcaatagccc cgactgggct ccggcccggt ccggggtagg gcgccggccg tgagcggcag 180
aactttatag tcatcgcggc gagtgggcta tctgggatca actcccacat agcctactcg 240
ccgcgcgggt ggggatccac tgattctcac ctgtgaaaaa aaatccttta tacgccatac 300
tcgtatactt taaagttgtc atttttacgg accactttca atcaatcaag tactaagtat 360
tcgtgacgac agaaatattg gtaataacta gtaatactat agattcttct tctttctaag 420
tcataaataa gatataatct tagtttaaat attttaaact aagattatat tatcttatta 480
tataattttg cttgtgaaca ttgtatatca acatttataa catcaatata acaaagaacg 540
ttccattata cttaataata gcccagtaca caaaaaatta ctaataccaa gcgaatagag 600
aggttaataa attgttttaa aattattcta catatttttt gtaacacagc aatagaaaaa 660
tatgtaaaca ggcgagggag atcactgccc acctcatata gtgaggccaa ccttaggaca 720
tgaggtctga atctcaattg taccttcaaa ccgaaacata ttagtacttt atggcagaag 780
tagacagggt ggtgatacct acccatactc aaaagatatt aaaccatcat aataatatac 840
atgtgtgtaa tataaaatca caagaacatt taacaaagag tagaacacta atgagaaaaa 900
atccatatgc atatggttcc aaattcaaat ctaatgctat ttttgctgta gcagtattta 960
tggccagact atgtaaatac gtctaatgat caccaagttc cataacattg catgaaaagt 1020
ccgcagtaca tactttcaac caaaaggttt tcaacagaac gagttctact tccattcaag 1080
gtggatattt agagcaaaac tacggcagct aaatctatgt atcttactca tttttctttc 1140
ttggttcagt gcacaccgta gtcacgtaat caatatttat agcttttggc aataaataga 1200
agtttcagga taagaaattt cacagaattg gaaaatttgt gtacgcaaaa tgtagaaatg 1260
tagagctccc tacaattttg caaaagttac gcactggttg gactaaagaa aacaaaaccg 1320
attttcatgc tattttacct acttttgtgt atcacgtgct ctgaatacaa gaaagagaac 1380
acggtgcctg ttttaaatct tcattaacat tttaattgtc ctcttctatt catctagtct 1440
gcgacattga tggtttgttt cggtattacc gaacagcata ttttggcaca acaaagcgcc 1500
ttttataagg cgtgccacat tctgaaaaat atcagttgtc ttccatcgat caccaggaaa 1560
gatg 1564
<210> 2
<211> 30
<212> DNA
＜213〉artificial sequence
<220>
＜223〉amplimer 274-F
<400> 2
ttgtcgacgt agcctttaca taatatgccg 30
<210> 3
<211> 27
<212> DNA
＜213〉artificial sequence
<220>
＜223〉amplimer 274-R
<400> 3
gaagatctct ttcctggtga tcgatgg 27
<210> 4
<211> 22
<212> DNA
＜213〉artificial sequence
<220>
＜223〉amplimer DsRed-F
<400> 4
atggtgcgct cctccaagaa cg 22
<210> 5
<211> 22
<212> DNA
＜213〉artificial sequence
<220>
＜223〉amplimer DsRed-R
<400> 5
ctacaggaac aggtggtggc gg 22
Claims (5)
1. silkworm epidermal protein BmCP274 promotor, is characterized in that, is comprised of nucleotide sequence shown in SEQ ID No.1.
2. the recombinant expression vector that contains the described silkworm epidermal protein of claim 1 BmCP274 promotor.
3. recombinant expression vector according to claim 2, it is characterized in that, described recombinant expression vector is pBac[BmCP274-expressing gene-SV40, 3xP3EGFP], will be by silkworm epidermal protein BmCP274 promotor, the expression cassette [BmCP274-expressing gene-SV40] that expressing gene sequence and SV40 termination signal form inserts in the multiple clone site of shuttle vectors pSLfa1180fa, obtain recombinant vectors pSL[BmCP274-expressing gene-SV40], use again Asc I from recombinant vectors pSL[BmCP274-expressing gene-SV40] downcut [BmCP274-expressing gene-SV40] fragment, with the carrier pBac[3xP3-EGFPafm that cuts through Asc I enzyme equally] be connected, and get final product.
4. recombinant expression vector according to claim 3, is characterized in that, described expressing gene is red fluorescent protein DsRed gene.
5. silkworm epidermal protein BmCP274 promotor claimed in claim 1 is as the application of silkworm anterior division of silkgland specificity promoter.
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| CN104513821B (en) * | 2013-09-27 | 2017-06-06 | 西南大学 | The human acid fibroblast growth factor gene and its recombinant vector of transformation and application |
| CN103555761B (en) * | 2013-10-28 | 2015-05-13 | 西南大学 | Silkworm calcium ion-ATP (adenosine triphosphate) enzyme gene recombination expression vector 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 |
| CN112359061A (en) * | 2020-11-16 | 2021-02-12 | 西南大学 | Method for improving mechanical property of silk fiber and product thereof |
| CN112522266B (en) * | 2020-12-10 | 2022-06-10 | 西南大学 | Promoter of bombyx mori pebrine induced expression gene BmPGT 2 and application thereof |
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| CN101139598A (en) * | 2007-08-07 | 2008-03-12 | 西南大学 | Promoter for cultivated silkworm pupal period specification expression gene |
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Non-Patent Citations (6)
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| A LARVAL SERUM PROTEIN OF THE SILKWORM,BOMBYX MORI:cDNA SEQUENCE AND DEVELOPMENTAL SPECIFICITY OF THE TRANSCRIPT;YOSHIHIRO FUJIWARA et al.;《Insect Biochem》;19911231;第21卷(第7期);735-742 * |
| BmLSP基因启动子驱动DsRed在转基因家蚕中的表达分析;邓党军等;《蚕业科学》;20111231;第37卷(第2期);200-205 * |
| YOSHIHIROFUJIWARAetal..ALARVALSERUMPROTEINOFTHESILKWORM BOMBYX MORI:cDNA SEQUENCE AND DEVELOPMENTAL SPECIFICITY OF THE TRANSCRIPT.《Insect Biochem》.1991 |
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| 彭云等.带有丝素重链信号肽序列的家蚕丝胶蛋白启动子驱动DeRed的瞬时分泌表达.《昆虫学报》.2009,第52卷(第11期),1177-1182. |
| 邓党军等.BmLSP基因启动子驱动DsRed在转基因家蚕中的表达分析.《蚕业科学》.2011,第37卷(第2期),200-205. |
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