CN102604953B - BmCP274 promoter of bombyx mori cuticular protein, recombinant expression vector and application of the promoter - Google Patents

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

Bombyx mori epidermal protein BmCP274 promoter and its recombinant expression vector and application

technical field

The invention belongs to the field of biotechnology, and relates to a tissue-specific promoter, its expression vector and its application.

Background technique

The silk fiber produced by the silkworm under natural conditions is a fiber material widely used in textile industry, biomedicine and other fields. Spiders and silkworms have similar spinning bodies, but the silk fibers produced by them have stronger hardness and better toughness than silkworm silk. The formation of silk fibers in living organisms is an extremely complicated process, which has not been fully elucidated so far. Existing synthetic fibers are artificially simulated in the natural silk production process of insects and synthesized in vitro, but their mechanical properties are far from being comparable to spider silk fibers. If the silkworm can be efficiently and specifically produced a large amount of high-quality spider silk-like fibers, it will be of great significance for the acquisition of high-quality fiber materials. The anterior silk gland of the silkworm is the site for the formation of nematic liquid crystals, and the ionic strength plays an important role in the formation of silk fibers. The overexpression of exogenous proteins such as some ion pathway proteins in the anterior silk gland of the silkworm by using the specific promoter of the anterior silk gland of the silkworm may change the properties of silk fibers and obtain better silk fibers.

Contents of the invention

In view of this, the purpose of the present invention is to provide a kind of Bombyx mori epidermal protein promoter and its recombinant expression vector and application, this promoter can make exogenous protein express specifically in silk gland of silkworm anterior part, utilize this promoter in silkworm front Regulating the expression of some ion pathway proteins in the external silk glands may change the properties of silk fibers and obtain better silk fibers.

To achieve the above object, the present invention provides the following technical solutions:

1. The Bombyx mori epidermal protein BmCP274 promoter consists of the nucleotide sequence shown in SEQ ID No.1.

2. A recombinant expression vector containing the promoter of the silkworm epidermal protein BmCP274.

Further, the recombinant expression vector is pBac[BmCP274-expression gene-SV40, 3xP3EGFP], which is to insert the expression frame [BmCP274-expression gene-SV40] composed of the silkworm epidermal protein BmCP274 promoter, expression gene sequence and SV40 termination signal into the shuttle In the multiple cloning site of the vector pSLfa1180fa, the recombinant vector pSL[BmCP274-expression gene-SV40] was obtained, and the [BmCP274-expression gene-SV40] fragment was cut out from the recombinant vector pSL[BmCP274-expression gene-SV40] with Asc I , connected with the vector pBac[3xP3-EGFPafm] which was also digested with Asc I, to obtain.

Further, the expressed gene is the red fluorescent protein DeRed gene.

3. The use of the silkworm epidermal protein BmCP274 promoter as a silkworm anterior silk gland-specific promoter.

The beneficial effect of the present invention is that: the present invention identifies an epidermal protein BmCP274 that is specific and highly expressed in the anterior silk gland of the silkworm by means of proteomics, clones its promoter sequence, and constructs it using the DsRed gene as a representative gene The DsRed expression vector regulated by the BmCP274 promoter was obtained, and the expression vector was microinjected into the silkworm body to obtain a transgenic silkworm positive individual. The study of the transgenic silkworm positive individual found that the BmCP274 promoter drives DsRed to be specific in the anterior silk gland of the silkworm. Therefore, the BmCP274 promoter can be used as a silkworm anterior silk gland-specific promoter. Utilizing the BmCP274 promoter to regulate the expression of certain ion pathway proteins in the anterior silk gland of the silkworm may change the properties of silk fibers and obtain better silk fibers. The expression of pathway proteins to influence silkworm silking behavior and improve silk fiber performance has laid a foundation, and the recombinant expression vector containing BmCP274 promoter provided by the present invention provides a powerful tool for the above research.

Description of drawings

Figure 1 is the photos of the BmCP274 promoter transgenic silkworm at different stages under white light and fluorescent irradiation, where a and b are eggs under white light and fluorescent irradiation respectively, c and d are adults under white light and fluorescent irradiation respectively, e and f are respectively Bombyx mori cocoon shell under white light and fluorescent light.

Figure 2 is an anatomical diagram of the silk gland of BmCP274 promoter transgenic silkworm, where a and b are transgenic silk glands under white light and fluorescent irradiation, respectively, and c and d are non-transgenic silkworm silk glands under white light and fluorescent irradiation, respectively.

Figure 3 is the Southern blot analysis of BmCP274 promoter transgenic silkworm, in which 1 is Tran 2K plus DNA Marker, 2 is the non-transgenic silkworm DNA digested by Bgl Ⅱ, and 3 is the transgenic silkworm G1 generation silkworm digested by Bgl Ⅱ DNA, 4 is the enhanced green fluorescent protein (EGFP) gene (positive control).

Figure 4 is a Western blot analysis of the specific expression of DsRed in the anterior silk gland of the BmCP274 promoter transgenic silkworm, in which 1 is the anterior silk gland protein of the transgenic silkworm, and 2 is the anterior silk gland protein of the non-transgenic silkworm.

Detailed ways

In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings. For the experimental methods not specified in the examples, the conventional conditions are generally followed, such as the conditions described in the Molecular Cloning Experiment Guide (Third Edition, J. Sambrook et al.), or the conditions suggested by the manufacturer.

The silkworm species used in the examples is Dazao, provided by the Silkworm Gene Resource Bank of Southwest University of China.

1. Cloning of Bombyx mori epidermal protein BmCP274 promoter

According to the sequence nscaf1681 in the silkworm genome database, a pair of upstream and downstream primers were designed. The designed primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The primer sequences are as follows:

274-F: 5'-tt gtcgac gtagcctttacataatatgccg-3' (SEQ ID No.2, the underlined part is the Sal I restriction site);

274-R: 5'-ga agatct ctttcctggtgatcgatgg-3' (SEQ ID No. 3, the underlined part is the restriction site of Bgl II).

Using Dazao genomic DNA on the third day of the fifth instar as a template, primers 274-F and 274-R were used to amplify the BmCP274 promoter fragment (SEQ ID No.1). The PCR reaction conditions were: pre-denaturation at 94°C for 4 minutes; then denaturation at 94°C for 40 seconds, annealing at 51°C for 40 seconds, extension at 72°C for 1.5 minutes, a total of 24 cycles; and finally extension at 72°C for 10 minutes. The PCR product was separated by agarose gel electrophoresis, the target fragment was recovered and purified by cutting the gel, and then cloned into the vector pMD19-T Simple (TaKaRa) to obtain the recombinant vector pMD19-BmCP274.

2. Construction of DsRed expression vector regulated by BmCP274 promoter

The recombinant vector pMD19-BmCP274 was digested with Sal I and Bgl II, and the BmCP274 promoter fragment was recovered, and then connected with the vector pSL[MCS-DsRed-SV40] which was also digested with Sal I and Bgl II, to obtain the recombinant vector pSL[BmCP274-DsRed-SV40]. The vector pSL[MCS-DsRed-SV40] is constructed by referring to the literature method (Horn and Wimmer, 2000), using pSLfa1180fa to clone the shuttle vector, and adopting a two-step cloning method. For the specific construction method, see Chinese patent application 20110423280.3.

The recombinant vector pSL[BmCP274-DsRed-SV40] was digested with Asc I, and the BmCP274-DsRed-SV40 fragment was recovered, and then ligated with the vector pBac[3xP3-EGFPafm] also digested with Asc I to obtain the recombinant vector pBac[BmCP274 -DsRed-SV40, 3xP3EGFP]. The vector pBac[3xP3-EGFPafm] was constructed according to the literature method (Horn and Wimmer, 2000).

3. Obtaining of DsRed expression vector transgenic silkworm regulated by BmCP274 promoter

The recombinant vector pBac[BmCP274-DsRed-SV40, 3xP3EGFP] was mixed with the transgene helper vector pHA3PIG encoding piggyBac transposase in an equimolar ratio, and microinjected into Dazao early embryos that had been released from diapause (2-5 hours after oviposition , G0 generation), the injected silkworm eggs were sealed with non-toxic glue, accelerated to hatch at 25°C, and the hatched larvae were fed with artificial feed, and then selfed for seed production after adulthood. The obtained silkworm eggs on the seventh day of the egg stage Eggs (G1 generation) were detected with green fluorescence under a macroscopic stereoscopic fluorescence microscope (Olympus MVX10) using excitation light with a wavelength of 460-490nm, and the transgene-positive individuals that specifically excited green fluorescence in the eyes or nerves were screened out (Fig. 1 a-d), namely Obtain BmCP274 promoter transgenic silkworm.

The BmCP274 promoter transgenic silkworm G1 silkworm moth was randomly selected, and the genomic DNA was extracted for Southern blot detection. The results are shown in Figure 3. The insertion copy number of EGFP is 1, and DsRed and EGFP are in the same transposon region. The copy number was also 1, indicating that the piggyBac transposable element carrying DsRed had a transposition event in the genome of the BmCP274 promoter transgenic silkworm.

4. Specific expression of the DsRed expression vector regulated by the BmCP274 promoter in the anterior silk gland of the transgenic silkworm

1. Fluorescent observation of the period and tissue specificity of DsRed in transgenic silkworms

The BmCP274 promoter transgenic silkworm was randomly selected, and continuous fluorescence detection was carried out at the later stage of embryonic development. It was found that red fluorescence could be observed from the anterior silk gland in the ant silkworm, and then with the development of the transgenic silkworm, the red fluorescence became more and more intense. stronger.

After dissecting the BmCP274 promoter transgenic silkworm on the first day of the fifth instar, it was found that the red fluorescence was emitted from the anterior silk gland and there was no gender difference, while no red fluorescence was observed in other tissues such as the midgut and gonad. No red fluorescence was observed in the cocoon shells (Fig. 1e, f). After dissecting the silk glands of BmCP274 promoter transgenic silkworms and non-transgenic silkworms, red fluorescence could only be detected in the anterior silk glands of BmCP274 promoter transgenic silkworms (Figure 2).

2. RT-PCR detection of the stage and tissue specificity of DsRed gene in transgenic silkworm

The individual transgenic silkworms from the fifth instar to the second day of the pupal stage, and the tissue samples of transgenic silkworms on the third day of the fifth instar were taken respectively, and were quickly ground in liquid nitrogen to extract total RNA, reverse transcribe into cDNA, and use DsRed gene-specific primers [DsRed-F: 5'-atggtgcgctcctccaagaacg-3' (SEQ ID No.4); DsRed-R: 5'-ctacaggaacaggtggtggc-gg-3' (SEQ ID No.5)] for RT-PCR detection , with Actin3 as the control. The PCR reaction conditions were: pre-denaturation at 94°C for 4 minutes; then denaturation at 94°C for 15 seconds, annealing at 65°C for 30 seconds, and extension at 72°C for 1 minute, a total of 24 cycles; finally, extension at 72°C for 10 minutes. PCR products were detected by 1% agarose gel electrophoresis. The results showed that, in terms of stage specificity, the DsRed gene was highly expressed from the fifth instar silkworm stage to 12 hours in the stacking, and the expression level decreased sharply after 12 hours in the stacking; in terms of tissue specificity, the DsRed gene was only expressed in the silkworm Anterior silk gland expression.

3. Western blot detection of the expression of DsRed in the anterior silk gland of the transgenic silkworm

The anterior silk glands of BmCP274 promoter transgenic silkworms and non-transgenic silkworms on the third day of the fifth instar were taken respectively, quickly ground in liquid nitrogen and dissolved in 8mol/L urea buffer, placed at 4°C for 1 hour, centrifuged to obtain the supernatant, Determine the total protein concentration, then mix with 5× loading buffer, denature at 100°C for 10 minutes, and perform gel electrophoresis on 10% SDS-polyacrylamide gel. After electrophoresis, use Anti-DsRed antibody as the primary antibody and Tubulin antibody as the Internal reference antibody was used for Western blot analysis. The results are shown in Figure 4. DsRed was expressed in the anterior silk gland of the BmCP274 promoter transgenic silkworm, but not in the anterior silk gland of the non-transgenic silkworm.

Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described with reference to the preferred embodiments of the present invention, those skilled in the art should understand that it can be described in the form Various changes may be made in matter and details thereof without departing from the spirit and scope of the invention as defined in the appended claims.

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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 tccatcaag 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

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ttgtcgacgt agcctttaca taatatgccg 30

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gaagatctct ttcctggtga tcgatgg 27

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Claims (5)

1. Bombyx mori epidermal protein BmCP274 promoter, is characterized in that, is made up of nucleotide sequence shown in SEQ ID No.1. 2. The recombinant expression vector containing the Bombyx mori epidermal protein BmCP274 promoter of claim 1. 3. recombinant expression carrier according to claim 2, is characterized in that, described recombinant expression carrier is pBac[BmCP274-expression gene-SV40, 3xP3EGFP], is to be terminated by silkworm epidermal protein BmCP274 promotor, expression gene sequence and SV40 The expression cassette [BmCP274-expressed gene-SV40] composed of the signal was inserted into the multiple cloning site of the shuttle vector pSLfa1180fa to obtain the recombinant vector pSL[BmCP274-expressed gene-SV40], and then Asc I was used to obtain the recombinant vector pSL[BmCP274-expressed gene-SV40]. -SV40], the [BmCP274-expression gene-SV40] fragment was cut out, and connected with the vector pBac[3xP3-EGFPafm] which was also digested with Asc I, to obtain final product. 4. The recombinant expression vector according to claim 3, wherein the expression gene is a red fluorescent protein DsRed gene. 5. The application of the silkworm epidermal protein BmCP274 promoter as claimed in claim 1 as the silkworm anterior silk gland-specific promoter.

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