CN102191270B - Pseudobagrus-fulvidraco-richardson-origin transgenic vectors constructed with elements of pseudobagrus fulvidraco richardson [beta]-actin promoter and growth hormone gene, and application thereof - Google Patents

Abstract

The invention discloses a beta-actin promoter, a growth hormone gene and its 3'-untranslated region sequence of the yellow catfish and a native transgenic carrier of the yellow catfish constructed using the elements. The present invention obtains the β-actin promoter sequence and the sequence of exon 1 and part of exon 2 of the yellow catfish, and also obtains the amino acid coding sequence and the 3'-untranslated region of the growth hormone gene of the yellow catfish sequence, and the obtained yellow catfish β-actin promoter, growth hormone coding gene and 3'-untranslated region were used as elements to construct the yellow catfish native growth hormone transgene vector. The present invention constructs the transgenic vector by using the promoter of the β-actin of the yellow catfish itself to drive the expression of its own growth hormone gene, which can effectively avoid the biological safety problems faced by the transgenic fish. Breeding and artificial propagation have great practical significance and application prospects.

Description

The original transgenic vector of yellow catfish constructed with the promoter of β-actin and growth hormone gene of yellow catfish and its application

technical field

The present invention relates to the cloning, recombination and promoter activity analysis and application of the promoter of the β-actin of the yellow catfish, the growth hormone gene and the 3'-untranslated region sequence, and constructs the original transgene of the yellow catfish as an element The carrier belongs to the field of genetic engineering. the

Background technique

Yellow catfish (Pseudobagrus fulvidraco Richardson) is commonly known as Angzi, yellow wax ding. Belongs to Siluriformes, Cullidae, Peeled catfish. The fish is a warm-water fish that lives on the bottom. The individual is usually about 30-100 grams, and it is widely distributed in inland waters of my country. Its body surface is smooth and phosphorus-free, the meat is tender, without intermuscular spines, the protein content is 15.37%, the total amino acid content is 14.19%, the essential amino acid index reaches 73.34, the lysine content is high, the taste is delicious, and it has high nutritional value. Its meat tastes sweet and flat, and has the effects of expelling wind and diuresis, and is very popular among consumers. From the perspective of aquatic economy, yellow catfish is a high-quality and high-grade fish with a mature consumer market at home and abroad. In recent years, its average price has been rising steadily, generally ranging from 20 to 40 yuan/kg. The annual sales volume in the Nanjing market alone has reached more than 700,000 kilograms, and the sales volume in Suxichang and Shanghai is even greater. In the international market, yellow catfish is very popular among consumers in Southeast Asian countries. South Korea, in particular, imports hundreds of tons of yellow catfish from my country every year. It is an excellent variety for export to earn foreign exchange. However, the yellow catfish currently on the market mainly comes from the fishing of wild resources. Because the fish grows slowly and the individual size is relatively small, it usually takes two years to reach sexual maturity. Commercial fish is not only small in quantity but also small in size (mostly about 50 grams), which is far from satisfying the demand of the market. the

In order to solve these problems, domestic scholars have made many beneficial attempts, mainly focusing on human breeding and domestication, species improvement and optimization of breeding environment. Transformation and directional cultivation of new breeds are the most promising method; what is more important is that the bait coefficient of transgenic fish with growth hormone is low, the breeding cycle is short, and it can be marketed in the same year, which can significantly reduce the cost of fish farming. At present, the growth hormone gene-transformed fish has shown good effects in accelerating the growth of the fish body, increasing the size of the fish body, and improving the utilization rate of bait. In fish genetic engineering breeding, the "whole fish" technology is adopted, that is, all the transgenes come from the recipient fish's own strains, and the large-scale transgenic fish produced are considered to ensure the biosafety, ecological environment safety, food safety The best choice after factors such as safety and consumer recognition. At present, many European countries have approved the application of genetically modified fish in feed, and my country's genetically modified carp has also been successfully produced. However, the research and development of transgenic yellow catfish has not yet been involved in literature and research, so the construction of the original transgenic vector of yellow catfish has a broad prospect of economic benefits. the

Contents of the invention

The purpose of the present invention is to provide the promoter of the yellow catfish β-actin, the growth hormone gene and its 3'-untranslated region sequence, and the native growth hormone transgene vector of the yellow catfish constructed using these elements. the

The purpose of the present invention is achieved through the following technical solutions:

1. Cloning of the β-actin promoter of yellow catfish:

(1) The present invention designs an upstream primer P1 in the homologous conserved region according to the existing fish β-actin promoter sequence on GenBank, and its position is near the CAAT box. Afterwards, the sequences of the exons were compared, and a downstream primer P2 was designed in the conserved region of exon 3 of β-actin, and the β-actin promoter of the yellow catfish was cloned through this pair of primers . the

(2) Extract the genomic DNA of the pituitary tissue of the yellow catfish, use it as a template, and use P1 and P2 as primers to amplify the β-actin promoter sequence of the yellow catfish. The promoter sequence includes β-actin gene expression control sequence, exon 1 and part of exon 2 sequences, and the full length is 1756bp. the

(3) The β-actin promoter sequence amplified by PCR was cloned into pMD18-T vector, named pActinproexon-T. the

(4) In order to prove the activity of the yellow catfish β-actin promoter, insert the β-actin promoter into the yellow fluorescent protein vector to see if it can drive the expression of fluorescent protein. Through the in vivo experiment of transgenic zebrafish, it was proved that the β-actin promoter can effectively drive the widespread expression of yellow fluorescent protein in zebrafish. the

2. Cloning of the growth hormone gene and its 3'-untranslated region sequence of yellow catfish:

(1) Referring to the cDNA sequence of the fish growth hormone gene published in GenBank, a pair of degenerate primers (P3 and P4) were designed in the homologous conserved region to amplify the open reading frame (ORF) sequence of the gene. the

(2) Extract the total RNA of the brain tissue of the yellow catfish and obtain the cDNA template for PCR reaction by reverse transcription. Subsequently, P3 and P4 were used as primers to amplify the open reading frame sequence of the growth hormone gene of the yellow catfish, that is, the protein coding sequence. The full length of the sequence is 603bp, encoding 200 amino acids. the

(3) The growth hormone open reading frame sequence amplified by PCR was cloned into pMD18-T vector, named pGHORF-T. the

(4) In order to obtain the 3'-untranslated region sequence (3'-UTR) of the growth hormone gene of yellow catfish by 3'-RACE reaction, the primer P5 was designed at 100 bp upstream of the stop codon. Another primer is the universal primer UPM containing linker sequence. the

(5) Extract the mRNA of the brain tissue of the yellow catfish and obtain the cDNA template for PCR reaction by reverse transcription. Subsequently, P5 and UPM were used as primers to amplify the 3'-untranslated region sequence of the growth hormone gene of the peltaceae. The full length of the sequence is 687bp. the

(6) The growth hormone gene 3'-untranslated region sequence amplified by PCR was cloned into the pMD18-T vector, named pGH3-UTR-T. the

(7) Since there is a 160bp overlapping region between the open reading frame sequence and the 3'-untranslated region sequence, the recombination of the open reading frame sequence and the 3'-untranslated region sequence of the growth hormone gene of yellow catfish was mediated by OverlappingPCR. According to the obtained cDNA sequence of the yellow catfish, two pairs of Overlapping PCR primers P6, P7 and P8, P9 were designed. the

(8) Use the recombinant plasmid pGH ORF-T as a template and use P6 and P7 as primers to perform the first round of PCR amplification; use the recombinant plasmid pGH3-UTR-T as a template and use P8 and P9 as primers to perform the first round of PCR amplification PCR amplification. Then use the PCR product amplified by the above two pairs of primers as a common template, and use P6 and P9 as primers to perform a second round of PCR amplification to obtain the open reading frame and 3'-untranslated region sequence of the yellow catfish growth hormone gene. growth hormone cDNA. The full length of the sequence is 1088bp. the

(9) Cloning the cDNA of Pelida catfish growth hormone obtained by Overlapping PCR recombination into pMD18-T vector and named it pGH-cDNA-T. the

3. Construction of the transgenic vector of native growth hormone of yellow catfish:

(1) In order to make the expression-related sequences of the entire transgene expression vector from the promoter to the tailing signal all be the original sequence of the yellow catfish, the BamH I site was introduced into the β-actin promoter by designing primer P10 5'-end of the subsequence. Using P10 and P11 as primers and pActin proexon-T as a template, the β-actin promoter fragment introduced into the BamH I site was amplified by PCR, and cloned into the pMD18-T vector, named pActin Bproexon -T. the

(2) The β-actin promoter fragment and the growth hormone cDNA fragment (including the open reading frame and 3'-untranslated region sequence). These two elements are subsequently cloned into the pBluescript-SK plasmid through the BamH I site, and the correct positive clones are screened by enzyme digestion. The original transgenic element drives the expression of the growth hormone gene of the yellow catfish itself through the β-actin promoter of the yellow catfish itself. the

Advantages of the present invention

The present invention obtains the promoter sequence of the β-actin gene of the yellow catfish from the genomic DNA of the yellow catfish, and expands the promoter types of the transgenic fish; the present invention obtains the growth hormone of the yellow catfish from the total RNA of the yellow catfish Gene open reading frame and 3'-untranslated region sequence expand the gene pool of transgenic fish; the present invention constructs yellow fluorescent protein expression vector driven by yellow catfish β-actin gene promoter, and successfully expresses it in transgenic zebra Stable expression in fish, the recombinant vector has great biological and bioengineering significance for fish transgenic research; the native transgenic vector of yellow catfish obtained in the present invention does not contain any bacterial source when applied to the breeding and production of transgenic yellow catfish Nucleotide, its microinjection gene fragment is a whole yellow catfish original gene fragment. the

Description of drawings

Fig. 1. PCR electrophoresis of the β-actin promoter of yellow catfish. Lane 1 is the DNA Marker, and lane 2 is the PCR result, the band size is 1756bp. the

Figure 2. Fluorescent micrographs of transgenic zebrafish larvae expressing yellow fluorescence driven by the yellow catfish β-actin promoter at 96 hours. the

Fig. 3. PCR electrophoresis image of the open reading frame of the growth hormone gene of the yellow catfish. Lane 1 is the DNA Marker, and lane 2 is the PCR result, with a band size of 603bp. the

Figure 4. PCR electrophoresis of the 3'-RACE of the growth hormone gene of yellow catfish. Lane 1 is the DNA Marker, and lane 2 is the PCR result, with a band size of 687bp. the

Fig. 5. Overlapping PCR electrophoresis of growth hormone gene in peltaceae. Figure A is the PCR electrophoresis of the open reading frame, lane 1 is DNAMarker, and lane 2 is the PCR result, the band size is 603bp; Figure B is the PCR electrophoresis of the 3'-untranslated region sequence. Lane 1 is DNAMarker, lane 2 is the PCR result, the band size is 687bp; Figure C is the overlappingPCR electrophoresis image, lane 1 is the PCR result, the band size is 1088bp, and lane 2 is DNAMarker. the

Figure 6. Enzyme digestion identification diagram of the original transgenic vector pActin promoter-GH of yellow catfish. Lane 1 is DNAMarker, lane 2 is the electrophoresis band of pActin promoter-GH plasmid, lane 3 is the electrophoresis band of the result of BamHI and Nco I double digestion, and lane 4 is the electrophoresis band of BamHI digestion. the

Fig. 7. Schematic diagram of the native transgenic vector pActin promoter-GH of yellow catfish. the

Detailed ways

The cloning of embodiment 1 yellow catfish β-actin promoter

1. Primer design: compare the β-actin promoter sequences of fish such as stingcatfish and tilapia published on GenBank, and design an upstream primer P1 in the homologous conserved region, which is located near the CAATbox. Afterwards, the sequences of the exons were compared, and a downstream primer P2 was designed in the conserved region of exon 3 of β-actin, and the β-actin promoter of the yellow catfish was cloned through this pair of primers . the

P1: 5'-GTGWGTGACGCMGGACCAATC-3' (W=A+T, M=T+C)

P2: 5'-CCATXTCXTCCCAGTTGGTYACAAT-3'(X＝A+G，Y＝G+C) 

2. Genomic DNA extraction: Take the adult yellow catfish pituitary gland and cut it into 1.5ml sterilized centrifuge tube, add a certain amount (m/v=1/10, preferably 10ml per g tissue) of digestion buffer (10mM Tris-Cl, pH8.0, 1% SDS, 100μg/ml proteinase K) and placed in a water bath at 37°C for 5 hours with slight shaking from time to time until the tissue block was completely digested; then an equal volume of phenol: chloroform: iso Pentanol (25:24:1) was extracted twice, the aqueous phase was transferred to a clean 1.5ml sterilized centrifuge tube, and 3M sodium chloride was added to a final concentration of 0.3M; water and ethanol, shake the centrifuge tube gently until the solution is completely fused, and a white flocculent precipitate can be observed; it is centrifuged at 3000g for 10 minutes; the precipitate is washed with 70% ethanol and dried at room temperature, and placed in 100μl TE (10mM Dissolve in Tris-Cl, pH8.0, 1mM EDTA); after dissolving, add one-tenth volume of 3M sodium acetate (pH=5.6), add (nuclease-free) RNaseA to a concentration of 100μg/ml; place at 37°C for 5 minutes, Add 10% SDS to a final concentration of 0.2%; add equal volumes of phenol: chloroform: isoamyl alcohol (25: 24: 1) to extract twice, and transfer the aqueous phase to a clean 1.5ml sterilized centrifuge tube, And add 3M sodium chloride to a final concentration of 0.3M; then add 2.5 times the volume of absolute ethanol, shake the centrifuge tube gently until the solution is completely fused, and a white flocculent precipitate can be observed, centrifuge at 1200g for 10 minutes, dry, 20μl 10mM Dissolve in TE and store at -80°C until use. the

3. PCR amplification: Using the above-mentioned genomic DNA as a template, use P1 and P2 to amplify the promoter of the β-actin gene of the yellow catfish. The PCR reaction system is shown in Table 1:

Table 1. PCR reaction system

PCR amplification reaction conditions: pre-denaturation time at 95°C for 5 minutes, each cycle includes denaturation at 94°C for 1 minute, renaturation at 58°C for 45 seconds, extension at 72°C for 2 minutes, 30 cycles, and at the end of the last cycle, extension at 72°C for 10 seconds minute. After the reaction, 5 μl of the PCR product was taken, and the PCR amplification result was detected by 0.8% agarose gel electrophoresis (Fig. 1). the

4. T-carrier connection of PCR product: Carry out PCR product and pMD-18T carrier connection according to Takara pMD18-T carrier instructions, the system is shown in Table 2:

Table 2: Connection system

After the reaction product was mixed, it was ligated at 16°C for 2 hours. the

5. PCR detection of bacterial liquid of recombinant bacteria: the ligation product of PCR product and pMD-18T vector was transformed into competent Escherichia coli DH5α strain, and cultured upside down at 37°C overnight. Pick a single colony with a sterilized gun tip and put it into a centrifuge tube added with 1ml LB liquid medium (containing ampicillin, 50μg/ml). After incubating at 37°C for 10 hours, use primers P1 and P2 to perform bacterial liquid PCR to select recombinant transformants. The PCR reaction system is shown in Table 3 below:

Table 3: Bacterial liquid PCR reaction system

See step 3 of this embodiment for PCR amplification conditions. Take 5 μl of PCR product, and detect the PCR amplification result by 0.8% agarose gel electrophoresis. As a result of agarose gel electrophoresis, 5 of the 10 recombinant colonies could amplify 1700bp fragments, which were initially judged as positive clones. The DNA fragment includes the yellow catfish β-actin promoter, exon 1, exon 2 and part of exon 2. the

6. Enzyme digestion identification: Take a tube of bacterial liquid from the 5 positive clones in the PCR results of the bacterial liquid, and extract the recombinant plasmid according to the operation method in the Qiagen plasmid extraction kit manual. The endonucleases EcoR I and Sal I were used to identify the plasmid DNA by double digestion, and the results of the digestion showed two expected bands (~2600bp and ~1700bp), confirming that the recombinant plasmid was inserted into the target fragment. The obtained recombinant plasmid was named pActin proexon-T, and the strain of the recombinant plasmid was sent to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing to obtain sequence information. the

The identification of embodiment 2 yellow catfish β-actin promoter activity

1. The connection of the promoter and the yellow fluorescent protein YFP carrier: in order to prove the activity of the yellow catfish β-actin promoter, the plasmid (pActin proexon-T) constructed in Example 1 is extracted, the promoter is separated, and inserted into In the yellow fluorescent protein carrier, see if it can drive the expression of fluorescent protein. In the pActin proexon-T plasmid, the pMD18-T vector has its own BamHI site; and at the end of the inserted yellow catfish β-actin promoter, it is the beginning of encoding β-actin At the codon (ATG), it has an Nco I site. Double digestion with BamH I and Nco I can obtain a complete promoter fragment without actin sequence. In addition, the pCYP26A1-2.5keYFP plasmid was digested with BamH I and Nco I to obtain the yellow fluorescent protein vector. The digestion system is: 10×buffer 2.5 μl, plasmid 4 μl, BamH I and Nco I 1 μl each, add MilliQ H ₂ O to 25 μl, and react at 37°C for 2 hours. After the digested fragments were separated by agarose gel electrophoresis, the corresponding molecular weight bands were cut under ultraviolet light, and the gel recovery kit was used to recover according to the instructions. The ligation system is: 1 μl of 10×ligation buffer, 2 μl of DNA carrier, 6 μl of target DNA fragment, 1 μl of T4 DNA ligase, and the reaction products are mixed and ligated at 16°C for 2 hours. The ligation product was transformed into Escherichia coli DH5α strain, and after transformation and amplification, the recombinant plasmid was extracted according to the operation method in the instruction manual of Qiagen plasmid extraction kit. After the above target fragments are successfully connected to the vector, the vector of the yellow fluorescent protein gene driven by the β-actin promoter is obtained. The new recombinant plasmid was named pActin-promter-eYFP.

2. Detection of the activity of the β-actin promoter in yellow catfish by microinjection: for microinjection, it is first necessary to make a microinjection needle. The production method is as follows: firstly pull a micro-moving capillary with an inner diameter of 0.5 mm and an outer diameter of 1.4 mm on a needle pulling device, and then use a needle breaking device to cut a slanted opening of 5-10 μm as an injection needle. A 60mm petri dish is used for the microinjection plate, and a piece of glass slide is inserted at an angle of 10-20 degrees with melted agarose. After the agar solidifies, remove the glass slide and place the embryo in the groove of the agarose for visualization microinjection. When injecting, with the help of agar gel with strips of grooves and gravity, gently hold the eggs in place. Use a surgical needle to adjust the position of the egg while turning it into the proper orientation for injection. The target fragment for microinjection was obtained by double digestion of the corresponding plasmid with BamH I and Not I. The target fragment concentration for injection is 20-50ng/μl. The injection volume is 2-3 nl. Injected embryos should be 1-cell fertilized eggs. After the injection, the fertilized eggs were taken out, transferred to a petri dish filled with water and incubated at 28°C. Observe every day, remove dead embryos in time, and change the water to reduce bacterial infection. By microinjecting the linearized pActin-promter-eYFP plasmid into zebrafish single-cell embryos, we obtained zebrafish embryos expressing yellow fluorescent protein driven by the yellow catfish β-actin promoter, microinjected for 96 hours Finally, it was seen under a fluorescent microscope that the whole body of the zebrafish larvae emitted yellow fluorescence, and the results are shown in Figure 2. This indicated that the yellow catfish β-actin promoter was active. the

The cloning of embodiment 3 yellow catfish growth hormone gene open reading frame (ORF)

1. Primer design: Design a pair of degenerate primers (P3 and P4) in the homologous conserved region with reference to the growth hormone gene cDNA sequences of fish sting catfish (AF147792), giant catfish (L27835) and channel catfish (AF267989) published in GenBank ) amplifies the open reading frame (ORF) sequence of the gene. the

P3: 5'ATGGCTXGAGYYTTMGTG3'(X=A, C; Y=G, T; M=G, A);

P4: 5' CTACAGPGTGCAGTTGGAATC3' (P = G, A). the

2. Reverse transcription reaction: Take about 1 gram of brain tissue of yellow catfish and extract total RNA. For specific steps, refer to the instructions of Qiagen RNA extraction kit. The obtained total RNA was detected by 0.8% agarose gel electrophoresis and stored at -20°C for future use. The reverse transcription of RNA was carried out according to the instructions of Promega's M-MLV reverse transcriptase, and the first-strand synthesis was performed using the total RNA of yellow catfish as a template. First add 0.5 μg of oligonucleotide primer (Oligo dT, 12-18primer) 1 μl, 2 μg of yellow catfish total RNA in order, and use DEPC water to make the volume to 13 μl; after mixing, bathe in 70°C water for 5 minutes , to open the secondary structure of the RNA, and then quickly transfer to ice and place it for 5 minutes; then, add 4 μl of 5×first strand buffer in sequence, 0.4 μl of ldNTP at a final concentration of 1 mM, and 0.5 μl of 200 units of reverse transcriptase ( SuperScript II RNaseH-Reverse Transcriptase), 1 μl 40 units of RNase Inhibitor (RNase Inhibitor), mix well and put in a water bath at 37°C for 1h. The obtained cDNA products were stored in aliquots at -80°C until use. the

3. PCR amplification: using the above-mentioned reverse transcription product as a template, P3 and P4 were used to amplify the open reading frame (ORF) of the growth hormone gene of the yellow catfish. The PCR reaction system is shown in Table 1 (see Example 1). PCR amplification reaction conditions: pre-denaturation time at 95°C for 2 minutes, each cycle includes denaturation at 94°C for 1 minute, renaturation at 56°C for 45 seconds, extension at 72°C for 1 minute, 30 cycles, and finally extension at 72°C for 10 minutes. After the reaction, 5 μl of the PCR product was taken, and the PCR amplification result was detected by 0.8% agarose gel electrophoresis (Fig. 3). the

4. T-vector ligation of the PCR product: Ligate the PCR product and the pMD-18T vector according to the instructions of the T-vector, and the system is shown in Table 2 (see Example 1). After the reaction product was mixed, it was ligated at 16°C for 2 hours. the

5. PCR detection of bacterial liquid of recombinant bacteria: the ligation product of PCR product and pMD-18T vector was transformed into Escherichia coli DH5α strain, and cultured upside down at 37°C overnight. Pick a single colony with a sterilized gun tip and put it into a centrifuge tube added with 1ml LB liquid medium (containing ampicillin, 50μg/ml). After incubating at 37°C for 10 hours, use primers P3 and P4 to perform bacterial liquid PCR to select recombinant transformants. The PCR reaction system is shown in Table 3 (see Example 1). See step 3 of this example for the conditions of the PCR amplification reaction. Take 5 μl of PCR product, and detect the PCR amplification result by 0.8% agarose gel electrophoresis. 8 of the 10 recombinant colonies could amplify 600bp fragments, which were preliminarily judged as positive clones. the

6. Enzyme digestion identification: Take a tube of bacterial liquid from the 8 positive clones in the PCR results of the bacterial liquid, and extract the recombinant plasmid according to the operation method in the instruction manual of the Qiagen plasmid extraction kit. Using EcoR I and Hind III to identify the plasmid DNA by double digestion, the result of digestion showed two expected bands (~2600bp and ~600bp), confirming that the recombinant plasmid was inserted into the target fragment. The obtained recombinant plasmid was named pGH ORF-T, and the strain of the recombinant plasmid was sent to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing to obtain sequence information. the

Cloning of embodiment 4 yellow catfish growth hormone cDNA 3'-untranslated region sequence

1. Primer design: An upstream primer P5 was designed according to the ORF sequence of the growth hormone gene in Example 3 above, and a universal primer UPM of the SMART ^TM RACE cDNA Amplification Kit was used as the downstream primer.

P5: 5'-GGCTCCGCCATTCGAGGATTTCTACC-3';

UPM: 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3'. the

2. Isolation and reverse transcription of mRNA: Take some (about 0.25 mg) of total RNA extracted in Example 3 above, and extract mRNA. For specific steps, refer to the instructions of Qiagen's Oligotex mRNA extraction kit. Using the extracted mRNA as a template and Oligo(dT) with a SMART oligonucleotide sequence universal linker primer as a locking primer, synthesize the first strand of 3'-RACE according to the instructions of BD's SMART ^TM RACE cDNA Amplification Kit cDNA.

3.PCR amplification

Using the above reverse transcription product as a template, using P5 and UPM as primers, the Advantage2polymerasemix of BD Company was used to amplify the 3'-untranslated region sequence of the yellow catfish growth hormone gene cDNA. The PCR reaction system is shown in Table 4:

Table 4: 3’-RACE PCR reaction system

PCR amplification reaction conditions: denaturation at 94°C for 30 seconds, renaturation at 68°C for 30 seconds, extension at 72°C for 2 minutes, and 20 cycles; then denaturation at 94°C for 30 seconds, renaturation at 68°C for 30 seconds, extension at 72°C for 3 minutes, Do another 20 cycles. After the reaction, 5 μl of the PCR product was taken, and the PCR amplification result was detected by 0.8% agarose gel electrophoresis (Fig. 4). the

4. T-vector ligation of the PCR product: Ligate the PCR product and the pMD-18T vector according to the instructions of the T-vector, and the system is shown in Table 2 (see Example 1). After the reaction product was mixed, it was ligated at 16°C for 2 hours. the

5. Detection of the recombinant bacterial plasmid: the ligation product of the PCR product and the pMD-18T vector was transformed into Escherichia coli DH5α strain, and cultured upside down at 37°C overnight. Pick a single colony with a sterilized gun tip and put it into a centrifuge tube with 1ml of LB liquid medium (containing ampicillin, 50μg/ml), incubate at 37°C for 8 hours, and centrifuge the bacterial solution at a speed of 12,000g for 30 seconds Aspirate the supernatant, resuspend the bacteria with 40 μl STE solution, and then add 40 μl equal volume mixed phenol and chloroform solution. After strong shaking and mixing, centrifuge at a speed of 12,000g for 15 minutes, take 10 μl of supernatant, and use 0.8% agarose gel electrophoresis to detect the rapid extraction results and perform agarose gel electrophoresis. Seven of the 16 recombinant colonies are significantly larger than pMD-18T Carrier, initially judged as a positive clone. the

6. Enzyme digestion identification: Take a tube of bacteria liquid from the 7 positive clones in the electrophoresis results, and extract the recombinant plasmid according to the operation method in the instruction manual of the Qiagen plasmid extraction kit. Using EcoR I and Hind III to carry out double-digestion plasmid DNA identification, the result of restriction enzyme digestion showed two expected bands (~2600bp and ~680bp), confirming that the recombinant plasmid was inserted into the target fragment. The obtained recombinant plasmid was named pGH3-UTR-T, and the strain of the recombinant plasmid was sent to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing to obtain sequence information. the

Example 5 Recombination of open reading frame sequence and cDNA3'-untranslated region sequence of yellow catfish growth hormone gene

Since there is a 160bp overlapping region between the open reading frame sequence and the 3'-untranslated region sequence of the cDNA, in order to obtain the cDNA of the 3'-UTR containing the growth hormone of the yellow catfish, the present invention uses the Overlapping PCR method to mediate the growth hormone of the yellow catfish Recombination of gene open reading frame sequence and 3'-untranslated region sequence. the

1. Splicing of the open reading frame sequence and 3'-untranslated region of the growth hormone gene of the yellow catfish: the open reading frame sequence and the 3'-untranslated region sequence of the growth hormone gene of the yellow catfish were spliced with Vector software to obtain the growth of the yellow catfish The cDNA sequence of the hormone gene, the full length is 1088bp, see SEQ ID NO.1 for the sequence. According to the analysis by Jellyfish software, the open reading frame (ORF) of the gene is 603bp long, and the 3' non-coding region is 485bp, encoding a total of 200 amino acids. The sequence is shown in SEQ ID NO.2. The encoded amino acids have more than 90% identity with the growth hormone amino acids of catfish (sting catfish, giant catfish, walking catfish, channel catfish), among which the identity with giant catfish is as high as 96.5%. the

2. Primer design: According to the cDNA sequence of the above-mentioned yellow catfish, 2 pairs of Overlapping PCR primers (P6, P7 and P8, P9) were designed. Do the second round of PCR. the

P6: 5'-CATGCCATGGCTAGAGGGTTAGTG-3' (marked as Nco I site in bold);

P7: 5'-CTACAGGGTGCAGTTGGAATC3';

P8: 5'-GGGAAACCTGAGGAAGAGCTTCCGTC-3';

P9: 5'-CGGGATCCCAGGGATTAGGTTTATT-3' (BamH I site marked in bold). the

3. PCR amplification of the open reading frame sequence and 3'-untranslated region sequence of the yellow catfish growth hormone gene: using the plasmid containing the open reading frame sequence as a template, and using P6 and P7 as primers to amplify the yellow catfish growth hormone gene open reading frame sequence. Using the plasmid containing the cDNA 3'-untranslated region of the growth hormone gene of the peliotus peliotus as a template, the cDNA 3'-untranslated region of the growth hormone gene of the yellow catfish was amplified with primers P8 and P9. The PCR reaction system is shown in Table 1 (see Example 1). PCR amplification reaction conditions: pre-denaturation time at 95°C for 2 minutes, each cycle including denaturation at 94°C for 1 minute, renaturation at 56°C for 45 seconds, extension at 72°C for 1 minute, 30 cycles, and finally extension at 72°C for 10 minutes. The electrophoresis results of the PCR products are shown in Figure 5A, B. the

4. PCR-mediated recombination: Take 1 μl of each of the above two PCR products and mix them evenly as a template for the second round of PCR to amplify the open reading frame and 3'-untranslated region of the recombined growth hormone gene. The PCR reaction system is shown in Table 5:

Table 5: Recombinant PCR reaction system

PCR amplification reaction conditions: pre-denaturation time at 95°C for 2 minutes, each cycle including denaturation at 94°C for 15 seconds, renaturation at 55°C for 1 minute, extension at 72°C for 2 minutes, 5 cycles. Add 1 μl each of primers P6 and P9, and perform the following PCR amplification reaction conditions: 95°C pre-denaturation time for 1 minute, each cycle includes denaturation at 94°C for 30 seconds, renaturation at 50°C for 45 seconds, extension at 72°C for 2 minutes, cycle 25 times, and finally extended at 72°C for 10 minutes. After the reaction, 5 μl of the PCR product was taken, and the PCR amplification result was detected by 0.8% agarose gel electrophoresis (FIG. 5C). the

5. T-vector ligation of the PCR product: Ligate the PCR product and the pMD-18T vector according to the instructions of the T-vector, and the system is shown in Table 2 (see Example 1). After the reaction product was mixed, it was ligated at 16°C for 2 hours. the

6. PCR detection of bacterial liquid of recombinant bacteria: the ligation product of PCR product and pMD-18T vector was transformed into Escherichia coli DH5α strain, and cultured upside down at 37°C overnight. Use a sterilized pipette tip to pick colonies and put them in a centrifuge tube with 1ml LB liquid medium (containing ampicillin, 50μg/ml). After incubating at 37°C for 10 hours, use primers P6 and P9 to perform bacterial liquid PCR to select recombinants. Turn. The PCR reaction system is shown in Table 3 (see Example 1). See step 3 of this embodiment for the conditions of the PCR amplification reaction. After the reaction, 5 μl of the PCR product was taken, and the PCR amplification result was detected by 0.8% agarose gel electrophoresis. Seven of the 12 recombinant colonies could amplify 1100bp fragments, which were preliminarily judged as positive clones. the

7. Enzyme digestion identification: Take a tube of bacterial liquid from the 7 positive clones in the electrophoresis results, and extract the recombinant plasmid according to the operation method in the Qiagen plasmid extraction kit manual. Using EcoR I and HindIII to identify the double-digested plasmid DNA, the results of the enzyme digestion showed two expected bands (~2600bp and ~1100bp), confirming that the recombinant plasmid had inserted the target fragment. The obtained recombinant plasmid was named pGH-cDNA-T, and the strain of the recombinant plasmid was sent to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing to obtain sequence information. the

Embodiment 6 Construction of the native growth hormone gene transgenic vector of yellow catfish

1. Introduction of enzyme cleavage sites for the promoter of yellow catfish β-actin: Through the Jellyfish software analysis of the sequencing results of the above-mentioned Example 1, it was found that the introduction of 5 bases in front of the promoter of actin can form a BamH I enzyme cleavage site site, the introduced non-native sequence can be removed by enzyme digestion, so that the expression-related sequences of the entire transgene expression vector from the promoter to the tailing signal are all native sequences of the yellow catfish; therefore, primers P10 and P11 were redesigned Amplify the actin promoter sequence. the

P10: 5' G TGAGTGCGCCGGACCAATC-3' (The 5 bases added are marked in bold; the BamHI site is underlined)

P11: 5'-CCATATCATCCCAGTTGGTGACAAT-3'

Using P10 and P11 as primers, using the pActinproexon-T recombinant plasmid as a template, through PCR amplification, according to the method described in Example 1, a new recombinant plasmid was constructed, named pActin Bproexon-T. the

2. Construction of the native vector: the recombinant plasmid pGH-cDNA-T obtained in Example 5 is needed to construct the native vector,

The recombinant plasmid pActin Bproexon-T obtained in Example 6, and the commercial plasmid pBluescript-SK. The recombinant plasmid was extracted according to the operation method in the instruction manual of Qiagen plasmid extraction kit. the

(1) In the pActin Bproexon-T plasmid, the pMD18-T vector has its own BamHI site; and at the tail end of the inserted yellow catfish β-actin promoter, it is encoding β-actin At the initiation codon (ATG) of the gene, it has an NcoI site. Double digestion with BamH I and Nco I can obtain a complete promoter fragment without actin sequence. The enzyme digestion system is: 10×buffer 2.5 μl, plasmid 4 μl, BamH I 1 μl, Nco I 1 μl, MilliQ H ₂ O 16.5 μl; enzyme digestion reaction at 37°C for 2 hours. After the digested fragments were separated by agarose gel electrophoresis, the corresponding molecular weight bands were cut under ultraviolet light, and the gel recovery kit was used to recover according to the instructions. This step obtains the promoter fragment of β-actin.

(2) Digest pGH-cDNA-T recombinant plasmid with BamH I and Nco I. The enzyme digestion system is: 10×buffer 2.5 μl, plasmid 4 μl, BamH I 1 μl, Nco I 1 μl, MilliQH ₂ O 16.5 μl; enzyme digestion reaction at 37°C for 2 hours. After the digested fragments were separated by agarose gel electrophoresis, the corresponding molecular weight bands were cut under ultraviolet light, and the gel recovery kit was used to recover according to the instructions. This step obtains a fragment containing growth hormone cDNA, including the complete growth hormone coding sequence (ORF) and 3'-untranslated region sequence (3'-UTR).

(3) Digest pBluescript-SK plasmid with BamH I. The enzyme digestion system is: 10×buffer 2.5 μl, plasmid 4 μl, BamHI 1 μl, MilliQ H ₂ O 17.5 μl; enzyme digestion reaction at 37°C for 2 hours. After the digested fragments were separated by agarose gel electrophoresis, the corresponding molecular weight bands were cut under ultraviolet light, and the gel recovery kit was used to recover according to the instructions. Afterwards, dephosphorylate the recovered enzyme-cut vector with alkaline phosphatase CIAP to prevent self-ligation. The reaction system is: 20 μl of recovered DNA, 5 μl of 10×buffer, 1 μl of CIAP enzyme, 24 μl of MilliQ H ₂ O, and react at 37°C for 1 hour , The reaction product was recovered with a gel recovery kit according to the instructions. This step yields a DNA fragment for the vector to accommodate the β-actin promoter and the growth hormone gene.

(4) 3 μl of β-actin promoter DNA fragment obtained above, 3 μl of growth hormone gene DNA fragment, 2 μl of pBluescript-SK vector, 1 μl of T4ligation buffer, and 1 μl of T4ligase, in a total volume of 10 μl. Ligation overnight at 16°C. Then the ligation product was transformed into Escherichia coli DH5α bacteria, and cultured upside down at 37°C overnight. Pick a single colony with a sterilized gun tip and put it into a centrifuge tube with 3ml of LB liquid medium (containing ampicillin, 50μg/ml), incubate at 37°C for 8 hours, and centrifuge the bacterial solution at a speed of 12,000g for 30 seconds Aspirate the supernatant, resuspend the bacteria with 40 μl STE solution, and then add 40 μl equal volume mixed phenol and chloroform solution. Shake and mix vigorously and centrifuge at 12,000g for 15 minutes, take 10 μl of supernatant, and perform agarose gel electrophoresis on 0.8% agarose gel electrophoresis to detect the rapid extraction results. Four of the 10 recombinant colonies are significantly larger than pBluescript-SK Carrier, initially judged as a positive clone. Refer to the operation method in the instruction manual of the Qiagen plasmid extraction kit to extract the recombinant plasmid of the bacteria liquid with positive electrophoresis results. Digestion with BamHI, or double digestion with BamHI and NcoI to confirm the successful construction of the vector, named pActin promoter-GH. The results of enzyme digestion identification are shown in Figure 6. the

3. Digest the pActinpromoter-GH recombinant plasmid (see Figure 7 for the schematic diagram) with BamHI to obtain the original growth hormone gene driven by the β-actin promoter of the yellow catfish with the polyA tail letter of the growth hormone gene number of DNA fragments. All the sequences of the DNA fragment are the native sequences of the yellow catfish, and can be used for the construction of the transgenic yellow catfish by microinjection, so as to avoid the safety problem of the transgenic organism caused by the introduction of the foreign DNA sequence. the

sequence listing

Claims (4)

1. Yellow catfish beta-actin promotor, is characterized in that, its nucleotide sequence consists of 5 ' of sequence shown in SEQ ID NO:1-end 1 to 1017 bit base.

2. the carrier for expression of eukaryon that contains Yellow catfish beta-actin promotor described in claim 1.

3. the recombinant microorganism that contains Yellow catfish beta-actin promotor described in claim 1.

4. the application of Yellow catfish beta-actin promotor in Yellow catfish gene breeding described in claim 1.

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