CN110684769B

CN110684769B - Ostrea gigas EF-1 alpha promoter, recombinant vector and application thereof

Info

Publication number: CN110684769B
Application number: CN201910975861.4A
Authority: CN
Inventors: 李琪; 岳晨阳; 于红
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2019-10-15
Filing date: 2019-10-15
Publication date: 2022-04-05
Anticipated expiration: 2039-10-15
Also published as: CN110684769A

Abstract

The invention discloses a crassostrea gigas EF-1 alpha promoter, a recombinant vector and application thereof. The EF-1 alpha promoter provided by the invention is a high-expression housekeeping gene promoter, and can be expressed in the early development stage of the crassostrea gigas embryo. The concrete expression is as follows: designing a specific primer, amplifying an EF-1 alpha gene promoter fragment of the crassostrea gigas, inserting the promoter fragment into a vector containing a GFP reporter gene by utilizing In-Fusion connection, and introducing the recombinant vector into fertilized eggs of the crassostrea gigas to efficiently drive the GFP reporter gene to express In the embryos and larvae of the crassostrea gigas. The invention obtains the general efficient promoter sequence of the crassostrea gigas and provides an important technical basis for promoting the development of the crassostrea gigas CRISPR/Cas9 targeted gene editing technology.

Description

Ostrea gigas EF-1 alpha promoter, recombinant vector and application thereof

Technical Field

The invention relates to the fields of genetic engineering and molecular biology, and particularly relates to an EF-1 alpha promoter of crassostrea gigas, a recombinant vector and application thereof.

Background

The crassostrea gigas has important economic value as a marine shellfish distributed worldwide. In recent years, genetic breeding work of crassostrea gigas is greatly advanced in China, new species of crassostrea gigas, such as 'Hailao No. 1' and 'Hailao No. 2', are successively cultured, and the adopted method is mainly the traditional artificial selective breeding. In recent years, the application of the CRISPR/Cas9 targeted gene editing technology in genetic improvement and variety breeding of animals and plants is receiving wide attention. By using a method of introducing Cas9 protein + sgRNA into fertilized eggs through microinjection, efficient and accurate target gene editing can be realized on crassostrea gigas. However, this method is not suitable for mass treatment of fertilized eggs, and the method of introducing plasmid DNA containing CRISPR/Cas9 system into fertilized eggs by electrotransfection or chemical transfection has the advantages of high throughput and low cost. However, the promoter sequences contained in the above plasmid DNAs are derived from viruses or model animals in many cases, and are not completely suitable for use in crassostrea gigas, and promoters capable of efficiently and stably driving the expression of foreign genes are still lacking in the studies of the gene functions of shellfish.

The promoter is a non-coding DNA sequence located upstream of the 5' end of the transcribed gene and capable of correctly and efficiently initiating transcription, and as a cis-acting element in the transcription process, it occupies a very important position in the regulation of gene expression. The promoter sequence mainly comprises a core promoter region and an upstream regulatory region. The core promoter region functions to recruit transcription factors and transcription enzymes to the promoter core region, recognize the transcription initiation site, and then the transcription enzymes perform functions, and the upstream regulatory region sequence has a stimulating effect on the transcription regulation process, and can control the transcription efficiency by combining with the corresponding transcription factor. The promoter acts like a "switch", and the core promoter element, upstream regulatory element and response element, etc. act together to determine the activity of the gene. The promoter elements and positions vary from gene to gene, and different combinations and sequence variations of these elements constitute a vast number of diverse promoters. Obviously, the development of a suitable promoter is a key link for achieving a suitable transcription efficiency of the target gene.

The high-efficiency promoter can promote the high-level expression of exogenous genes, and the housekeeping gene promoter of a certain species is usually required to obtain the constitutive expression of target genomes in the species. Housekeeping genes are an indispensable class of genes for maintaining minimal cell function, and are expressed in all cell types. Housekeeping gene expression levels are less influenced by environmental factors, are consistently and stably expressed in most, or almost all, tissues during various growth stages of an individual, and are only influenced by promoter sequences or the interaction of promoters with RNA polymerase, and are not regulated by other mechanisms. The number of housekeeping genes in organisms is large, a plurality of choices are provided for developing constitutive promoters, and at present, the efficient promoter suitable for crassostrea gigas is still not successfully developed.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide the crassostrea gigas EF-1 alpha promoter, a recombinant vector and application thereof.

In order to achieve the purpose, the invention adopts the following specific technical scheme:

an EF-1 alpha promoter of crassostrea gigas has a sequence shown in SEQ ID NO. 1.

The specific positions of the crassostrea gigas EF-1 alpha are as follows: the candidate promoter region was flanked by approximately 3500bp of the ATG front including the first exon and the first intron.

A recombinant expression vector comprising a nucleotide fragment of which the sequence is SEQ ID NO. 1.

The construction method of the recombinant expression vector is characterized by comprising the following specific steps of:

1) designing a Pair of specific amplification primers of an EF-1 alpha gene upstream promoter of the crassostrea gigas according to a crassostrea gigas genome sequence, and adding plasmid homologous ends, wherein the sequences are shown as SEQ ID NO. 2 and SEQ ID NO. 3;

2) taking the genomic DNA of the crassostrea gigas as a template, carrying out PCR amplification, and recovering a product;

3) digesting the plasmid and recovering the product;

4) carrying out In-Fusion connection on the enzyme digestion linearized plasmid and the recovered PCR product In the step 2), introducing the connection product into escherichia coli competent cells, carrying out plate screening, and selecting monoclonal shake bacteria for culture;

5) extracting bacteria liquid plasmids, carrying out enzyme digestion, selecting plasmids which accord with expectation according to the enzyme digestion result, sequencing, and naming the plasmids with correct sequencing as pEGFP-CgEF-1 alpha, namely the recombinant expression vector.

Further, the PCR system in step 2) is: 5x Phusion HF Buffer, 10. mu.l; 2.5mM dNTPs, 4. mu.l; 2.5. mu.l of upstream primer (10 uM); 2.5. mu.l of downstream primer (10 uM); 1. mu.l of template DNA (100 ng/. mu.l); phusion High-Fidelity DNA Polymerase 0.5. mu.l; 29.5. mu.l of ultrapure water. The amplification procedure was: pre-denaturation at 98 ℃ for 30 s; denaturation at 98 ℃ for 10s, annealing at 64 ℃ for 30s, extension at 72 ℃ for 1min for 20s, and 35 cycles; extension at 72 ℃ for 10 min.

Further, the enzyme cutting system in the steps 3) and 5) is as follows: 10 × CutSmart Buffer 5 μ l, plasmid DNA (150 ng/. mu.l) 6 μ l, BamHI enzyme (20U/. mu.l) 0.5 μ l, ultra pure water 37.5 μ l. The reaction program was 37 ℃ for 2 h.

Further, the In-Fusion linker In step 4) is: 5 XIn-Fusion HD Enzyme Premix 2. mu.l, linearized pEGFP-1 plasmid DNA (43 ng/. mu.l) 1.5. mu.l, and recovered product (16 ng/. mu.l) 6.5. mu.l. The reaction program is 50 ℃ for 15 min.

Further, in the step 1), plasmid pEGFP-1 is selected.

The specific application method of the recombinant expression vector is characterized in that the recombinant expression vector and phenol red mixture are introduced into fertilized eggs of crassostrea gigas by a microinjection method, and are continuously observed under a fluorescence microscope, so that green fluorescence can be observed in various cells of mulberries, blastocysts, gastral embryos, trochophores and D-shaped larvae, namely EGFP gene expression.

Further, the final concentration of the microinjected recombinant vector was 200 ng/. mu.l.

The crassostrea gigas EF-1 alpha promoter is applied to crassostrea gigas gene breeding.

The invention has the advantages and beneficial effects that:

the invention successfully develops the Elongation factor-1 alpha (EF-1 alpha) gene promoter sequence from a plurality of promoter sequences of the housekeeping genes of the crassostrea gigas, and the EF-1 alpha promoter provided by the invention is a high-expression housekeeping gene promoter and can be expressed at the early development stage of the crassostrea gigas embryo. The invention drives the constitutive expression of exogenous genes in the early embryo of the crassostrea gigas, further perfects the gene editing technology of the crassostrea gigas and provides a technical basis for developing the research on the gene functions of the crassostrea gigas.

Drawings

FIG. 1 is a diagram of a recombinant vector drawn by using SnapGene software.

FIG. 2 is a graph showing the results of EGFP expression in early embryos and larvae of crassostrea gigas injected with recombinant vectors.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings, in which the experimental methods without specific conditions being noted are all conventional experimental conditions. The examples are given for the purpose of better illustration of the invention and the invention is not limited to these examples.

Example 1:

and (3) screening the promoter fragment of the crassostrea gigas housekeeping gene.

1) Preliminarily selecting 6 crassostrea gigas housekeeping genes such as EF-1 alpha, ACT,18S rRNA, 28S rRNA, RL7 and the like according to published crassostrea gigas genome data, further screening the housekeeping genes with stable and high expression in the crassostrea gigas embryonic period by combining the existing transcription group data, and selecting the EF-1 alpha gene with the highest expression quantity for further analysis;

2) specifically, the transcription factor binding site in the first ATG front flanking sequence of the crassostrea gigas EF-1 alpha is analyzed, and finally, the flanking sequence of about 3500bp in front of the ATG including the first exon and the first intron is used as a candidate promoter region.

Example 2:

and (3) amplifying the EF-1 alpha promoter of the crassostrea gigas.

1) The genomic DNA of the crassostrea gigas is extracted by a phenol chloroform method, specific primers are designed according to the sequence of a candidate EF-1 alpha gene promoter region, and 16 basic groups of vector pEGFP-1 homologous ends are added to the upstream primer and the downstream primer respectively, as shown in Table 1.

TABLE 1 primer sequences for the amplification of oyster EF-1. alpha. Gene promoter

The 16 base homologous end of the vector pEGFP-1 is underlined.

2) PCR amplification was carried out using a Phusion High-Fidelity DNA Polymerase from Thermo Scientific as a template. The PCR system is shown in Table 2. The PCR reaction program is: pre-denaturation at 98 ℃ for 30 s; denaturation at 98 ℃ for 10s, annealing at 64 ℃ for 30s, extension at 72 ℃ for 1min for 20s, and 35 cycles; extension at 72 ℃ for 10 min.

TABLE 2 PCR reaction System for the amplification of oyster EF-1. alpha. Gene promoter

3) The PCR amplification product was separated by 1% agarose Gel electrophoresis to obtain a band of about 3500bp in size, which was then purified and recovered by Gel cutting using the GeneJET Gel Extraction Kit (K0692) from Thermo Scientific.

Example 3:

and (3) constructing a pEGFP-CgEF-1 alpha recombinant expression vector.

1) Plasmid pEGFP-1 was digested with the New England BioLabs restriction enzyme BamH I, as shown in Table 3. The reaction program was 37 ℃ for 2 h.

TABLE 3 BamH I digestion of plasmid pEGFP-1 reaction System

2) The linearized pEGFP-1 was recovered as a cut gel, and subjected to In-Fusion ligation with the PCR-recovered product (In-Fusion HD Cloning Kit, TaKaRa). The In-Fusion ligation system is shown In Table 4, and the reaction procedure is 50 ℃ for 15 min.

TABLE 4 In-Fusion ligation System

3) Taking out Escherichia coli DH5 alpha competent cell, storing at-80 deg.C, placing on ice, adding 10 μ l ligation product into 100 μ l competent cell, mixing, placing on ice for 30min, heat-shocking at 42 deg.C for 60-90s, and placing on ice for 2 min. Add 500. mu.l SOC medium and incubate at 37 ℃ for 1h at 150 rpm. Centrifuging at 3000rpm for 2-3min, sucking 450 μ l of supernatant, and suspending the bacterial suspension with the residual culture medium. The resuspended suspension was spread on LB plates containing kanamycin antibiotics and cultured for 16h at 37 ℃ in an inverted manner.

4) Single colonies were picked, cultured in 3ml LB broth containing kanamycin antibiotic for 16h, plasmids were extracted using the Endo-Free Plasmid Mini Kit I (D6948) from OMEGA Bio-tek, and digested with BamH I, selecting plasmids that cleaved two DNA bands of about 4100bp and about 3500 bp. The sequencing primers were CgEF-1. alpha.F and CgEF-1. alpha.R, and the plasmid with the correct sequencing was designated as pEGFP-CgEF-1. alpha (as shown in FIG. 1).

Example 4:

EGFP expression in crassostrea gigas embryos and larvae.

1) Preparing the extracted pEGFP-CgEF-1 alpha recombinant vector into an injection: recombinant vector 200 ng/. mu.l, phenol red 0.05%.

2) Selecting oysters with mature gonads, collecting sperms and ova by an anatomical method, carrying out artificial insemination, and collecting fertilized ova for microinjection.

3) Placing the fertilized eggs of the crassostrea gigas collected in the step 1) in the center of a polypropylene culture dish with the diameter of 60mm, dripping 1 drop of seawater, placing under an inverted microscope, and adjusting the fertilized eggs to the center of the visual field. Using a Microloader (eppendorf), 1. mu.l of the injection solution was aspirated and injected into the injection needle, which was connected to a nitrogen-pressurized quantitative microinjection system (WARNER, PLI-100A). The pressure of the suction needle is adjusted to be negative pressure by utilizing a pneumatic manual microinjection instrument, and one fertilized egg is sucked and fixed. Using a microinjection system, 0.1nL of the injection was injected into fertilized eggs of crassostrea gigas. After the injection, the injected eggs are discharged by adjusting the internal pressure of the holding needle to be positive pressure, the position of the holding needle is changed, and other fertilized eggs are held by the internal pressure of the holding needle, so that the injection is carried out one by one. And culturing the fertilized eggs in sterile seawater at 23-24 ℃.

4) After microinjection, embryos and larvae are observed under a fluorescence microscope every other hour, and the result shows that green fluorescence can be observed after microinjection is carried out for four hours, which indicates that the crassostrea gigas EF-1 alpha promoter can very early start EGFP gene expression. FIG. 2 shows embryos, trochophores and D-type larvae of the Mulberry stage, the blastocyst stage, the gastral stage and the gastral stage of the Ostreea gigas which emit green fluorescence under a fluorescence microscope.

As can be seen from FIG. 2, green fluorescence can be observed in various types of cells of the crassostrea gigas embryo and the larva, and the EGFP gene expression has no tissue specificity, so that the recombinant vector can drive the constitutive expression of the target gene in the early stage of the crassostrea gigas embryo and the larva.

Sequence listing

<110> China oceanic university

<120> crassostrea gigas EF-1 alpha promoter, recombinant vector and application thereof

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tatttcttac ggaaacttat agttaattca tagctctata gaaacagcca gactgaaatc 180

tacacgccct atttatcgat tggtcgaaat ctacagcggc tgaaagtgac aggacagaaa 240

ttgacacgcc ctgtttatcg attggtctga acctacagcg actttagtaa aagcccggac 300

tgcacgaaat aatcatgacg atgccagact caaagtctca caagagacct ctaccatcgg 360

ctcatacatt cagagaggaa cgaacgacaa ctctgcatgg gagattgacg attttgctgt 420

ttcttttagc taacgtactt ttgtacatta acagtaattt agtgaatttt actaactttt 480

cataatcagt ttattaatta gttaaagata gatgttaata taaacaataa aatgctttct 540

ttgatggttc atgcgggtaa tgaagatagc gatcattgca ggaaaaatgt acataacccg 600

ctacgtattt ttccagcaat gtcgctacct tcatatcccg aatgaatcac caaagaaagc 660

attttattgt ttaaataaat gcaaactaaa catcaataaa tattttacaa agcgtgcaaa 720

aaaatagttt tttaagctca aaactgcatg taaaagcatt ggttaaacat gttaatcatt 780

ttgagtacta ttatagtatt gatatataca atattatata aatggtacac gaaaaaaatc 840

aacaaaaaga attcagcaag atataaaaac tatttaacgt ttctataata tagaattatt 900

tgtcagttgc aatcacataa ccggcttaca ggtacaggtt aaccttgcaa ctctgacttc 960

taaaaggaat ttaaatttac aagatgtagg tatagcaaac taactactag tatttaaata 1020

gattcctctt gcaaaaaatt gcttaaattt tctgaatttc ctgtgttcct acaaaaaata 1080

tagaaaactt acgatgtgtg atgaaagcat tgtgtagtga cggacttaaa aggcatttga 1140

taaacatatt gtgtattcag tattgcaatc tgattaaaat acagatctct acataagcgc 1200

acagcgcaga gagatcatct ctctgcgctg tgcgcttatg tagagatctg tataaaactt 1260

ttttttatta gattggttaa aattggtact attggtggga tctggatttc gcacttcgag 1320

gcggtggtaa agggccgaag aaaaaaatca tagtttatcg tatatcgcaa attacgccat 1380

gaaaattaac atactgtaca taccttcttg attcatattt aaatttcgaa tggttttatg 1440

ttatcaatga ggctagctta aacttacttt ttctataaat ttcaattcaa tataataaca 1500

cgtaaactgt cccaacacat gtacagatta aatactcgaa tgtataacaa gtaaatgagt 1560

agatttaaat aatggtcgtt ttttaaaagt atatatcaga aacacccggc cggtctatgt 1620

gcatacagct cttgtaatac aatacatatt ttaagtatat agttgtatga aaatacacat 1680

tattcaagtg atctttcatt gtccaatgaa agaaaaaacc cactgtactt tctcacatta 1740

atttacaaat tttgacaggt attatatatt ctttttaatc taagggcatc attttacatg 1800

ctttttcaca cgtggccctc cagagtggtt gtcatcaacg tacctttggg gtgaattaaa 1860

caacaaccat aaaacaaaca ttttagtgac taggacttat tcgataagct atttttatac 1920

tttgacgtat tttgacggtg tgcataaaaa caggaaataa tcttcaataa aattataaat 1980

aaagaaagat attaagataa catgtatgaa caaaaatcaa gataaacaat ccagaaattg 2040

aaaaacatac ttttgtatta tgagtaaggt atatatttat ttcaagctga aaaaagaaac 2100

atcacgtcag gcgcccattc ttaggatgaa ggtgatgaat tataagatta cttacacttt 2160

gcaagaaaaa tatgtttcaa aaataacttt atatcatgta ttattaacag tatcgaataa 2220

aacagagagt ttttgaaaga atagtattgt atttttgcta aagtatcttt tttgtctcac 2280

ttaaccattt tctggttact agtcatatcg ggtaatcctt aaccgtgacg ttatcaacac 2340

gtgacaatcc tctctctttc ctttttgctg tggcgaactc tgagagtaag tacagtgtgg 2400

aatttctgtt aaaatccaat ggatatcgct atgattaaat gacatccatt taccatttct 2460

gtatttaatg tatcatcagg acattaagat ttgttattct tgtaagattt ttgtttatta 2520

agtgtatttt agaagatttt gccaagatta aaatttgtgg tagccggcta gacatattgg 2580

tgtcgccatc ttggaatttc atcataatgt agcaatattt tttatgaaca ttacttatat 2640

attgaagtta atatgtatat atttaagtaa tttatatcat ttaattctta aaatgttaag 2700

ttaaaacgcc aagttgtact ttgaactctt tggtgcactc tgggtcatgt ccaaagtatg 2760

attatgttaa gtaatatcaa tataacatac ttaaaaggaa aatattttcg gtaaactgtg 2820

tgtaatacat agttcttgat aaccttttat tgaaatttta ttttaaaaac gttcgtaaat 2880

tccgacacaa cttcatacaa aagcagtagt tcgactcggt ccaaattggt caatatcatg 2940

aaaatgtcat ttgattctat tgaaatatgt ttgttttgtt taacctggaa aacattttaa 3000

acgttttaaa tcataattat cattttgtaa caaaatataa gtaaacaaag cggtgacagg 3060

cattatagca cacgtggtca agatggcggc cattttgatt tgtacaatct gttgtgtggt 3120

attgagtaat tgaatagggc cgcttataat tctttcttaa aaaagtaatg gcgcactgaa 3180

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aaggttcccc ctgatgttta tgatgaccat agtagttgta atttagttaa ttgcaaattt 3420

cttttaaaat tacagtatct agagtaataa aaacaaggag caacg 3465

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cgcgggcccg ggatccacta tatgacggac tggacaac 38

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<213> Artificial Sequence (Artificial Sequence)

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ggcgaccggt ggatcccgtt gctccttgtt tttattactc 40

Claims

1. An oyster EF-1 alpha promoter is characterized in that the sequence of the promoter is shown as SEQ ID NO. 1.

2. A recombinant expression vector comprising a nucleotide fragment having the sequence of SEQ ID NO. 1.

3. The method for constructing the recombinant expression vector of claim 2, which comprises the following steps:

3) digesting the plasmid and recovering the product;

4. The construction method according to claim 3, wherein the PCR system in step 2) is: 5x Phusion HF Buffer, 10. mu.l; 2.5mM dNTPs, 4. mu.l; upstream primer 10uM, 2.5. mu.l; downstream primer 10uM, 2.5. mu.l; template DNA 100 ng/. mu.l, 1. mu.l; phusion High-Fidelity DNA Polymerase 0.5. mu.l; 29.5. mu.l of ultrapure water; the amplification procedure was: pre-denaturation at 98 ℃ for 30 s; denaturation at 98 ℃ for 10s, annealing at 64 ℃ for 30s, extension at 72 ℃ for 1min for 20s, and 35 cycles; extension at 72 ℃ for 10 min.

5. The construction method according to claim 4, wherein the enzyme cutting system in steps 3) and 5) is as follows: 10x CutSmart Buffer 5 ul, plasmid DNA 150 ng/ul, 6 ul, BamHI enzyme 20U/ul, 0.5 ul, ultra pure water 37.5 ul; the reaction program was 37 ℃ for 2 h.

6. The method of claim 3, wherein the In-Fusion linker system In step 4) is: 5 XIn-Fusion HD Enzyme Premix 2. mu.l, linearized pEGFP-1 plasmid DNA 43 ng/. mu.l, 1.5. mu.l, recovered product 16 ng/. mu.l, 6.5. mu.l; the reaction program is 50 ℃ for 15 min.

7. The method of claim 3, wherein the plasmid pEGFP-1 is selected in step 1).

8. The use of the crassostrea gigas EF-1 α promoter of claim 1 in crassostrea gigas gene breeding.

9. The method for using the recombinant expression vector of claim 3, wherein green fluorescence, i.e., the EGFP gene expression, is observed in each cell type of Mulberry stage, blastocyst stage, gastral stage embryo, trochophore and D-shaped larva by continuous observation under a fluorescence microscope after the mixture of the recombinant expression vector and phenol red is introduced into fertilized eggs of Ostreidae by microinjection.