CN117051039B - Plasmid containing LoxP locus at two ends and expressing GFP protein, construction method and application thereof - Google Patents
Plasmid containing LoxP locus at two ends and expressing GFP protein, construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a plasmid containing LoxP loci at two ends and expressing GFP protein, a construction method and application thereof, wherein the construction method of the plasmid comprises the following steps: s1: two different types of homodromous LoxP sites are designed on two sides of the GFP expression element; s2: respectively connecting two different LoxP sites into forward and reverse primers, and amplifying an expression element of a GFP gene by taking a pEGFP-N3 plasmid as a template, wherein the expression element comprises a promoter, a coding region and a 5' UTR region; s3: connecting to a pMD18-T vector, designing forward and reverse primers from the outer side of an insertion sequence after positive detection is correct, and amplifying a GFP gene expression element combination with LoxP sites at two ends; s4: this combination of elements was ligated into pTol2-MCS plasmid at the multiple cloning site to construct the pTol2-GFP plasmid. After the plasmid is transferred into the mandarin fertilized ovum, the mandarin transgenic chassis family with green fluorescence is obtained, so that the site-directed editing site of the host genome is obtained, the plasmid is suitable for the same pond efficient screening and construction of different transgenic target families, the assistance is provided for fish genetic engineering breeding, and the fish genetic breeding efficiency is greatly improved.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a plasmid containing LoxP sites at two ends and expressing GFP, a construction method and application thereof.
Background
The Cre-loxP mediated recombination system can generate efficient and specific recombination at the loxP site without influencing the expression regulation of other genes, so that the Cre-loxP mediated recombination system is widely applied to transgenic research. Cre recombinase is a class of enzymes encoded by the Cre gene derived from the P1 phage, belonging to the Int family, recognizing a specific target site (loxP site) and catalyzing cleavage and recombination at that site. Specific recombination between two loxP sites mediated by Cre recombinase is classified into three cases according to the difference of sequence positions and directions: when two identical loxP sites are positioned on the same DNA chain and have the same direction, cre recombinase can knock out the DNA fragment between the two loxP sites; when two identical loxP sites are positioned on the same DNA chain and the directions are opposite, the Cre recombinase can reverse the DNA fragment between the two loxP sites; when two identical loxP sites are located on different DNA strands, cre recombinase is able to mediate exchange of DNA strands or chromosomal translocation between the loxP sites. In the experimental design, an experimenter can select a corresponding sequence construction mode according to different research requirements so as to achieve a corresponding purpose.
The green fluorescent protein (Green fluorescent protein, abbreviated as GFP) is a protein consisting of about 238 amino acids, and can be excited by blue light to ultraviolet light to emit green fluorescence. In cell biology and molecular biology, the Green Fluorescent Protein (GFP) gene is commonly used as a reporter gene. Through genetic engineering techniques, the Green Fluorescent Protein (GFP) gene can be transferred into the genome of different species for sustained expression in offspring. Now, the Green Fluorescent Protein (GFP) gene has been introduced and expressed in cells of many species including bacteria, yeast and other fungi, fish (e.g., zebra fish), plants, flies, and even mammals such as humans.
Mandarin (SINIPERCA CHUATSI) is an important fresh water famous excellent cultured fish in China, and belongs to the genus of perciformes, the family of lipid and the genus of mandarin. The siniperca chuatsi belongs to 7 species including siniperca chuatsi, siniperca scherzeri, siniperca chuatsi and siniperca chuatsi. The mandarin siniperca chuatsi used for pond culture at present mainly comprises siniperca chuatsi, siniperca scherzeri and siniperca scherzeri, wherein the growth speed of siniperca chuatsi is the fastest, and the economic value is the highest. With the rapid development of the mandarin fish breeding industry in recent years, the breeding density is continuously increased, and the disease occurrence of the breeding is frequent, so that the healthy development of the mandarin fish breeding industry is seriously hindered. The new disease-resistant cultivation variety is a basic guarantee for the healthy development of the mandarin cultivation industry.
The siniperca chuatsi disease-resistant related genetic engineering breeding research is one of effective ways for cultivating disease-resistant siniperca chuatsi cultivars. Because mandarin baits are used as food, the large number of groups is required for gene editing, fry opening, group cultivation, editing site identification and the like of more candidate disease resistance or susceptibility genes, and pond cultivation and multi-generation screening are required, so that great difficulty is brought to pond cultivation management. In order to screen suitable and efficient gene editing sites in genome in advance and facilitate co-pool cultivation and subsequent identification, development of a plasmid which expresses GFP protein and can perform subsequent site-directed replacement is needed to construct a mandarin GFP transgenic chassis family.
Disclosure of Invention
The invention provides a plasmid containing LoxP sites at two ends and expressing GFP protein, a construction method and application thereof. The identification of the transgenic families of the plasmids constructed by the method of the invention obtains the efficient site-directed editing sites of the host genome, and is suitable for the subsequent co-pool efficient screening and construction of different transgenic target families. The plasmid constructed by the method develops a mandarin GFP transgenic chassis family, and integrates a plurality of GFP gene copies on the genome. Through the application of red fluorescent protein (Red fluorescent protein, RFP for short) to GFP fixed-point replacement, the family can be used as a visual marker chassis family for subsequent mandarin transgenic breeding research, can be used as a high-efficiency visual screening tool for effectively integrating disease-resistant gene families in mandarin genetic engineering breeding, can also provide assistance for genetic engineering breeding of other fishes, and greatly improves the efficiency of fish genetic breeding.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a construction method of a plasmid containing LoxP loci at two ends and expressing GFP, which comprises the following steps:
s1: two different types of homodromous LoxP sites are designed on two sides of the GFP expression element;
S2: respectively connecting two different LoxP sites into forward and reverse primers, and amplifying an expression element of a GFP gene by taking a pEGFP-N3 plasmid as a template, wherein the expression element comprises a promoter, a coding region and a 5' UTR region;
S3: connecting to a pMD18-T vector, designing forward and reverse primers from the outer side of an insertion sequence after positive detection is correct, and amplifying a GFP gene expression element combination with LoxP sites at two ends;
S4: the element combination is connected to the polyclone site of pTol2-MCS plasmid to construct pTol2-GFP plasmid, namely the plasmid which contains LoxP site at the two ends and expresses GFP protein.
In the above technical solution, in S1: lox66 has the nucleotide sequence shown as SEQ ID NO.1 at the upstream side and Lox71 has the nucleotide sequence shown as SEQ ID NO.2 at the downstream side.
In the above technical solution, in S2: the promoter has a nucleotide sequence shown as SEQ ID NO.7, the coding region has a nucleotide sequence shown as SEQ ID NO.8, and the 5' UTR region has a nucleotide sequence shown as SEQ ID NO. 9.
In the above technical solution, in S2: the forward and reverse primers are F1 with a nucleotide sequence shown as SEQ ID NO.3, and R1 with a nucleotide sequence shown as SEQ ID NO. 4.
In the above technical solution, in S3: the forward and reverse primers are F2 with a nucleotide sequence shown as SEQ ID NO.5, and R2 with a nucleotide sequence shown as SEQ ID NO. 6.
The invention provides a plasmid for expressing GFP protein, which contains LoxP sites at two ends, and the plasmid is constructed by adopting the construction method.
The invention also provides application of the plasmid in genetic engineering breeding.
As one example, the plasmid is applied to construction of fish transgenic GFP gene chassis pedigree.
As one example, the plasmid is applied to the construction of a mandarin GFP transgenic chassis family, and the construction method comprises the following steps:
S1: in vitro transcribing Tol2 mRNA, microinjecting the Tol2 mRNA and pTol2-GFP into mandarin embryos together to construct a P0 generation transgenic population;
s2: after the P0 generation is sexually mature, the strain is subjected to test cross with a wild parent fish, and green fluorescence-emitting embryos are screened, so that the stable mandarin fish GFP transgenic chassis family is obtained.
In the technical scheme, the process of sexual maturity of the P0 generation is as follows: when the P0 generation transgenic population grows to 12-36 h, placing the microinjected embryo under an inverted fluorescent microscope for observation, selecting the embryo with strong luminous signals for incubation, simultaneously breeding the megalobrama amblycephala bait fish, and after the transgenic population moves smoothly, culturing the megalobrama amblycephala bait fish with an opening of the bait fish, and culturing the megalobrama amblycephala bait fish to sexual maturity after one month of age.
The invention has the beneficial effects that: compared with the prior art, the method uses the advantages of the Cre-LoxP recombinase system for constructing the transgenic family of the fish for the first time, and the basic steps comprise conventional plasmid construction, microinjection, fluorescent observation, family passage and the like, so that the whole process is simple and easy, and the aim of constructing the transgenic family of the target in a follow-up efficient manner can be achieved. The mandarin transgenic chassis family provided by the invention can be directly used for mandarin genetic engineering breeding research, simplifies mandarin cultivation facilities and cultivation periods, and greatly improves the efficiency of mandarin genetic breeding.
(1) The plasmid construction is not different from the conventional plasmid construction in difficulty, and is simple and easy to implement. The reagent materials are easy to purchase, and operators can operate without complicated scientific research training, so that the applicability is high;
(2) The detection of the expression condition of the exogenous GFP gene is more visual, the expression effect of the transgenic gene can be judged by observing the fluorescence brightness, and the screening is convenient. The conventional detection of the expression of exogenous genes requires the detection of integration sites, and the detection of the expression efficiency of genes cannot be judged on the basis of integration into the genome. According to the invention, through fluorescent observation of two-generation embryo samples, transgenic chassis families which are stably integrated and efficiently expressed can be screened out in the embryo development period, so that the detection difficulty is greatly reduced;
(3) After the establishment of the chassis family is completed, the establishment of the follow-up formal multi-class transgenic family can be realized by replacing the chassis family on the basis. The construction of the conventional transgenic family requires a large number of generations of family construction, pond-separating culture, sample collection, detection of exogenous target genes and the like, and the invention can judge the integration site and the expression efficiency of the exogenous target genes through observation of the fluorescent replacement condition of the subsequent generation transgenic embryos, thereby greatly reducing the expensive cost caused by construction, culture and manual management of a large number of economic fish families .
Drawings
FIG. 1 is an image of 3 green fluorescing F1 chassis families selected and a non-emitting control family.
FIG. 2 is a graph showing red fluorescence observations at various time points after transfer of the Mandarin Chassis family into red fluorescent protein.
Detailed Description
The invention will be further described with reference to specific examples for better illustrating the objects, technical solutions and advantages of the invention. This invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art, and the present invention will only be defined by the appended claims.
The invention provides a plasmid containing LoxP sites at two ends for expressing GFP protein, a construction method and application thereof, wherein the plasmid containing LoxP sites at two ends for expressing GFP protein is constructed as follows:
To avoid the occurrence of subsequent intramolecular recombination events at LoxP sites, two different types of LoxP sites were designed on both sides of GFP expression element. The method is to connect two different LoxP sites into forward and reverse primers respectively, and to amplify the expression element of GFP gene, including promoter, coding region and 5' UTR region, by using pEGFP-N3 plasmid as template. Then, the mixture was ligated to pMD18-T vector, and after positive detection, primers were designed from the outside of the insert sequence (on pMD18-T vector), and GFP gene expression element combinations comprising LoxP sites at both ends were amplified. This combination of elements was ligated into pTol2-MCS plasmid at the multiple cloning site to construct the pTol2-GFP plasmid.
The application of the plasmid containing LoxP locus for expressing GFP protein in constructing transgenic chassis family, the specific steps of constructing the transgenic chassis family by using the plasmid are as follows:
Tol2 mRNA was transcribed in vitro, and Tol2 mRNA and pTol2-GFP were annotated together into Mandarin embryos to construct P0 generation transgenic chassis families. After the P0 generation is sexually mature, the strain is subjected to test cross with a wild parent fish, and green fluorescence-emitting embryos are screened, so that the strain is a chassis family for stably transferring the LoxP-GFP gene.
As one example, the invention provides a plasmid construction method for expressing GFP protein with LoxP sites at two ends, which comprises the following specific steps:
1) Two different types of cognate LoxP sites (Lox 66: ATAACTTCGTATAGTATACATTATACGAACGGTA upstream) were designed on either side of the GFP expression element; downstream is Lox71: TACCGTTCGTATAGCATACATTATACGAAGTTAT. ).
2) Two LoxP sites were ligated into forward and reverse primers (F1:ATAACTTCGT ATAGTATACATTATACGAACGGTATAGTTATTAATAGTAATCAATT ACGGG;R1:ATAACTTCGTATAATGTATGCTATACGAACGGTAAC AAACCACAACTAGAATGCAGTG),, respectively, using pEGFP-N3 plasmid (purchased from Clontech) as a template, to amplify expression elements of GFP gene, including promoter, coding region and 5' utr region.
The nucleotide sequence of the promoter is ：TAGTTATTAATAGTAATCAATTACGG GGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGCGCTACCGGACTCAGATCTCGAGCTCAAGCTTCGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCATCGCCACC(SEQ ID NO.7);
The nucleotide sequence of the coding region is ：ATGGTGAGCAAGGGCGAGGAGCTG TTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA(SEQ ID NO.8);
The nucleotide sequence of the 5' UTR region is ：AGCGGCCGCGACTCTAGATCAT AATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGT(SEQ ID NO.9).
3) To pMD18-T vector (purchased from Takara corporation), after positive detection was correct, primers were designed from outside the insert (on pMD18-T vector) (F2: GC CTCTTCGCTATTACGCCAGC; r2: CCAATACGCAAACCGCCTCT CC), a GFP gene expression element combination including LoxP sites at both ends was amplified. This combination of elements was ligated into pTol2-MCS plasmid at the multiple cloning site to construct the pTol2-GFP plasmid.
As one embodiment, the invention provides a construction method of a mandarin GFP transgenic chassis family, which comprises the following specific steps:
1) Using the CZP13 plasmid purchased from the national zebra fish resource center as a template, tol2 mRNA was transcribed using an sp6 in vitro transcription kit (purchased from Thermo FISHER SCIENTIFIC company), and Tol2 mRNA (100 ng/ul) and pTol2-GFP (20 ng/ul) were annotated together with Mandarin embryos to construct a P0 generation transgenic population.
2) When the embryo grows for 12-36 h, placing the microinjected embryo under a fluorescence inverted microscope for observation, and selecting the embryo with strong luminous signals for incubation in an incubation barrel. And simultaneously, breeding megalobrama amblycephala bait fish, and culturing with a bait fish opening after the mandarin transgenic population swims smoothly. And after one month of age, the culture is carried out in a pond until the sexual maturity.
3) After sexual maturity of the transgenic P0 generation of one month, the breeding season is tested and crossed with the wild parent fish, and the embryo which emits green fluorescence is observed and screened under a fluorescence inversion microscope when the fertilized ovum grows to 12-36 hours, namely the chassis family which stably transfers LoxP-GFP genes.
4) 3 Families with stronger fluorescence are selected as the chassis families for seed conservation, and after the swimming is smoothed, the families are cultivated by using the bait fish openings. And after one month of age, the pond culture is carried out until sexual maturity, and the constructed transgenic chassis family is obtained (figure 1).
Experimental example
Verification experiment of transgenic chassis family target gene replacement:
1) The adeno-associated viral plasmid pAV-4in1shRNA-CMV-RFP was purchased from Shandong Uygur autonomous biosciences, inserted into two LoxP sites, and modified into pAV-lox66-4in1shR NA-CMV-RFP-polyA-lox71 plasmid.
2) After the transgenic chassis families are sexually mature, the male parent fish and the male parent fish in the F6 family (with the strongest green fluorescence intensity) are selfed in the breeding season to obtain chassis embryos for the transgenesis, cre enzyme (100 ng/ul) and pAV-lox66-4in1shRNA-CMV-RFP-polyA-lox71 (20 ng/ul) are subjected to microscopic annotation on mandarin embryos together, and the families of the transgenic RFP (replaced by the GFP gene) genes are constructed.
3) When the embryo developed to 24-96 h, the microinjected embryo is observed under a fluorescence inversion microscope, and as a result, 10% of the embryo only emits red fluorescence (figure 2), and the rest emits green fluorescence, which proves that 10% of the embryo successfully generates fluorescent protein replacement (green reddening), and the mandarin transgenic GFP gene chassis family designed and constructed based on the Cre-LoxP recombinase system is verified to be successful.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (4)
1. The application of the plasmid containing LoxP loci at two ends for expressing GFP in genetic engineering breeding is characterized in that the plasmid is applied to the construction of a mandarin GFP transgenic chassis family, and the construction method comprises the following steps:
S1: in vitro transcribing Tol2 mRNA, microinjecting the Tol2 mRNA and pTol2-GFP into mandarin embryos together to construct a P0 generation transgenic population;
s2: after the P0 generation is sexually mature, the strain is subjected to test cross with a wild parent fish, and green fluorescence-emitting embryos are screened to obtain the stable mandarin fish GFP transgenic chassis family;
Wherein, the pTol2-GFP construction method comprises the following steps:
S11: designing two different types of homodromous LoxP sites on two sides of a GFP expression element, wherein the upstream LoxP site is Lox66, the nucleotide sequence of the Lox66 is shown as SEQ ID NO.1, the downstream LoxP site is Lox71, and the nucleotide sequence of the Lox71 is shown as SEQ ID NO. 2;
S12: respectively connecting two different LoxP sites into forward and reverse primers, and amplifying an expression element of a GFP gene by taking a pEGFP-N3 plasmid as a template, wherein the expression element comprises a promoter, a coding region and a 5' UTR region; the nucleotide sequence of the promoter is shown as SEQ ID NO.7, the nucleotide sequence of the coding region is shown as SEQ ID NO.8, and the nucleotide sequence of the 5' UTR region is shown as SEQ ID NO. 9;
s13: connecting to a pMD18-T vector, designing forward and reverse primers from the outer side of an insertion sequence after positive detection is correct, and amplifying a GFP gene expression element combination with LoxP sites at two ends;
S14: the element combination is connected to the polyclone site of pTol2-MCS plasmid to construct pTol2-GFP plasmid, namely the plasmid which contains LoxP site at the two ends and expresses GFP protein.
2. The use according to claim 1, characterized in that in S12: the forward and reverse primers are F1 and R1 respectively, the nucleotide sequence of F1 is shown as SEQ ID NO.3, and the nucleotide sequence of R1 is shown as SEQ ID NO. 4.
3. The use according to claim 1, characterized in that in S13: the forward and reverse primers are F2 and R2 respectively, the nucleotide sequence of F2 is shown as SEQ ID NO.5, and the nucleotide sequence of R2 is shown as SEQ ID NO. 6.
4. The use according to claim 1, wherein the P0 generation sexual maturation is performed by: when the P0 generation transgenic population grows to 12-36 h, placing the microinjected embryo under an inverted fluorescent microscope for observation, selecting the embryo with strong luminous signals for incubation, simultaneously breeding the megalobrama amblycephala bait fish, and after the transgenic population moves smoothly, culturing the megalobrama amblycephala bait fish with an opening of the bait fish, and culturing the megalobrama amblycephala bait fish to sexual maturity after one month of age.
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