阅读说明：本技术 一种六盘山黄牛circR-UQCC1基因及其过表达载体、构建方法和应用 (CircR-UQCC1 gene of Lianshan cattle, over-expression vector, construction method and application thereof ) 是由 蔡翠翠 王健 李玉莲 万鹏 谢建亮 杨雪瑶 李毓华 余治家 贾宝光 佘萍 王维 于 2019-09-10 设计创作，主要内容包括：本发明公开了一种六盘山黄牛circR-UQCC1基因及其过表达载体、构建方法和应用,属于基因工程技术领域,本发明首次公开了环状的六盘山黄牛circR-UQCC1基因,并构建了能够表达六盘山黄牛circR-UQCC1基因的重组过表达载体,本发明所构建的载体,转染原代培养的牛肌肉细胞后,可获得circR-UQCC1 mRNA的高效表达,为circR-UQCC1基因功能鉴定和改造细胞,调控代谢和生长发育奠定了基础。(The invention discloses a circR-UQCC1 gene of a six-disc yellow bull, and an overexpression vector, a construction method and application thereof, belonging to the technical field of genetic engineering, and the invention discloses a circular circR-UQCC1 gene of the six-disc yellow bull for the first time, and constructs a recombinant overexpression vector capable of expressing the circR-UQCC1 gene of the six-disc yellow bull, and the constructed vector can obtain high-efficiency expression of the circR-UQCC1 mRNA after transfecting primary cultured bovine muscle cells, thereby laying a foundation for function identification and cell modification of the circR-UQCC1 gene, regulation of metabolism and growth and development.)

1. The circR-UQCC1 gene of the six-disk yellow bull is characterized in that the circR-UQCC1 gene of the six-disk yellow bull is circular DNA, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. a plasmid-type overexpression vector pCD2.1-circR-UQCC1 comprising the circR-UQCC1 gene of the six-pan yellow bull as described in claim 1.

3. the plasmid-based overexpression vector pCD2.1-circR-UQCC1, according to claim 2, wherein the circR-UQCC1 gene of the plasmid-based overexpression vector pCD2.1-circR-UQCC1 is linked below the shuttle plasmid CMV promoter and contains a prokaryotic ampicillin resistance gene in front of the start codon of the circR-UQCC1 gene.

4. The method for constructing plasmid-type overexpression vector pCD2.1-circR-UQCC1 of claim 2 or 3, comprising:

Amplifying a coding region sequence of the circR-UQCC1 gene by adopting high-fidelity enzyme PCR and connecting the sequence to a pMD-19T vector;

the recombinant plasmid pMD-19T-circR-UQCC1 and the over-expression vector pcD2.1 plasmid are subjected to double enzyme digestion by Kpn I and BamH I respectively;

carrying out enzyme-linked reaction on the circR-UQCC1 gene full-length sequence and pcD2.1 plasmid vector fragment by using T4 DNA Ligase;

the E.coli DH5 alpha competent bacteria were transformed with the enzyme-linked product to obtain plasmid-type overexpression vector pCD2.1-circR-UQCC 1.

5. The method for constructing plasmid-type overexpression vector pCD2.1-circR-UQCC1, according to claim 4, further comprising the step of determining whether the recombinant plasmid is successfully constructed:

after a plasmid type overexpression vector pCD2.1-circR-UQCC1 is transfected by liposome LIPOFECTAMINE 2000, the expression of the pCD2.1-circR-UQCC1 gene is detected in a cattle myoblast cell of Hexapany, and whether the construction of a recombinant plasmid is successful or not is determined.

6. use of the plasmid-type overexpression vector pCD2.1-circR-UQCC1 of claim 2 or 3 for identifying the function of the circR-UQCC1 gene of Flavobacterium Hexasporum.

7. use of the plasmid-type overexpression vector pCD2.1-circR-UQCC1 of claim 2 or 3 in cell engineering.

8. use of the plasmid-type overexpression vector pCD2.1-circR-UQCC1 of claim 2 or 3 for detecting differential expression of animal muscle growth and development marker genes.

9. The use of claim 8, wherein the animal muscle growth development marker genes are MyoD, MyoG, PCNA and MYHC.

Technical Field

the invention belongs to the technical field of genetic engineering, and relates to a six-pan yellow cattle circR-UQCC1 gene and a plasmid type overexpression vector pcD2.1-circR-UQCC1 thereof, a construction method and application.

Background

at present, epigenetics has become an important research direction for analyzing life activities such as growth and development, cancer development and treatment, immune response, and the like. Modulation of non-coding RNAs is one way of epigenetic regulation. Circular RNA is a type of covalently closed circular non-coding RNA molecule without a 5 'end cap and a 3' end poly-tail structure. With the rapid development of bioinformatics and the continuous innovation of high-throughput sequencing technology in recent years, a large number of circrnas which have stable endogenous structures, high abundance and show temporal-spatial expression specificity have been discovered in eukaryotic cells, and the circrnas may play an important role in regulating gene expression. In vitro expression of circular RNA is a prerequisite for experimental studies of its function.

in order to achieve overexpression of circular RNA, many plasmid vectors have been developed to achieve overexpression of circular RNA, and the key point is successful integration of the coding region of the target gene into the vector. The pcD2.1 vector is one of vectors for expressing circular RNA, and a CMV promoter strong promoter regulates and controls the expression of exogenous genes. The vector has high copy number and high expression amount. With green fluorescent label, without tag. Amp + prokaryotic screening resistance and Neo + eukaryotic screening resistance, and G418 can be used for screening stable cell strains. The size of the plasmid vector is 7.9 Kb. Has a multiple cloning site region containing restriction enzyme BamH I and Kpn I restriction sites, which determines that the target gene can be integrated on a plasmid vector to construct a recombinant plasmid by a double enzyme cutting method.

In molecular biology experiments, two different restriction enzymes are generally used to treat a target gene and a vector at the same time so as to prevent the self-ligation or reverse ligation of the target gene and the vector. The method utilizes the principle that restriction endonucleases can recognize and cut specific nucleotides, uses two different restriction endonucleases to cut a target gene to obtain target gene fragments with cohesive ends at the front and the back, and is connected with a linear vector with the same cohesive end, which is obtained by cutting through the two restriction endonucleases, under the action of T4 DNA ligase, so that gene cloning is realized.

Circular RNA (circRNA) is a newly discovered class of non-coding RNA molecules (part of the circRNA can be translated into small peptides). It is characterized by 5 'closure and 3' tail termination, and has a closed loop structure, which makes circRNA more stable. circRNA is more resistant to RNase R than other ncRNAs. Depending on the source of the circular RNA, the circular RNA can be divided into three types, exon circRNAs, intron circRNAs and exon-intron circRNAs. In general, most exon circrnas localize in the cytoplasm and can act as sponges of micrornas (mirnas) to affect post-transcriptional regulation, meaning that the circrnas can play a competitive role as endogenous RNAs (cernas). For example, cirS-7/CDR1as is considered to be an inhibitor of miR-7, which modulates hepatocellular carcinoma. The CircRNAs are widely involved in various vital activities such as cell proliferation, differentiation, muscle development, cancer development, immune response, and the like. CircRNA is highly enriched in striated muscle. Recent studies have shown that circRNA acts as a new regulatory network, contributing to skeletal muscle development, muscle production and regeneration, etc. It was reported that a part of circRNA in monkey muscle was significantly inhibited as muscle senescence. Circ-ZNF609 can be translated into peptides and regulate myogenesis. Thus, circRNA regulates skeletal muscle function through miRNA sponges and other means. However, the function and mechanism of most circrnas in skeletal muscle are still unclear.

The circR-UQCC1 gene of the six-pan yellow bull is located on chromosome 13 and is formed by circularization of the 5 th, 6 th and 7 th exons of the ubiquinol-cytochrome c reductase complex assembly factor 1(ubiquinol-cytochrome c reductase complex assembly factor 1, UQCC1) gene. High throughput sequencing results showed that circR-UQCC1 was specifically expressed during adult radishes in the six-disc mountain, with little expression during the embryonic phase. The online software RNAhybrid predicts that circR-UQCC1 can adsorb miR-186, miR-186 can promote myoblast differentiation by inhibiting myogenin, and in addition, miR-186 can inhibit prostate cancer cell proliferation, which suggests that circR-UQCC1 is likely to participate in muscle cell proliferation and differentiation, and further influences muscle function.

Disclosure of Invention

The invention aims to provide a circR-UQCC1 gene of a six-pan yellow bull, an overexpression vector thereof, a construction method and application thereof.

in order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses a circR-UQCC1 gene of a six-disk yellow bull, wherein the circR-UQCC1 gene of the six-disk yellow bull is circular DNA, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

The invention also discloses a plasmid type overexpression vector pCD2.1-circR-UQCC1 containing the circR-UQCC1 gene of the six-pan yellow cattle. The circR-UQCC1 gene in the plasmid type overexpression vector pCD2.1-circR-UQCC1 is connected below a shuttle plasmid CMV promoter, and contains a prokaryotic ampicillin resistance gene in front of the initiation codon of the circR-UQCC1 gene.

The invention also discloses a construction method of the plasmid type overexpression vector pCD2.1-circR-UQCC1, which comprises the following steps:

(1) Amplifying a coding region sequence of the circR-UQCC1 gene by using high-fidelity enzyme PCR, adding A into a PCR product, purifying and connecting the PCR product to a pMD-19T vector, converting, coating a plate, selecting a monoclonal and sequencing; carrying out double enzyme digestion on the recombinant plasmid pMD-19T-circR-UQCC1 and the over-expression vector pCD2.1 plasmid through Kpn I and BamH I respectively, carrying out enzyme ligation on a circR-UQCC1 gene coding region sequence and a pCD2.1 plasmid vector fragment by utilizing T4 DNA Ligase, and transforming E.coli DH5 alpha competent bacteria by using an enzyme ligation product to obtain the pCD2.1-circR-UQCC1 recombinant plasmid;

(2) Transfecting the recombinant plasmid pCD2.1-circR-UQCC1 to a cattle myogenic cell of the six-dribbling cattle, detecting the mRNA level and protein level change of the circR-UQCC1 gene after 24 hours, and determining whether the recombinant plasmid is constructed successfully.

specifically, after the circR-UQCC1 is amplified by PCR, the amplified circR-UQCC1 and pDNA2.1 shuttle plasmid are subjected to double enzyme digestion through Kpn I and BamH I, a gel recovery kit is used for recovering a circR-UQCC1 gene fragment and a pDNA2.1 vector fragment, and the recovered vector fragment and a target gene fragment T4 DNA Ligase are subjected to enzyme ligation reaction. Transforming E.coli DH5 alpha competent cells by enzyme ligation products, coating plates, selecting single clone, sequencing, extracting pCD2.1-circR-UQCC1 recombinant shuttle plasmid from a bacterial liquid purification kit with correct sequencing, and obtaining correct pCD2.1-circR-UQCC1 recombinant plasmid through Kpn I and BamH I double enzyme digestion identification.

The invention also discloses application of the plasmid type overexpression vector pCD2.1-circR-UQCC1 in identifying the function of the circR-UQCC1 gene of the cattle in Hexapan.

specifically, a recombinant overexpression plasmid pCD2.1-circR-UQCC1 is transfected into a primary bovine muscle cell, the influence of an overexpression circR-UQCC1 gene on the proliferation and differentiation of the bovine muscle cell and the expression quantity of a muscle development related gene is detected, and the plasmid type overexpression vector pCD2.1-circR-UQCC1 is used for the functional identification of the circR-UQCC1 gene of the cattle with six-disc mountain.

The invention also discloses application of the plasmid type overexpression vector pCD2.1-circR-UQCC1 in cell modification.

specifically, the recombinant overexpression plasmid pCD2.1-circR-UQCC1 of the six-disc yellow cattle circR-UQCC1 gene can be used for transfecting cell lines such as cattle muscle cells, fat cells, 293 cells, C2C12 cells, 3T3L cells and the like, the influence of the cell lines on the corresponding cell structure and function is detected, and the plasmid type overexpression vector pCD2.1-circR-UQCC1 is applied to cell modification.

the invention also discloses application of the plasmid type overexpression vector pCD2.1-circR-UQCC1 in detecting differential expression conditions of animal muscle growth and development marker genes.

Specifically, a recombinant plasmid pCD2.1-circR-UQCC1 is transfected into a bovine primary muscle cell, differential expression conditions of an over-expressed circR-UQCC1 gene on proliferation and differentiation of the muscle cell and muscle development marker genes (MyoD, MyoG, PCNA, CDK2 and MYHC) in the bovine muscle cell are detected through real-time quantitative PCR, and the plasmid type over-expression vector pCD2.1-circR-UQCC1 is used for regulating and controlling muscle growth and development.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses a circular six-disc yellow bull circR-UQCC1 gene for the first time, and constructs a recombinant overexpression vector capable of expressing the six-disc yellow bull circR-UQCC1 gene, and the constructed vector can obtain high-efficiency expression of circR-UQCC1 mRNA after transfecting primary cultured bovine muscle cells, thereby laying a foundation for the functional identification and cell modification of the circR-UQCC1 gene, and the regulation of metabolism and growth and development.

Drawings

FIG. 1 is a schematic structural diagram of a recombinant overexpression vector of the circR-UQCC1 gene of the six-pan yellow cattle constructed by the invention;

FIG. 2 shows PCR amplification electrophoresis detection of pCD2.1-circR-UQCC1 recombinant plasmid;

FIG. 3 shows the double restriction enzyme identification of recombinant plasmid Kpn I and BamH I of pCD2.1-circR-UQCC 1;

FIG. 4 shows the detection of the mRNA level overexpression efficiency of the pCD2.1-circR-UQCC1 recombinant plasmid;

FIG. 5 shows the detection of mRNA of the marker gene for proliferating and differentiating the primary cells of the six-pan Huangniu muscle after transfection; wherein (a) is PCNA, (b) is CDK2, (c) is MyoD, (d) is MyoG, and (e) is MYHC.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

the nucleotide sequence of the circR-UQCC1 gene of the cattle with six discs provided by the invention is shown in SEQ ID NO. 1.

referring to FIG. 1, FIG. 1 is a structural diagram of a plasmid type overexpression vector pCD2.1-circR-UQCC1 constructed according to the present invention and containing the above-described circR-UQCC1 gene, which is constructed from the circR-UQCC1 gene of the six-discorea cattle by a double enzyme cutting method, wherein the circR-UQCC1 gene of the plasmid type overexpression vector pCD2.1-circR-UQCC1 is linked under a shuttle plasmid CMV promoter, and contains an ampicillin resistance gene in front of the circR-UQCC1 gene.

The plasmid-type overexpression vector pCD2.1-circR-UQCC1 and the construction method thereof of the present invention will be further described below with reference to specific test examples.