CN110004234B - Chicken anti-salmonella infection related miRNA and application thereof - Google Patents
Chicken anti-salmonella infection related miRNA and application thereof Download PDFInfo
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Abstract
The invention provides miRNA related to chicken salmonella infection resistance and application thereof. According to the invention, a second-generation high-throughput sequencing technology is utilized, and a miRNA gga-miR-1306-5p for regulating and controlling the chicken salmonella infection key gene Tollip is screened by combining a miRNA-mRNA joint analysis method, is significantly and differentially expressed between salmonella susceptible group samples and control group samples, and can regulate and control the release of proinflammatory factors in the chicken salmonella infection process by down-regulating the expression of the Tollip gene. The miRNA disclosed by the invention can be used as a molecular marker related to salmonella infection of chickens, and the miRNA marker can be used for resisting salmonella infection of chickens for assisted breeding and has important practical application value in genetic assisted disease-resistant breeding.
Description
Technical Field
The invention belongs to the technical field of genetic breeding for chicken disease resistance, relates to screening and application of molecular markers related to salmonella infection of chicken, and particularly relates to miRNA of a Tollip gene which is a key gene for regulating and controlling salmonella infection and application of the miRNA.
Background
It is known that Salmonella Enteritidis (SE) is the only Salmonella serotype which can cause human diseases by egg contamination, and seriously threatens human food and health. Although salmonella contamination can be effectively controlled by various management measures in the chicken industry breeding industry, reports on salmonella infection are frequently made in recent years. In addition, salmonella has developed severe resistance to a variety of antibacterial agents, posing a significant threat to the prevention and treatment of animal and human diseases. In order to further explore the salmonella infection mechanism, protect animal welfare, reduce economic loss of poultry breeding industry and maintain food health, effective prevention and treatment methods need to be developed urgently, and a new field is developed to carry out strategic planning and future research.
miRNA is a non-coding, single-stranded small RNA molecule (18-25 nucleotides), and can regulate various biological processes by reducing gene expression at the level after transcription, and respectively play an important regulation role in physiological processes of cell proliferation, differentiation, apoptosis, tumor formation, inflammation and the like, and 60 percent of protein coding genes are regulated and controlled by miRNA to a certain extent. There is increasing evidence that mirnas are widely involved in host immune salmonella infection and are indispensable regulators in human and animal disease resistance. If miRNA of key genes related to host disease resistance can be obtained as molecular markers, the progress of disease resistance genetic selection can be accelerated.
The regulation and control effect of miRNA on animal disease immunity is realized by regulating and controlling specific target genes, and the target genes are predicted only by means of bioinformatics software, which is undoubtedly about the great sea fishing needle. By means of simultaneous deep sequencing analysis of miRNA and mRNA in a specific tissue at a specific period, according to the principle that miRNA and mRNA are in negative correlation expression and the result predicted by target gene software, the screening range of target miRNA target genes can be greatly reduced; through miRNA-mRNA joint analysis, a brand new means is provided for identifying and controlling key genes and miRNA with complex traits like salmonella infection resistance.
In conclusion, under the large background that the disease resistance difficulty of the current poultry is high and the breeding cost is dramatically increased, the related mechanism of the complex character of resisting salmonella infection by using a molecular marker assisted selection method is explored, and then related regulatory factors and key target genes thereof are screened out, so that the cost of disease-resistant line breeding can be saved for solving host immune salmonella infection.
Disclosure of Invention
The invention aims to provide a chicken anti-salmonella infection related miRNA and application thereof.
The invention screens a molecular marker gga-miR-1306-5p for regulating and controlling the infection of salmonella in a chicken host through a high-throughput sequencing technology and miRNA-mRNA combined analysis, wherein a target gene of the molecular marker gga-miR-1306-5p is Tollip. The molecular marker is significantly and differentially expressed between a salmonella infection group and a control group. Further molecular biology verification results show that gga-miR-1306-5p can effectively regulate and control the expression and release of downstream proinflammatory factors by inhibiting the expression of target genes Tollip.
Based on the miRNA molecular marker, the miRNA molecular marker related to chicken salmonella infection resistance is provided, and the RNA sequence of the miRNA molecular marker is shown in SEQ ID NO. 1.
The biological material containing the molecular marker belongs to the protection scope of the invention, and the biological material is expression box, transposon, carrier, host cell, transgenic cell line and engineering bacteria.
The invention provides an expression promoter of the miRNA molecular marker gga-miR-1306-5p, or an application of the biological material in targeted regulation of Tollip gene expression quantity. The invention discovers that the target gene of gga-miR-1306-5p is the Tollip gene and can negatively regulate the Tollip gene.
The invention also provides an expression promoter of the miRNA molecular marker gga-miR-1306-5p, or an expression promoter of gga-miR-1306-5p, or an application of the biological material in promoting expression and release of proinflammatory factors.
The skilled person in the art should understand that the application of the expression inhibitor of the miRNA molecular marker gga-miR-1306-5p in reducing the expression and release of proinflammatory factors, the application of the expression inhibitor in preparing anti-Salmonella infection medicines, and the application in improving the resistance of chickens to Salmonella infection also belong to the protection scope of the invention.
Furthermore, the invention provides a specific primer for detecting the gga-miR-1306-5p molecular marker, and the nucleotide sequence of the specific primer is shown in SEQ ID NO. 2.
The invention provides a kit for detecting chicken infected by salmonella, which contains a specific stem-loop primer for reverse transcription of gga-miR-1306-5p and/or a specific primer for detecting the expression quantity of gga-miR-1306-5 p.
In the kit provided by one embodiment of the invention, the nucleotide sequence of the specific stem-loop primer is shown as SEQ ID NO.3, and the specific primer for detecting the expression level of gga-miR-1306-5p is shown as SEQ ID NO. 2.
In the embodiment of the invention, the determination method of the miRNA molecular marker gga-miR-1306-5p is provided as follows:
(1) Extracting total RNA containing small RNA in the spleen of the chicken to be detected by using the kit;
(2) Carrying out specific reverse transcription of gga-miR-1306-5p by a miRNA stem-loop method, wherein specific stem-loop primers of the gga-miR-1306-5p are as follows: GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACACTGGAC (SEQ ID No. 3); and an internal reference downstream primer: TACTAACCGAGCCCGACCCT (SEQ ID NO. 4) together undergo a specific inversion.
(3) The quantification of gga-miR-1306-5p is carried out by using miRNA specific forward primer gACCACCTCCCCTGCAA (SEQ ID NO. 2), universal primer CAGTGCGTGTCGTGGAGT (SEQ ID NO. 5) and a commercial fluorescent quantitative PCR kit.
Preferably, the step of detecting the expression level of gga-miR-1306-5p comprises the following steps: (1) Extracting total RNA (Cat No. DP 419) from spleen tissue of chicken to be detected by using total RNA extraction kit of Beijing Tiangen company (2), using specific stem-loop primer and internal reference downstream primer, and performing Invitrogen reaction
III Reverse Transcription Kit (product number 1876472) carries out specific Reverse Transcription of miRNA; (3) The fluorescence quantitative kit (the product number is KK 4601) of miRNA specific forward primer gACCACCTCCCCTGCAA, universal primer CAGTGCGTGTCGTGGAGT and KAPA is adopted to carry out the quantitative determination of gga-miR-1306-5 p.
Specifically, the detection kit for detecting the microRNA marker provided by the invention is suitable for all types of fluorescent quantitative gene amplification instruments in the market at present, and has the advantages of high sensitivity, high speed and accuracy in quantification, good stability and good application prospect. The components of the fluorescent quantitative PCR kit for detecting the expression level of gga-miR-1306-5p prepared by the invention comprise: the kit comprises a reverse transcription reagent, a specific stem-loop reverse transcription primer, an internal reference downstream primer, a quantitative specific upstream primer, a quantitative universal primer and a fluorescent quantitative PCR reaction solution. Wherein the sequence of the specific stem-loop primer is as follows: GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACACTGGAC (SEQ ID No. 3); the quantitative specific upstream primer is as follows: gACCACCTCCCCTGCAA (SEQ ID NO. 2).
Furthermore, the invention provides application of the miRNA molecular marker gga-miR-1306-5p, or the biological material containing the miRNA molecular marker gga-miR-1306-5p, or a specific primer for detecting the miRNA molecular marker gga-miR-1306-5p, or the kit in detection of chickens with the ability of resisting salmonella infection.
The invention provides application of a miRNA molecular marker gga-miR-1306-5p, or the biological material containing the miRNA molecular marker gga-miR-1306-5p, or a specific primer for detecting the miRNA molecular marker gga-miR-1306-5p, or the kit in molecular genetic-assisted disease-resistant chicken breeding.
The invention has the beneficial effects that the miRNA gga-miR-1306-5p for regulating and controlling the salmonella infection key gene Tollip is screened by utilizing a new-generation high-throughput sequencing technology and combining a miRNA-mRNA joint analysis method, the significant differential expression between samples of a salmonella susceptible group and a control group can be realized, the release of proinflammatory factors in the salmonella infection process can be regulated and controlled by reducing the expression of the Tollip gene, specifically, the expression of the Tollip gene is reduced, the release of the proinflammatory factors is promoted, if the expression quantity of the gga-miR-1306-5p is inhibited, the quantity of the proinflammatory factors is significantly reduced in the salmonella infection process of chickens, the correlation between the gga-miR-1306-5p and the disease resistance character of chicken salmonella infection resistance is proved to be good, the miRNA gga-miR-1306-5p can be used for disease resistance genetic breeding of the salmonella infection of chickens, and early auxiliary selection can be realized. The gga-miR-1306-5p provided by the invention can be used as a molecular marker related to salmonella infection of chickens, and through detecting the miRNA marker, salmonella infection resistant chickens can be subjected to assisted breeding, so that the miRNA marker has an important practical application value in genetic assisted disease resistance breeding.
Drawings
FIG. 1 is a schematic representation of the binding site of the 3' UTR of Tollip to gga-miR-1306-5 p.
FIG. 2 is a graph showing the results of the relative expression amounts of gga-miR-1306-5p in the Salmonella control group and the Salmonella infected group.
FIG. 3 is a graph showing the results of the relative expression amounts of Tollip in the Salmonella control group and the infection group.
FIG. 4 shows the target gene dual luciferase assay of gga-miR-1306-5 p.
FIG. 5 is a graph showing the expression levels of pro-inflammatory cytokines NF-. Kappa.b, TNF-. Alpha., IL-6, and IL-1. Beta. Following transfection of a gga-miR-1306-5p mimic, inhibitor.
Detailed Description
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1 screening of miRNA associated with Salmonella infection resistance in chickens Using bioinformatic techniques
1) Experimental animals and sample preparation
White-skinned henry chicken (SPF chicken, beijing research center for laboratory animals) without specific pathogen is used as the source of experimental materials. Feeding in a totally-enclosed environment control box of the experiment center of Chinese agriculture university with constant conditions. At 3 days of age, 150 SPF chickens were orally administered with 1ml of 10 8 cfu Salmonella enterica in PBS and an additional 75 SPF chickens orally administered 1ml PBS, respectively, as controls. Slaughtering, taking blood and spleen samples, measuring the bacteria-carrying quantity of the blood, and storing the rest samples at-80 ℃.
From the phenotypic data of the amount of bacteria carried and the severity of the clinical symptoms (diarrhea, wingdrop and death), the susceptible groups (S, salmonella infection, mild clinical symptoms, > 10) were distinguished 7 cfu/10. Mu.L blood), disease resistant group (R, salmonella infection, severe clinical symptoms, < 10 5 cfu/10. Mu.L blood), control group (C, no Salmonella infection). Selecting 3 samples with typical characteristics from 3 groups of SPF chickens; the selected samples of each group were subjected to extraction of total RNA from spleen tissue using a commercial kit, and RNA was analyzedAnd detecting the total amount and the purity, and respectively constructing cDNA libraries for sequencing miRNA and mRNA. Sent to sequencing company for sequencing.
2) Analysis of differentially expressed mirnas and mrnas based on high throughput sequencing results
RPM > 3, p<0.05 (Student t-test) is defined as differentially expressed mirnas; miR-1306-5p is significantly and differentially expressed in three phenotype groups (a susceptible group, an anti-disease group and a control group); differentially expressed mRNA is defined as: | log 2 fold change|>0.58、p<0.05 (Cuffdiff test).
3) Key miRNA screening by differential expression miRNA and mRNA combined analysis
And performing combined analysis on all the differential expression miRNA and the differential expression mRNA by utilizing a miRanda and Targetscan algorithm based on the Ensemble database of the chicken, namely performing target gene prediction on the differential expression miRNA. The predicted target gene Tollip plays an important regulating role in the immune response of organisms to salmonella. The possible mechanism is that the gga-miR-1306 is combined with a Tollip gene in a targeted manner to influence a TLR-MyD88 signal pathway, so that the regulation and control of proinflammatory factor expression are realized, the immune response of an organism for resisting the infection of salmonella is further activated, and the subsequent work needs to further verify the action mechanism of the targeted regulation and control of Tollip by the gga-miR-1306-5 p.
The target gene prediction results indicated that Tollip 3' UTR region and gga-miR-1306-5p seed sequence are complementarily combined by multiple bases (FIG. 1). The Tollip gene is a down-regulated gene, the gga-miR-1306-5p is an up-regulated gene, and the function expectation of miRNA negative regulation is met, namely most of miRNA has negative regulation effect on target gene expression, so that the miRNA, miR-1306-5p, which is differentially expressed is presumed to participate in the regulation of Tollip expression.
Example 2 validation of the correlation of the gga-miR-1306-5p marker with Salmonella infection Using different populations
And performing amplification sample verification on the expression of key gga-miR-1306-5p and Tollip identified and obtained in miRNA and mRNA high-throughput sequencing by using real-time fluorescent quantitative PCR (qRT-PCR).
1) Preparation of Experimental samples
With white-skinned henry chicken (SPF) free of specific pathogensChicken, beijing research center for experimental animals) as the source of experimental materials. Feeding in a totally-enclosed environment control box of the experiment center of Chinese agriculture university with constant conditions. At 3 days of age, 30 SPF chickens were orally administered with 1ml of 10 capsules 8 PBS solution of cfu Salmonella enterica was used as the infection group, and 1ml PBS solution was orally administered to another 30 SPF chickens as a control. Slaughtering, taking blood and spleen samples, measuring the bacteria-carrying quantity of the blood, and storing the rest samples at-80 ℃.
Based on the phenotypic data of the amount of bacteria and the severity of clinical symptoms (diarrhea, wing drop and death), a total of 12 individuals (6 individuals per group) with typical phenotype values were selected for the control and infected groups.
2) Differential expression verification of gga-miR-1306-5p
Extracting total RNA of spleen tissues: total RNA extraction kit method of Beijing Tiangen company
Conditions for quantitation of Tollip mRNA were: the Kit miScript II RT Kit (Qiagen, germany) carries out reverse transcription; kit Quantifast SYBR Green PCR Kit (Qiagen, germany) the qRT-PCR primers were (F: ATGATCGCATTGCTTGGACA; R: AAAGACGTGTATGACATCACC). 3 parallel tube reactions were set for each assay, with beta-action as the internal reference. The PCR procedure was: 30s at 95 ℃;40 cycles (95 ℃ 3s,60 ℃ 34 s).
The conditions for quantifying gga-miR-1306-5p are as follows: reverse transcription was performed using a kit method (Invitrogen,
III Reverse Transcription Kit) and quantitative detection (KAPA, fluorescent quantitative Kit), wherein the specific primer for amplifying the gga-miR-1306-5p is gACCACCTCCCCTGCAA. The expression detection of miRNA sets 3 parallel tube reactions each time, and gga-5s-rRNA (F: CCATACCACCCTGGAAACGC; R: TACTAACCGAGCCCGACCCT) is used as an internal reference. The PCR procedure was: 30s at 95 ℃;40 cycles (95 ℃ 3s,60 ℃ 34 s).
Statistical analysis: analysis was performed using SAS 8.0 software (SAS inst. Cary, NC, USA). Statistical method the comparison between the mean values adopts t test, P is less than 0.05, the difference significance is determined, the statistical significance is achieved, and the expression difference between the phenotype groups of each group is analyzed.
The qRT-PCR result shows that the expression level difference of miR-1306-5p in an SPF chicken infection group and a control group is very obvious (figure 2); tollid also differed significantly in the SPF chicken infected group and the control group (fig. 3).
Example 3 Regulation of the proinflammatory response to Salmonella/LPS stimulation and Tollip Gene expression by miRNA gga-miR-1306-5p
1) Construction of plasmid and synthesis of miRNA
Construction of wild-type reporter vectors for Tollip: primers were designed based on the 3'UTR (NM-001006471) sequence of Tollip from chicken on Genbank, and the full-length 3' UTR sequence of Tollip was amplified by PCR using cDNA of chicken spleen tissue as a template. The target fragment is cloned to psiCHECK-2 through the steps of recovery, purification and the like TM In a carrier. The specific experimental steps include: analyzing enzyme cutting sites of a Tollip full-length 3' UTR sequence, and selecting two restriction enzyme cutting PCR products of XhoI and NotI according to the sequence information of the vector; purifying, enzyme-cutting and recovering a target fragment by using the kit; the PCR product was reacted with psiCHECK-2 TM Carrier connection; and carrying out colony PCR and sequencing identification to obtain the Tollip wild-type vector.
Construction of mutant reporter vectors for tollid: according to the binding Site of the 3' UTR region of Tollip and the corresponding miRNA seed region, the construction of a mutation vector is completed by using a Kit Fast Site-Directed Mutagenesis Kit (Tiangen, china), and a related primer is designed for mutation. The primers are as follows: gga-Tollip-mut-F TGGTTGCCAAATGGTTTATGTTTGAGGGCATCTCTGAAGG; gga-Tollip-mut-R ACCAACGGTTTACCAAATACAAACTCCCGTAGAGACTTCC; wherein the italicized bold part is a sequence of abrupt sites.
2) Dual-luciferase assay to verify targeted binding of Tollip and gga-miRNA-1306-5p
Taking HEK293T cells as test vectors, co-transfecting a Tollip dual-luciferase wild type reporter gene vector (Tollip 3'-UTR WT) and a mutant type reporter gene vector (Tollip 3' -UTR MT) with control gga-miR-NC or gga-miR-1306-5pmimics respectively by adopting a liposome transfection method, and transferring the Tollip dual-luciferase wild type reporter gene vector and the mutant type reporter gene vector into the cells.
The test was divided into four groups, specifically as follows: (1) co-transfection of gga-miR-NC and Tollip 3' -UTR WT; (2) co-transfection of gga-miR-1306-5p mimics with Tollip 3' -UTR WT; (3) co-transfection of gga-miR-NC and Tollip 3' -UTR MT; (4) co-transfection of gga-miR-1306-5pmimics with Tollip 3' -UTR MT.
One day before transfection, HEK293T cells were seeded into 12-well plates for overnight culture, and transfection was performed when the cell confluence reached 60%. Using the transfection reagent Lipofectamine 3000 TM (Invitrogen) Co-transfection experiments were performed using the following specific transfection system: the final concentration is 100nM gga-miR-NC or gga-miR-1306-5p mimics +3 mug of Tollip 3'-UTR WT or Tollip 3' -UTR MT.
After 36h of transfection, luciferase activities of the above four groups were detected using Dual-luciferase reporter assay system (Promega). The results in FIG. 4 show that compared with the gga-miR-NC transfection group, the co-transgenic gga-miR-1306-5p mimics and Tollip 3' -UTR WT group has obviously reduced relative luciferase activity. And after the binding site of the Tollip and the gga-miR-1306-5p seed sequence is mutated, the activity of the luciferase is not changed obviously. The results indicate that Tollip is the target gene of gga-miR-1306-5 p.
3) Effect of gga-miR-1306-5p on proinflammatory response of host infected with Salmonella
The gga-miR-1306-5p is over-expressed by a method of transfection and addition of gga-miR-1306-5p mimics in a chicken macrophage cell line HD11, and then LPS is used for stimulating macrophages to detect the expression level change of downstream inflammatory cytokines NF-kappa b, TNF-alpha, IL-6, and IL-1 beta. Results when gga-miR-1306-5p was overexpressed, the increase in proinflammatory cytokines was significantly promoted compared to the gga-miR-NC group (FIG. 5).
The expression of the gga-miR-1306-5p is reduced by a method of adding a gga-miR-1306-5p Inhibitor (Ribobio, available from Sharp synthesis company, guangzhou) into a chicken macrophage line HD11 by transfection, and then LPS is used for stimulating the macrophages to detect the expression level change of downstream inflammatory cytokines NF-kappa b, TNF-alpha, IL-6 and IL-1 beta. Results compared to the gga-miR-NC group, the increase of proinflammatory cytokines was significantly down-regulated when the expression of gga-miR-1306-5p was inhibited (FIG. 5).
Example 4 kit for detecting gga-miR-1306-5p in salmonella infection process
Obtaining small spleen RNA: qiagen miRNease Mini Kit
The reverse transcription and qRT-PCR reagents and primers are shown in Table 1, and a kit for detecting gga-miR-1306-5p related to chicken salmonella infection is constructed.
TABLE 1
Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (1)
- The application of the miRNA molecular marker gga-miR-1306-5p with the RNA sequence shown in SEQ ID NO.1 in the targeted regulation of the Tollip gene expression quantity; the miRNA molecular marker gga-miR-1306-5p is related to chicken salmonella infection resistance.
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