CN104017888A - Identification method for campylobacter jejuni infection-associated chicken microRNA - Google Patents
Identification method for campylobacter jejuni infection-associated chicken microRNA Download PDFInfo
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Abstract
The invention discloses an identification method for campylobacter jejuni infection-associated chicken microRNA. The identification method comprises the following steps of: a first step of collecting chicken meconium, and detecting a positive rate of campylobacter jejuni; a second step of collecting cecum content for counting campylobacter jejuni and cecum tissue samples for RNA extraction; a third step of extracting total RNA of samples, detecting quality, and mixing in a tank; a fourth step of constructing a sequencing sample library; a fifth step of carrying out Solexa sequencing; a sixth step of carrying out subsequent analysis; a seventh step of predicating a target gene of the microRNA; an eighth step of obtaining campylobacter jejuni cfu infection-assocaited chicken microRNAs according to the microRNA and the target gene information thereof. The identification method disclosed by the invention regulates the target gene of the microRNA to indirectly participate in a plurality of immune-associated signal channels, so that important regulation effect is achieved in campylobacter jejuni infection; the microRNAs can be important candidate microRNAs in anti-campylobacter jejuni infection breeding, and foundation is laid up for deeply researching an acting mechanism of the microRNAs in campylobacter jejuni in future, so that theoretical basis and scientific basis are provided for molecular disease-resistant breeding of chicken.
Description
Technical field
The present invention relates to biological technical field, especially relate to the authentication method that a kind of campylobacter jejuni infects relevant chicken microRNA.
Background technology
Campylobacter jejuni, a kind of important foodborne bacterial pathogens, causes livestock and poultry morbidity, causes acute human enteritis, to the mankind, has brought serious financial loss and food-safety problem, caecum is that it mainly breeds position.Host's genetic background is resisted in campylobacter jejuni and is played an important role chicken, and there were significant differences to the susceptibility/resistance of campylobacter jejuni for the chicken of different lines (kind).
MicroRNA (miRNA) is the little RNA of endogenous non-coding of the about 22nt of a class length, by being combined with target gene 3 ' UTR, suppressing the translation of target gene or target gene is cut, in a lot of biological procedureses such as immunity and disease, play a significant role, comprise bacterium, virus infection.
In recent years, Solexa high throughput sequencing technologies becomes the powerful of each species microRNA Analysis and Identification, and the existing research of the chicken microRNA relevant to bacterium, virus infection, there is no the research of infecting relevant microRNA by Solexa sequencing technologies Analysis and Identification and campylobacter jejuni at present.This is that large-scale Analysis and Identification infects relevant microRNA to chicken campylobacter jejuni first, this lays the foundation further investigate the mechanism of action of microRNA in campylobacter jejuni for us, for the molecule breeding for disease resistance of chicken is provided fundamental basis and scientific basis.
Also for campylobacter jejuni, do not infect the research that relevant microRNA identifies at present, do not exist and identify that campylobacter jejuni infects the research of relevant microRNA.
Summary of the invention
The object of the invention is to design the authentication method that a kind of novel campylobacter jejuni infects relevant chicken microRNA, address the above problem.
To achieve these goals, the technical solution used in the present invention is as follows:
Campylobacter jejuni infects an authentication method of relevant chicken microRNA, comprises that step is as follows:
The first step, gathers 1 age in days chicken meconium, by defined medium, detects campylobacter jejuni positive rate;
Second step, inoculates approximately 10 to the negative chicken of campylobacter jejuni
8cfu (mushroom formation unit) campylobacter jejuni, control group inoculation equivalent PBS is more than or equal to 4 time points collection cecal contents and extracts for RNA for campylobacter jejuni counting, collection caecum tissue sample after inoculation;
The 3rd step, according to the metainfective positive rate of campylobacter jejuni and dynamic change result, selects chicken to infect responsive time point to campylobacter jejuni, gathers chicken caecum and is organized as experiment material; Adopt mirVana
tMmiRNA extracts test kit and extracts the total RNA of sample, and the total RNA of extracting gained carries out quality inspection through agilent bio-analyser 2100; In treatment group and control group group, mix respectively pond, in each group, all have 2-3 mixed pond;
The 4th step, according to " the little RNA preparation of samples of TruSeq guide ", the total RNA after purifying is carried out to the connection of 3 ' end connector, the connection of 5 ' end connector, reverse transcription, amplification and cDNA purification step, build order-checking sample library, treatment group >=2 wherein, control group >=2, and quality inspection is carried out in described order-checking sample library;
The 5th step, carries out Solexa order-checking to the described order-checking sample library after purifying;
The 6th step, carries out subsequent analysis, comprises the total indicator reding distribution situation of little RNA order-checking output, the length distribution statistics of comparison database information, little RNA comparison database information and little RNA;
The 7th step, utilize bioinformatics method Analysis and Identification infect with non-infected chicken caecum tissue in the microRNA of differential expression, predict its target gene, and the functional classification of target gene and the signal path that participates in analyzed;
The 8th step, Gene Ontology term and the signal path result of the significant enrichment that comprehensive the 6th step is relevant to the significance of difference, target gene that microRNA in the 7th step expresses, draw to campylobacter jejuni and infect relevant microRNAs.
According to being more than or equal to 4 bacteria contents in different time points caecum after campylobacter jejuni infected chicken, determine that chicken infects 1 responsive time point of reaction to campylobacter jejuni;
Utilize Solexa method to mix pond order-checking, mixed pond >=2 in each group to being more than or equal to 4 individualities in treatment group and control group;
According to the CHARACTERISTICS IDENTIFICATION campylobacter jejuni of microRNA itself and target gene thereof, infect relevant microRNA.
The present invention relates to the authentication method that a kind of campylobacter jejuni infects relevant chicken microRNA, comprise that step is as follows:
The first step, gathers 1 age in days chicken meconium, by defined medium, detects campylobacter jejuni positive rate;
Second step, inoculates approximately 10 to the negative chicken of campylobacter jejuni
8cfu campylobacter jejuni, control group inoculation equivalent PBS is more than or equal to 4 time points after inoculation, gathers cecal content for campylobacter jejuni counting, gathers caecum tissue sample and extracts for RNA;
The 3rd step, according to the metainfective positive rate of campylobacter jejuni and dynamic change result, selects chicken to infect responsive time point to campylobacter jejuni, gathers chicken caecum and is organized as experiment material; Adopt mirVana
tMmiRNA extracts test kit and extracts the total RNA of sample, and the total RNA of extracting gained carries out quality inspection through agilent bio-analyser (Agilent Bioanalyzer) 2100; In treatment group and control group group, mix respectively pond, in each group, all have 2-3 mixed pond;
The 4th step, according to " the little RNA preparation of samples of TruSeq guide (TruSeq Small RNA Sample Preparation Guide) ", the total RNA after purifying is carried out to the connection of 3 ' end connector, the connection of 5 ' end connector, reverse transcription, amplification and cDNA purification step, build order-checking sample library, treatment group and control group at least comprise 2 libraries, and quality inspection is carried out in described order-checking sample library;
The 5th step, carries out Solexa order-checking to the described order-checking sample library after purifying;
The 6th step, carries out subsequent analysis, comprises total indicator reding (reads) distribution situation of little RNA order-checking output, the length distribution statistics of comparison database information, little RNA comparison database information and little RNA;
The 7th step, utilize bioinformatics method Analysis and Identification infect with non-infected chicken caecum tissue in the microRNA of significant difference, predict its target gene, and the functional classification of target gene and the signal path that participates in analyzed;
The 8th step, the Gene Ontology term (GO term) and signal path result of the significant enrichment that comprehensive the 6th step is relevant to the significance of difference, target gene that microRNA in the 7th step expresses, draw to campylobacter jejuni and infect relevant microRNAs.
The present invention mainly by new-generation sequencing method to infected group and not infected group microRNA express and to study, the function of differential expression microRNA target gene is analyzed, comprehensive microRNA itself and target gene information thereof are identified the microRNA that is correlated with.
MicroRNA (miRNA) is the little RNA of endogenous non-coding of the about 22nt of a class length, by being combined with target gene 3 ' UTR, suppressing the translation of target gene or target gene is cut, and in a lot of biological procedureses, plays a significant role.
There were significant differences to the susceptibility/resistance of campylobacter jejuni for the chicken of different lines (kind), caecum is that it mainly breeds position, the object of the invention is to be organized as example with chicken caecum, by Solexa high throughput sequencing technologies Analysis and Identification and campylobacter jejuni, infect relevant chicken microRNA, for we further investigate the mechanism of action of microRNA in campylobacter jejuni and lay the foundation from now on, for the molecule breeding for disease resistance of chicken is provided fundamental basis and scientific basis.
Cfu in the present invention is that mushroom forms unit.
Beneficial effect of the present invention can be summarized as follows:
Based on the above results, gga-miR-148a, gga-miR-30a-5p, gga-miR-30b, gga-miR-30c, gga-miR-1416-5p, gga-miR-146a-5p, gga-miR-155, gga-miR-181a etc. are by regulating and controlling its target gene, indirectly participate in the proteolysis (Ubiquitin mediated proteolysis) of ubiquitin degraded, mTOR signal path (mTOR signaling pathway), MAPK signal path (MAPK signaling pathway), insulin signaling pathway (Insulin signaling pathway), TGF-beta signal path (TGF-beta signaling pathway), a lot of Ia signal paths such as RNA degraded (RNA degradation), in infecting, chicken campylobacter jejuni plays important regulating effect, may be that anti-campylobacter jejuni infects the important candidate microRNAs in seed selection, for we further investigate the mechanism of action of microRNA in campylobacter jejuni and lay the foundation from now on, for the molecule breeding for disease resistance of chicken is provided fundamental basis and scientific basis.
Accompanying drawing explanation
Fig. 1: different time points positive rate result after infecting;
Fig. 2: agilent bio-analyser (Agilent Bioana lyzer) 2100 electrophoresis result of total RNA;
Fig. 3: the medium and small RNA sequence length of control group and treatment group distributes;
Fig. 4: Gene Ontology (GO) annotation of differential expression miRNAs target gene;
Embodiment
In order to make technical problem solved by the invention, technical scheme and beneficial effect clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
A kind of campylobacter jejuni as shown in Figures 1 to 4 infects the authentication method of relevant chicken microRNA, comprises that a kind of campylobacter jejuni infects the authentication method of relevant chicken microRNA, comprises that step is as follows:
The first step, gathers 1 age in days chicken meconium, by defined medium, detects campylobacter jejuni positive rate;
Second step, inoculates approximately 10 to the negative chicken of campylobacter jejuni
8cfu campylobacter jejuni, control group inoculation equivalent PBS, is more than or equal to 4 time points collection cecal contents and extracts for RNA for campylobacter jejuni counting, collection caecum tissue sample after inoculation;
The 3rd step, according to the metainfective positive rate of campylobacter jejuni and dynamic change result, selects chicken to infect responsive time point to campylobacter jejuni, gathers chicken caecum and is organized as experiment material; Adopt mirVana
tMmiRNA extracts test kit and extracts the total RNA of sample, and the total RNA of extracting gained carries out quality inspection through agilent bio-analyser 2100; In treatment group and control group group, mix respectively pond, in each group, all have 2-3 mixed pond;
The 4th step, according to " the little RNA preparation of samples of TruSeq guide ", the total RNA after purifying is carried out to the connection of 3 ' end connector, the connection of 5 ' end connector, reverse transcription, amplification and cDNA purification step, build order-checking sample library, treatment group >=2 wherein, control group >=2, and quality inspection is carried out in described order-checking sample library;
The 5th step, carries out Solexa order-checking to the described order-checking sample library after purifying;
The 6th step, carries out subsequent analysis, comprises the total indicator reding distribution situation of little RNA order-checking output, the length distribution statistics of comparison database information, little RNA comparison database information and little RNA;
The 7th step, utilize bioinformatics method Analysis and Identification infect with non-infected chicken caecum tissue in the microRNA of significant difference, predict its target gene, and the functional classification of target gene and the signal path that participates in analyzed;
The 8th step, Gene Ontology term and the signal path result of the significant enrichment that comprehensive the 6th step is relevant to the significance of difference, target gene that microRNA in the 7th step expresses, draw to campylobacter jejuni and infect relevant microRNAs.
In being more preferably embodiment, according to being more than or equal to bacteria content in 4 time point caecums after campylobacter jejuni infected chicken, determine that chicken infects 1 responsive time point of reaction to campylobacter jejuni;
Utilize Solexa method to mix pond order-checking, mixed pond >=2 in each group to being more than or equal to 4 individualities in treatment group and control group;
According to the CHARACTERISTICS IDENTIFICATION campylobacter jejuni of microRNA itself and target gene thereof, infect relevant microRNA.
In certain preferred embodiment, gather 1 age in days SPF chicken meconium, by defined medium, detect campylobacter jejuni positive rate, the negative chicken of campylobacter jejuni is inoculated to approximately 10
8cfu campylobacter jejuni, control group inoculation equivalent phosphoric acid buffer (PBS), gathers respectively cecal content for 4,8,12,16,20,24,48 hours after inoculation and extracts for RNA for campylobacter jejuni counting and caecum tissue sample.According to campylobacter jejuni infection positive rate result and metainfective Dynamic Variation Analysis, select the rear 8h SPF chicken caecum of campylobacter jejuni inoculation to be organized as experiment material (Fig. 1), each 4 samples for the treatment of group and control group, mix pond between two, employing mirVana
tMlittle RNA extracts test kit and extracts the total RNA of sample, and the total RNA of extracting gained carries out quality inspection (Fig. 2) through agilent bio-analyser 2100.According to " the little RNA preparation of samples of TruSeq guide ", the total RNA after purifying being carried out to 3 ' end connector connects, 5 ' end connector connects, reverse transcription, amplification, the steps such as complementary DNA (cDNA) purifying, build order-checking sample library, T1, T2, C1, C2, and quality inspection (table 1) is carried out in constructed library.Then Solexa order-checking is carried out in the library after purifying, subsequent analysis comprises total indicator reding (reads) distribution situation and the comparison database information (table 2) of little RNA order-checking output, little RNA comparison database information (table 3), and the length distribution of little RNA (Fig. 3), utilize the microRNA (table 4) of the significant difference in the infection of bioinformatics method Analysis and Identification and non-infected chicken caecum tissue, predict its target gene, and the functional classification of target gene and the signal path that participates in are analyzed to (Fig. 4, table 5), can draw based on the above results to campylobacter jejuni and infect relevant microRNAs.
The quality inspection result in the constructed library of table 1
Total indicator reding (reads) distribution situation and the comparison database information of the little RNA order-checking of table 2 output
Note: miRBase: little RNA database; Pirna, Rfam, ncrna is three non-coding databases
The little RNA comparison of table 3 database information
Note: miRBase: little RNA database; Pirna, Rfam, ncrna is three non-coding databases
Differential expression miRNA between table 4 infection and non-infection
The KEGG signal path of table 5 differential expression miRNA target gene is analyzed
More than by the detailed description of concrete and preferred embodiment the present invention; but those skilled in the art should be understood that; the present invention is not limited to the above embodiment; within the spirit and principles in the present invention all; any modification of doing, be equal to replacement etc., within protection scope of the present invention all should be included in.
Claims (2)
1. campylobacter jejuni infects an authentication method of relevant chicken microRNA, it is characterized in that, comprises that step is as follows:
The first step, gathers 1 age in days chicken meconium, by defined medium, detects campylobacter jejuni positive rate;
Second step, inoculates approximately 10 to the negative chicken of campylobacter jejuni
8cfu campylobacter jejuni, control group inoculation equivalent PBS, is more than or equal to 4 time points collection cecal contents and extracts for RNA for campylobacter jejuni counting and caecum tissue sample after inoculation;
The 3rd step, according to the metainfective positive rate of campylobacter jejuni and dynamic change result, selects chicken to infect responsive time point to campylobacter jejuni, gathers chicken caecum and is organized as experiment material; Adopt mirVana
tMmiRNA extracts test kit and extracts the total RNA of sample, and the total RNA of extracting gained carries out quality inspection through agilent bio-analyser 2100; In treatment group and control group group, mix respectively pond, in each group, all have 2-3 mixed pond;
The 4th step, according to " the little RNA preparation of samples of TruSeq guide ", the total RNA after purifying is carried out to the connection of 3 ' end connector, the connection of 5 ' end connector, reverse transcription, amplification and cDNA purification step, build order-checking sample library, treatment group >=2 wherein, control group >=2, and quality inspection is carried out in described order-checking sample library;
The 5th step, carries out Solexa order-checking to the described order-checking sample library after purifying;
The 6th step, carries out subsequent analysis, comprises the total indicator reding distribution situation of little RNA order-checking output, the length distribution statistics of comparison database information, little RNA comparison database information and little RNA;
The 7th step, utilize bioinformatics method Analysis and Identification infect with non-infected chicken caecum tissue in the microRNA of differential expression, predict its target gene, and the functional classification of target gene and the signal path that participates in analyzed;
The 8th step, the Gene Ontology term (GO Tterm) and signal path result of the significant enrichment that comprehensive the 6th step is relevant to the significance of difference, target gene that microRNA in the 7th step expresses, draw to campylobacter jejuni and infect relevant microRNAs.
2. campylobacter jejuni according to claim 1 infects the authentication method of relevant chicken microRNA, it is characterized in that:
According to being more than or equal to bacteria content in 4 different time points caecums after campylobacter jejuni infected chicken, determine that chicken infects 1 responsive time point of reaction to campylobacter jejuni;
Utilize Solexa method to mix pond order-checking, mixed pond >=2 in each group to being more than or equal to 4 individualities in treatment group and control group;
According to the CHARACTERISTICS IDENTIFICATION campylobacter jejuni of microRNA itself and target gene thereof, infect relevant microRNA.
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Cited By (3)
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| WO2016127345A1 (en) * | 2015-02-11 | 2016-08-18 | 深圳华大基因研究院 | Method for constructing and sequencing small rna library |
| CN110592085A (en) * | 2019-09-17 | 2019-12-20 | 山东省农业科学院家禽研究所 | H9N2 subtype avian influenza virus inhibitor and application thereof |
| CN113403403A (en) * | 2021-07-15 | 2021-09-17 | 山东农业大学 | Molecular marker of chicken infected campylobacter jejuni, detection method and application |
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| GB202117593D0 (en) * | 2021-12-06 | 2022-01-19 | Royal Veterinary College | Methods |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016127345A1 (en) * | 2015-02-11 | 2016-08-18 | 深圳华大基因研究院 | Method for constructing and sequencing small rna library |
| CN110592085A (en) * | 2019-09-17 | 2019-12-20 | 山东省农业科学院家禽研究所 | H9N2 subtype avian influenza virus inhibitor and application thereof |
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| CN113403403A (en) * | 2021-07-15 | 2021-09-17 | 山东农业大学 | Molecular marker of chicken infected campylobacter jejuni, detection method and application |
| CN113403403B (en) * | 2021-07-15 | 2022-07-01 | 山东农业大学 | Molecular marker of chicken infected campylobacter jejuni, detection method and application |
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