CN105463109B - A kind of the molecular labeling gga-miR-34a-5p and its detection method of chicken intestinal diorder salmonella infection resistance trait - Google Patents
A kind of the molecular labeling gga-miR-34a-5p and its detection method of chicken intestinal diorder salmonella infection resistance trait Download PDFInfo
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- 206010039438 Salmonella Infections Diseases 0.000 title claims abstract description 8
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- 206010039447 salmonellosis Diseases 0.000 title claims abstract description 8
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The present invention relates to gene engineering technology fields, provide a kind of chicken intestinal diorder salmonella infection resistance molecule label and its detection method, the resistance molecule is labeled as chicken miRNA gga-miR-34a-5p, inventor has found that, gga-miR-34a-5p can be by regulating and controlling infection of the expression regulation Bacterium enteritidis of NOTCH2 gene to chicken, therefore gga-miR-34a-5p can be used as the molecular labeling of Bacterium enteritidis infection Resistance detecting, inventor further provides its detection method, so that the resistant heredity breeding for chicken lays the foundation.
Description
Technical field
The present invention relates to gene engineering technology fields, provide a kind of molecule of chicken intestinal diorder salmonella infection resistance trait
Mark gga-miR-34a-5p and its detection method.
Background technique
Bacterium enteritidis (Salmonella enteritidis) is Gram-negative bacteria, is universally acknowledged food-borne
One of pathogenic bacteria not only cause significant impact to laying hen production, and it can by poultry by-product to the mankind health care belt come
Very big threat.It includes that poultry, egg and milk and dairy produce etc. cause in people that this germ, which mainly passes through animal product,
Poison or even death.Therefore how to obtain the high heredity individual of resistance becomes one of urgent problem to be solved.
Tiny RNA (miRNA) is the endogenous non-coding tiny RNA of a kind of length about 22nt, with 3 ' UTR (untranslated of target gene
Area) it is incorporated in the expression of post-transcriptional level regulation target gene, in cell Proliferation, differentiation, apoptosis, neurodevelopment, fat metabolism etc.
It plays a significant role in multiple biological processes.
Summary of the invention
The present inventor is directed to the case where above-mentioned prior art, in conjunction with studying for a long period of time and exploring, provides a breeder
Bacterium enteritidis infects the molecular labeling and its detection method of resistance trait, which is labeled as chicken miRNA gga-
MiR-34a-5p, inventor has found that gga-miR-34a-5p can pass through the expression regulation of regulation NOTCH2 gene
Infection of the Bacterium enteritidis to chicken, therefore gga-miR-34a-5p can be used as point of Bacterium enteritidis infection Resistance detecting
Son label, inventor further provide its detection method, so that the resistant heredity breeding for chicken lays the foundation.
The specific technical solution that inventor provides is as follows:
Inventor provides firstly a kind of molecular labeling of chicken intestinal diorder salmonella infection resistance trait, the resistance molecule mark
It is denoted as chicken miRNA gga-miR-34a-5p, nucleotide sequence is as shown in SEQ ID NO:1；
The detection method that inventor furthermore presents the molecular labeling is as follows:
The expression quantity of gga-miR-34a-5p detects
MiRNA reverse transcription
With One StepMiRNA cDNA Synthesis Kit (Perfect Real Time) examination
Agent box, stringent by specification carry out.Reverse transcription system is 20 μ L (being specifically shown in Table 1).Response procedures are as follows: 37 DEG C, 60min；85 DEG C,
5s；The cDNA obtained after the reaction was completed is placed in -20 DEG C and saves backup；
1 miRNA reverse transcription system of table (20 μ L)
Wherein the caecum total serum IgE is chicken caecum total serum IgE, is obtained using conventional method,
Gga-miR-34a-5p quantitative fluorescent PCR
According to gga-miR-34a-5p mature sequence design primer (table 2), closed by Shanghai Sheng Gong bioengineering Co., Ltd
At.Using set primer, using SYBR Green I dye method, according to the SYBR PrimeScriptTM of TaKaRa company
MiRNA RT-PCR Kit specification, is expanded, reaction system is such as on Stratagene MX3000P fluorescence quantitative PCR instrument
Shown in table 3.
2 miRNA quantitative detection primer of table
Wherein > gga-34a-5p and U6 is respectively as Forward qPCR Primer, and Uni-miR qPCR Primer
It selects and carries primer in kit；
Quantitative fluorescent PCR response procedures use two-step method: 95 DEG C of initial denaturation 30s, 1 circulation；95 DEG C of 5s, 60 DEG C of 30s, 40
A circulation；Solubility curve (95 DEG C of 1min, 62 DEG C of 30s, 95 DEG C of 30s) finally are added, 1 circulation, each cDNA sample repeats three
It is secondary.Using U6 as reference gene, with 2-ΔΔCtMethod calculates the relative expression quantity of miRNA, with single factor test side in SAS8.1 software
MiRNA expression difference is analyzed in difference analysis.
3 quantitative fluorescent PCR system of table (20 μ L)
Function of the inventor in order to verify above-mentioned gga-miR-34a-5p utilizes by the way of target gene detection
The software predictions such as TargetScan and miRanda are the target gene of gga-miR-34a-5p to NOTCH2.As a result, it has been found that by dividing
Do not compare Bacterium enteritidis infected group and the regulation direction for being uninfected by miRNA and target gene in group, it is known that gga-miR-34a-
5p is significantly lowered in infected group, and expression quantity of the target gene NOTCH2 in infected group, which is significantly higher than, is uninfected by group (such as Fig. 1 institute
Show), i.e. gga-miR-34a-5p and NOTCH2 regulation is contrary, interaction relationship is shown, with Bioinformatics Prediction
As a result consistent.Illustrate that gga-miR-34a-5p can be by regulating and controlling the expression regulation Bacterium enteritidis of NOTCH2 gene to chicken
Infection.Therefore gga-miR-34a-5p can be used as the molecular labeling of Bacterium enteritidis infection Resistance detecting.
In conclusion the present invention provides a kind of chicken intestinal diorder salmonella infection resistance molecule label and its detection method,
The resistance molecule is labeled as chicken miRNA gga-miR-34a-5p, inventor has found that gga-miR-34a-5p can be with
By regulating and controlling infection of the expression regulation Bacterium enteritidis of NOTCH2 gene to chicken, therefore gga-miR-34a-5p can be used as
Bacterium enteritidis infects the molecular labeling of Resistance detecting, and inventor further provides its detection method, thus for the anti-of chicken
Sick genetic breeding lays the foundation.
Detailed description of the invention
Fig. 1 is RNA gga-miR-34a-5p and target gene NOTCH2 in the opposite differential expression being uninfected by group of infected group
Multiple histogram,
As seen from the figure, gga-miR-34a-5p is significantly lowered in infected group, and expression quantity of the NOTCH2 in infected group is aobvious
It writes to be higher than and is uninfected by group, is i.e. gga-miR-34a-5p and NOTCH2 regulation is contrary, shows interaction relationship, explanation
Gga-miR-34a-5p can be by regulating and controlling infection of the expression regulation Bacterium enteritidis of NOTCH2 gene to chicken.
Specific embodiment
In following embodiments in addition to specified otherwise, used is state of the art；
Embodiment 1
The expression quantity of gga-miR-34a-5p detects
MiRNA reverse transcription
With One StepMiRNA cDNA Synthesis Kit (Perfect Real Time) examination
Agent box, stringent by specification carry out.Reverse transcription system is 20 μ L (table 1).Response procedures are as follows: 37 DEG C, 60min；85 DEG C, 5s；Instead
The cDNA that should be obtained after the completion is placed in -20 DEG C and saves backup；
1 miRNA reverse transcription system of table (20 μ L)
Wherein the caecum total serum IgE is chicken caecum total serum IgE, is obtained using conventional method,
Gga-miR-34a-5p quantitative fluorescent PCR
According to gga-miR-34a-5p mature sequence design primer (table 2), closed by Shanghai Sheng Gong bioengineering Co., Ltd
At.Using set primer, using SYBR Green I dye method, according to the SYBR PrimeScriptTM of TaKaRa company
MiRNA RT-PCR Kit specification, is expanded, reaction system is such as on Stratagene MX3000P fluorescence quantitative PCR instrument
Shown in table 3.
2 miRNA quantitative detection primer of table
Wherein > gga-34a-5p and U6 is respectively as Forward qPCR Primer, and Uni-miR qPCR Primer
It selects and carries primer in kit；
Quantitative fluorescent PCR response procedures use two-step method: 95 DEG C of initial denaturation 30s, 1 circulation；95 DEG C of 5s, 60 DEG C of 30s, 40
A circulation；Solubility curve (95 DEG C of 1min, 62 DEG C of 30s, 95 DEG C of 30s) finally are added, 1 circulation, each cDNA sample repeats three
It is secondary.Using U6 as reference gene, with 2-ΔΔCtMethod calculates the relative expression quantity of miRNA, with single factor test side in SAS8.1 software
MiRNA expression difference is analyzed in difference analysis.
3 quantitative fluorescent PCR system of table (20 μ L)
Embodiment 2
The expression quantity of target gene NOTCH2 detects
MRNA reverse transcription
It is required according to kit specification, utilizes TaKaRa Primer ScriptTM RT reagent kit
MRNA reverse transcription is cDNA by (Perfect Real Time) kit, is placed on -20 DEG C and is saved backup.Reverse transcription system such as table 4
It is shown.Reverse transcription step: 37 DEG C, 15min；85 DEG C, 5s.
4 reverse transcription system of table
NOTCH2 quantitative fluorescent PCR
According to ncbi database mRNA sequence (accession number: XM_001233595), designed with Primer Premier5.0 glimmering
Fluorescent Quantitative PCR primer (shown in table 5), primer are synthesized by Shanghai Sheng Gong bioengineering Co., Ltd.
5 primer sequence of table
Using I dye method of SYBR Green, according to the SYBR Premix Ex TaqTM specification of TaKaRa company,
It is expanded on Stratagene MX3000P fluorescence quantitative PCR instrument, reaction system is 20 μ L (table 6): SYBR Primer Ex
10 μ l, Forward Primer (10 μM) of TaqTM (2 ×) 0.4 μ l, Reverse Primer (10 μM) 0.4 μ l, ROX
0.4 μ L, cDNA template of Reference Dye II (50 ×), 2 μ L, sterilize distilled water (ddH2O) 6.8 μ L, 20 μ L of final volume.Reaction
Program is two-step method: 95 DEG C of initial denaturation 30s, PCR reactions are as follows: 95 DEG C, 5s；60 DEG C, 30s；Circulation 40 times；Add solubility curve:
95 DEG C, 1min；61 DEG C, 30s；95 DEG C, 30s；Each sample is repeated 3 times.Using β-act in as internal reference, with 2-ΔΔCtMethod is come
The relative expression quantity for calculating mRNA, analyzes mrna expression amount difference with one-way analysis of variance in SAS8.1 software.
6 quantitative fluorescent PCR system of table
Embodiment 3
The test of chicken inoculation Bacterium enteritidis
Salmonella feminine gender Bai Laihang laying hen is divided into infected group and is uninfected by group, every chicken inoculation of infected group 5.8 ×
108CFU Bacterium enteritidis is uninfected by group inoculation phosphate buffer (PBS), the 7th day acquisition caecal tissue sample after inoculation.
According to operation instruction, caecum total serum IgE is extracted with Trizol method.
The correlation analysis of gga-miR-34a-5p and Bacterium enteritidis infection
Using 1 the method for embodiment, caecal tissue gga-miR-34a-5p is expressed after detecting Bacterium enteritidis infection
Variation.Quantitative result shows that expression quantity of the gga-miR-34a-5p in infected group caecum, which is substantially less than, is uninfected by group (Fold
Change=-2.79).Illustrate that gga-miR-34a-5p has stronger correlation with Bacterium enteritidis infection.
The correlation analysis of NOTCH2 and Bacterium enteritidis infection
Using 2 the method for embodiment, detects caecum NOTCH2 and express the correlation infected with Bacterium enteritidis.As a result
It has been shown that, the expression quantity of infected group NOTCH2, which is significantly higher than, is uninfected by group, is 1.29 times (P < 0.05) for being uninfected by group.Explanation
NOTCH2 is related to chicken intestinal diorder salmonella infection.
Gga-miR-34a-5p and its target gene NOTCH2's mutually performs an analysis
Pass through the regulation direction for being respectively compared infected group Yu being uninfected by miRNA and target gene in group, it is known that gga-miR-
34a-5p is significantly lowered in infected group, and expression quantity of the NOTCH2 in infected group, which is significantly higher than, is uninfected by group (such as Fig. 1), i.e.,
Gga-miR-34a-5p and NOTCH2 regulation is contrary, shows interaction relationship.Illustrate that gga-miR-34a-5p can be with
By regulating and controlling infection of the expression regulation Bacterium enteritidis of NOTCH2 gene to chicken.Therefore gga-miR-34a-5p can be used as
The molecular labeling of Bacterium enteritidis infection Resistance detecting.
Claims (1)
1. application of the molecular labeling gga-miR-34a-5p in detection chicken intestinal diorder salmonella infection resistance trait, the molecule
Gga-miR-34a-5p is marked, nucleotide sequence is as shown in SEQ ID NO:1.
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| MHC B-L基因多态性与鸡白痢沙门氏菌易感性的关联分析;张泽樘 等;《中国兽医科学》;20121231;第42卷(第1期);第13-18页 |
| 鸡白痢沙门菌基因组差异片段的筛选与分析;徐耀辉 等;《中国兽医科学》;20071231;第37卷(第7期);第563-567页 |
| 鸡白痢的诊断与防治研究概况;刘知奇 等;《中国畜牧兽医文摘》;20131231;第29卷(第6期);第119-120页 |
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