Laying duck circRNA circ_13034 and detection reagent, method and application thereof

A detection kit and a technology for laying ducks are applied in the field of genetic engineering to achieve the effect of improving the fecundity of laying ducks

Active Publication Date: 2018-12-11
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on tissue-specific circular RNAs in laying ducks, especially the research on circular RNAs in laying duck follicles and its regulatory mechanism has not been reported.

Method used

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  • Laying duck circRNA circ_13034 and detection reagent, method and application thereof
  • Laying duck circRNA circ_13034 and detection reagent, method and application thereof
  • Laying duck circRNA circ_13034 and detection reagent, method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Laying duck circular RNA circ_13034 gene

[0035] 1. Laying duck circular RNA circ_13034

[0036] The cDNA sequence corresponding to the laying duck circular RNA circ_13034 described in this example is shown in SEQ ID NO: 1. The laying duck circular RNA circ_13034 of the present invention is based on the whole transcriptome sequencing result of the laying duck follicle tissue completed earlier by the applicant. A new circular RNA molecule was obtained through extensive bioinformatics analysis and screening of the sequencing results of two groups of follicular tissue samples at different developmental stages. The applicant named it egg-duck circular RNA circ_13034, and the length of the cDNA sequence corresponding to the egg-duck circular RNA circ_13034 was 1068 bp.

[0037] 2. Preparation of the full-length sequence fragment of circular RNA circ_13034 gene of laying duck

[0038] 1. Primer design and synthesis

[0039] According to the full-length fragment ...

Embodiment 2

[0084] Example 2 Fluorescent quantitative PCR detection primer pair and its detection kit and method for laying duck circular RNA circ_13034 gene

[0085] In this example, a pair of fluorescent quantitative PCR detection primers was designed to identify and identify the objective existence of the laying duck circular RNA circ_13034 described in Example 1 in the egg follicle granulosa cells of the laying duck. The identification method of this embodiment includes the following steps:

[0086] 1. Sample collection

[0087] With the method in embodiment 1.

[0088] 2. Extraction of total RNA from samples

[0089] Same as the RNA extraction method in Example 1.

[0090] 3. RNA sample quality detection and analysis

[0091] With the method in embodiment 1.

[0092] 4. Digestion of circular RNA (RNase R)

[0093] With the method in embodiment 1.

[0094] 5. Reverse transcription

[0095] With the method in embodiment 1.

[0096] 6. Fluorescent quantitative PCR reaction

[...

Embodiment 3

[0104] Example 3 Construction of egg duck circular RNA circ_13034 gene overexpression vector

[0105] This embodiment provides an overexpression vector of the egg duck circular RNA circ_13034 gene, and the vector includes the cDNA sequence corresponding to the egg duck circular RNA circ_13034. Vector construction specifically includes the following steps:

[0106] (1) Double digestion treatment of pLCDH-ciR vector

[0107] The schematic diagram of the structure of the pLCDH-ciR vector is shown in image 3 As shown, the vector contains restriction endonucleases EcoRI and BamHI cutting sites. The above carrier was double-digested with two restriction enzymes EcoRI and BamHI. The enzyme digestion reaction system was as follows, and the total volume of the system was 50 μL:

[0108] table 5

[0109]

[0110] The reaction conditions are: enzyme digestion at 37°C for 6 hours, followed by purification and recovery of the enzyme digestion products.

[0111] (2) Double enzyme d...

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Abstract

The invention belongs to the technical field of genetic engineering and provides laying duck circRNA circ_13034 and a detection reagent, a method and an application thereof. A cDNA sequence corresponding to the laying duck circRNA circ_13034 provided by the invention is shown in SEQ ID NO: 1. The invention further provides a fluorescent quantitative PCR detection primer pair and a detection kit ofthe laying duck circRNA. The detection primer pair has nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3. The invention further provides an overexpression vector of the laying duck circRNAcirc_13034 and a construction method thereof. The invention further provides application of the laying duck circRNA and the overexpression vector in differentiation of laying duck follicle developmentconditions and detection of cell proliferation and differentiation conditions of laying duck follicle granulosa cells as molecular markers. The application has an important significance in researching inheritance of proliferation character of laying duck and improving reproductivity of the laying duck.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to laying duck circular RNA circ_13034 and its detection reagent, method and application. Background technique [0002] Circular RNA (circular RNA, circRNA) is a newly discovered endogenous non-coding RNA, which is composed of a covalent bond to form a closed-loop RNA molecule, which is not affected by RNA exonuclease, and its expression is more stable and difficult to degrade . Studies have shown that most of circRNAs are non-coding RNAs, mainly derived from exons, and circRNAs are tissue-specific in animals. Recent studies have found that in a variety of biological cells and tissues, more than 10% of the expressed genes can produce circRNAs. In terms of function, recent studies have shown that circRNA has a high abundance and is rich in microRNA (miRNA) binding sites. It acts as a miRNA sponge in cells and can competitively inhibit the binding of miRNA to target gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/113
CPCC12Q1/686C12Q1/6888C12Q2600/124C12Q2600/158C12Q2600/178C12Q2563/107C12Q2545/114
Inventor 吴艳皮劲松梁振华潘爱銮张昊蒲跃进申杰杜金平孙静
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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