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MiRNA apla-mir-25-42 related to follicular development of laying ducks and detection primers, inhibitors and application thereof

A follicle development and inhibitor technology, applied in the field of genetic engineering, can solve the problem of lack of miRNA markers, and achieve the effects of inhibiting expression, improving genetically assisted breeding, and improving the fecundity of laying ducks

Active Publication Date: 2019-01-11
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

Studies have shown that poultry follicle development is an extremely complex biological process. At present, people have a certain understanding of the follicle development model of poultry. However, as an important factor that determines egg production, the specific regulatory mechanism of follicle development still needs to be further studied. Research
[0003] However, at present, there is a lack of miRNA markers to detect the follicular development of laying ducks

Method used

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  • MiRNA apla-mir-25-42 related to follicular development of laying ducks and detection primers, inhibitors and application thereof
  • MiRNA apla-mir-25-42 related to follicular development of laying ducks and detection primers, inhibitors and application thereof
  • MiRNA apla-mir-25-42 related to follicular development of laying ducks and detection primers, inhibitors and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Obtaining PCR of laying duck miRNA apla-mir-25-42 and its fluorescence quantitative detection kit and method

[0057] 1. Obtained from laying duck apla-mir-25-42

[0058] The nucleotide sequence corresponding to the laying duck apla-mir-25-42 described in this example is shown in SEQ ID NO: 1. The laying duck apla-mir-25-42 of the present invention is the laying duck completed by the applicant based on the previous stage The whole transcriptome sequencing results of follicle tissue, a new miRNA molecule obtained through a large number of bioinformatics analysis and screening of the sequencing results of two groups of follicle tissue samples at different developmental stages. The applicant named it the laying duck apla-mir-25-42.

[0059] 2. Fluorescent quantitative PCR detection primer pair and its detection kit and method for laying duck miRNA apla-mir-25-42

[0060] In this example, a pair of fluorescent quantitative PCR detection primers was designed to i...

Embodiment 2

[0100] Example 2 Application of apla-mir-25-42 as a molecular marker in detecting the proliferation and differentiation of egg follicle granulosa cells in laying ducks

[0101] 1. Isolation of egg follicle granulosa cells in laying ducks

[0102] The following steps are used for the isolation of laying duck follicular granulosa cells:

[0103] 1. Select laying ducks at the peak egg production period, and kill them by bleeding from the jugular vein; take out the entire ovarian tissue and place it in a sterile petri dish filled with pre-cooled PBS;

[0104] 2. Rinse blood stains with PBS buffer solution with double antibody, rinse 3 times;

[0105]3. Transfer the rinsed follicles to a plate filled with pre-cooled PBS buffer, and peel off the follicle adventitia, connective tissue and vascular network;

[0106] 4. Use a scalpel to make a 1-2cm long incision on the surface of the follicle (the action should be fast), release the yolk, rinse the remaining yolk liquid with PBS buf...

Embodiment 3

[0134] Example 3 Design and construction of apla-mir-25-42 target gene TGFB2 dual luciferase reporter gene detection system in laying ducks

[0135] First, for the selected miRNA apla-mir-25-42, association analysis was performed in the whole transcriptome sequencing results, and the target gene TGFB2 interacting with apla-mir-25-42 was screened out from the mRNA sequencing results. miRBase analysis showed that the binding site between apla-mir-25-42 and TGFB2 was ATCCCCA.

[0136] Subsequently, the 3'-UTR sequence of TGFB2 (that is, the inhibitor of apla-mir-25-42) was obtained from NCBI, and the ATCCCCA in the inhibitor of apla-mir-25-42 was mutated into CAGGCGC by mutation method, respectively The above two gene fragments were cloned into the pmirGLO vector to obtain the corresponding vector (pmirGLO-TGFB2-wt) containing the inhibitor sequence of apla-mir-25-42 and the vector containing the inhibitor mutation sequence of apla-mir-25-42 Vector (pmirGLO-TGFB2-mut). Vector c...

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Abstract

The invention discloses MiRNA apla-mir-25-42 related to follicular development of laying ducks and detection primers, inhibitors and application thereof, belonging to that field of genetic engineer technology. The nucleotide sequence of the MiRNA apla-mir-25-42 is shown in SEQ ID NO: 1. The research of the invention shows that the miRNA can regulate follicular development by targeting the TGFB2 gene, reduce the expression of the cell proliferation marker gene CyclinB2 and promote the expression of the cell apoptosis marker gene BCL2. The miRNA can be used as a marker to detect the proliferation and differentiation of granulosa cells in laying duck follicles. The technical scheme makes up the blank in the field, provides theoretical basis and scientific proof for analyzing the genetic mechanism of follicular development of laying ducks and is of great significance to study of the genetic nature of laying duck reproductive traits, improvement of the fecundity of laying duck and genetic assisted breeding of laying ducks.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to miRNA apla-mir-25-42 related to egg follicle development of laying ducks, detection primers, inhibitors and applications. Background technique [0002] Follicle development is closely related to the laying performance of laying ducks and directly affects the egg production of laying ducks. Studies have shown that poultry follicle development is an extremely complex biological process. At present, people have a certain understanding of the follicle development model of poultry. However, as an important factor that determines egg production, the specific regulatory mechanism of follicle development still needs to be further studied. Research. [0003] However, at present, there is a lack of miRNA markers to detect the follicular development of laying ducks. [0004] In view of this, the present invention is proposed. Contents of the invention [0005] In order to o...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/178
Inventor 吴艳肖红卫张昊梁振华皮劲松潘爱銮蒲跃进孙静申杰杜金平白丁平
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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