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Laying duck follicle growth-associated miRNA (miro ribonucleic acid) alpha-mir-145-4, detecting primer, inhibitor and application thereof

A technology of follicle development and inhibitors, which is applied in the field of genetic engineering, can solve the problems of lack of miRNA markers, etc., and achieve the effect of promoting expression and improving the fecundity of laying ducks

Active Publication Date: 2019-03-01
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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  • Laying duck follicle growth-associated miRNA (miro ribonucleic acid) alpha-mir-145-4, detecting primer, inhibitor and application thereof
  • Laying duck follicle growth-associated miRNA (miro ribonucleic acid) alpha-mir-145-4, detecting primer, inhibitor and application thereof
  • Laying duck follicle growth-associated miRNA (miro ribonucleic acid) alpha-mir-145-4, detecting primer, inhibitor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Obtaining PCR of laying duck miRNA apla-mir-145-4 and its fluorescence quantitative detection kit and method

[0057] 1. Obtained from laying duck apla-mir-145-4

[0058] The nucleotide sequence corresponding to the laying duck apla-mir-145-4 described in this example is shown in SEQ ID NO: 1. The laying duck apla-mir-145-4 of the present invention is the laying duck completed by the inventor 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-145-4.

[0059] 2. Fluorescent quantitative PCR detection primer pair and its detection kit and method for miRNA apla-mir-145-4 in laying ducks

[0060] In this example, a primer pair for fluorescent quantitative PCR was designed to identify...

Embodiment 2

[0100] Example 2 Application of apla-mir-145-4 as a molecular marker in detecting the proliferation and differentiation of follicular 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 buffe...

Embodiment 3

[0134] Example 3 Design and construction of apla-mir-145-4 target gene PIK3CA dual luciferase reporter gene detection system in laying ducks

[0135] First, for the selected miRNA apla-mir-145-4, association analysis was performed in the whole transcriptome sequencing results, and the target gene PIK3CA interacting with apla-mir-145-4 was screened out from the mRNA sequencing results. miRBase analysis showed that the binding site between apla-mir-145-4 and PIK3CA was ATCCCCA.

[0136] Subsequently, the 3'-UTR sequence of PIK3CA (that is, the inhibitor of apla-mir-145-4) was obtained from NCBI, and the AACTGGA in the inhibitor of apla-mir-145-4 was mutated into GTAATTC by mutation method, respectively The above two gene fragments were cloned into the pmirGLO vector to obtain the corresponding vector (pmirGLO-PIK3CA-wt) containing the inhibitor sequence of apla-mir-145-4 and the vector containing the inhibitor mutation sequence of apla-mir-145-4 Vector (pmirGLO-PIK3CA-mut). Ve...

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Abstract

The invention discloses a laying duck follicle growth-associated miRNA alpha-mir-145 and detecting primer, inhibitor and application thereof and belongs to the technical field of genetic engineering.The nucleotide sequence of the laying duck follicle growth-associated miRNA alpha-mir-145 is shown as SED ID NO: 1. Research shows that the laying duck follicle growth-associated miRNA alpha-mir-145 can regulate follicle growth through targeted PIK3CA genes, reduce expression of a cell proliferation marker gene CyclinB2 and enhance expression of a cell apoptosis marker gene BCL2. The laying duck follicle growth-associated miRNA alpha-mir-145 can serve as a marker for detecting proliferation of laying duck follicular cells, thereby filling the gap in the field, providing a theoretical basis anda scientific basis for analysis on the genetic mechanism of laying duck follicle growth and being of great significance to researching the genetic nature of duck reproduction traits, improving the reproductivity of laying ducks and achieving assisted genetic breeding.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to miRNA apla-mir-145-4 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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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12Q1/6888C12N15/11C12N5/071
CPCC12N5/0682C12N9/1205C12N15/113C12N2310/141C12N2501/65C12Q1/6888C12Q2600/124C12Q2600/158C12Q2600/178C12Y207/01153
Inventor 吴艳肖红卫梁振华张昊潘爱銮皮劲松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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