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Application of mirna-sc-mir-145 in the synthesis of fish lc-pufa

A mirna-sc-mir-145, 1. mirna-sc-mir-145 technology, applied in the field of fish molecular marker preparation, can solve the problem of affecting the growth and health of aquaculture fish, restricting the application of vegetable oil, and reducing the LC of fish - PUFA content and other issues

Active Publication Date: 2022-06-28
SHANTOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of LC-PUFA in vegetable oil, its substitution of fish oil not only affects the growth and health of aquaculture fish, but also significantly reduces the content of LC-PUFA in fish, which greatly limits the application of vegetable oil in compound feed.

Method used

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  • Application of mirna-sc-mir-145 in the synthesis of fish lc-pufa
  • Application of mirna-sc-mir-145 in the synthesis of fish lc-pufa
  • Application of mirna-sc-mir-145 in the synthesis of fish lc-pufa

Examples

Experimental program
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Effect test

Embodiment 1

[0022] To determine the changes in the expression level of sc-miR-145 in the liver tissue of S. maculatus under different fat source diet feeding conditions.

[0023] The yellow-spotted bluefish used in the present invention comes from wild fishing on Nan'ao Island, Shantou City, and is randomly divided into two groups after being domesticated and adapted to indoor brackish water (10ppt) for one month. Feed the compound feed with vegetable oil or fish oil as the main fat source. After 8 weeks of feeding, the liver tissue of the blue scorpion was collected, and the Trizol reagent provided by Roche was used to extract the total RNA in the tissue. Six parallel samples of liver tissue in the same treatment group were mixed in equal amounts, and two mixed samples of FO and VO were obtained. Sent to Shenzhen BGI Research Institute for HiSeq small RNA high-throughput sequencing.

[0024] The results of HiSeq small RNA high-throughput sequencing showed that the expression of sc-miR-1...

Embodiment 2

[0027] The targeted regulation of sc-miR-145 on Hnf4α gene

[0028] In this example, the precursor sequence of sc-miR-145 and its mature sequence were cloned and obtained by chromosome walking technology, wherein the mature sequence was: GUCCAGUUUUCCCAGGAAUCCC. The stem-loop structure of the precursor sequence of sc-miR-145 is shown in figure 2 shown, from figure 2 It can be seen that the sc-miR-145 precursor sequence folds into a stable stem-loop structure, which belongs to the typical secondary structure of miRNA precursors and conforms to the structural characteristics of miRNA precursors. According to the principle of complementary pairing between the target gene and the seed sequence, 9 consecutive base sequences that are completely complementary to the seed sequence of sc-miR-145 were found on the 3'UTR of the Hnf4α gene.

[0029] In order to prove whether there is an interaction effect between the two, a dual-luciferase reporter gene system experiment was carried out ...

Embodiment 3

[0033] Regulation of sc-miR-145 on Hnf4α and its downstream genes

[0034] Hnf4α was confirmed to be a transcription factor that regulates the expression of key enzyme genes △4Fad, △6△5Fad and Elovl5 in LC-PUFA synthesis in blue-spotted fish.

[0035] In order to verify the regulatory effect of sc-miR-145 on Hnf4α and its downstream genes, this example detected the protein and mRNA expression levels of Hnf4α and its downstream genes after overexpression or knockdown of sc-miR-145 in hepatocytes The change. The precursor sequence of sc-miR-145 and its stable inhibitor antagomir were synthesized in Guangzhou Ribo Bio Co., Ltd.

[0036] The results showed that after overexpression of sc-miR-145 precursor, the mRNA and protein expression levels of Hnf4α and the downstream gene △4Fad were significantly decreased, and the mRNA levels of △6△5Fad and Elovl5 were also significantly down-regulated. Conversely, after knockdown of sc-miR-145, the mRNA and protein expression levels of Hn...

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Abstract

The present invention relates to the application of miRNA-sc-miR-145 in the synthesis of LC-PUFA in fish, the fish is the yellow-spotted bluefish, and the sequence list of the miRNA-sc-miR-145 is shown in SEQ ID NO: 1 . The precursor sequence of the miRNA-sc-miR-145 is shown in SEQ ID NO: 2; the DNA sequence of the precursor sequence of the miRNA-sc-miR-145 is shown in SEQ ID NO: 3. The present invention verifies the response difference of miR‑145 to different fat sources by feeding macular bluefish with different fat source feeds, and concludes that miR‑145 and its precursor pre‑miR‑145 regulate the synthesis and metabolism of long-chain polyunsaturated fatty acids It plays an important role in LC-PUFA, and further carried out molecular biology experiment verification, which proves that the present invention's orchid miR-145 and its precursor pre-miR-145 play an important role in LC-PUFA synthesis, and orchid miR-145 and Knockdown of its precursor pre‑miR‑145 promotes LC‑PUFA synthesis in fish. The miR-145 of the present invention can be used as a molecular marker related to the long-chain polyunsaturated fatty acid synthesis ability of bluefish, and has important practical application value in genetic assisted breeding.

Description

technical field [0001] The invention belongs to the technical field of fish molecular marker preparation, in particular to the application of miRNA-sc-miR-145 in the synthesis of fish LC-PUFA. Background technique [0002] microRNA (miRNA) is a class of single-stranded non-coding small RNAs with a length of 18-25 nucleotides. Its transcription is initiated in the nucleus, and it is initially transcribed to form a length of about 60-90 nucleotides and has a typical stem-loop. Structural pre-miRNAs are then transported out of the nucleus and processed into mature miRNAs by Dicer enzymes in the cytoplasm. miRNA can recognize the 3' non-coding region (3' UTR) of a specific target mRNA through sequence complementation, make it degrade or inhibit translation, and participate in the post-transcriptional level regulation of genes. [0003] Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential fatty acids (EFAs) that maintain normal growth and development in vertebrates, m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12N15/113
CPCC12Q1/6888C12Q2600/178C12Q2600/158C12Q2600/124
Inventor 陈翠英王树启游翠红张媚胡宇
Owner SHANTOU UNIV
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