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CN-121991984-A - Method for improving carotenoid content in fish muscle

CN121991984ACN 121991984 ACN121991984 ACN 121991984ACN-121991984-A

Abstract

The invention belongs to the technical fields of aquatic biology, molecular breeding and gene expression regulation and control, and in particular relates to a method for improving carotenoid content in fish muscle. The method is that the expression of beta-carotene splitting oxygenase 2 (beta-carotene oxygenase 2, bco 2) gene or the functional homologous gene is regulated, so that the splitting efficiency of carotenoid in the body is reduced, and the accumulation of the carotenoid in fish muscle tissues is promoted, so that the improvement of the color and luster of the muscle and the improvement of the meat quality are realized. The technology of the invention can obviously reduce the expression level of bco2l gene, promote the increase of carotenoid content in muscle and improve the flesh color quality and economic value of cynoglossus semilaevis. The method is simple and efficient, and has good popularization and breeding application prospects.

Inventors

  • SHA ZHENXIA
  • LI TINGTING
  • WANG YANAN
  • SHI KUNPENG
  • Chang Mengyang
  • LIU HONGNING

Assignees

  • 青岛大学

Dates

Publication Date
20260508
Application Date
20260409

Claims (9)

  1. 1. A method for improving carotenoid content in fish muscle, which is characterized in that the expression of beta-carotene splitting oxygenase 2 (beta-carotene oxygenase 2, bco 2) gene or functional homologous gene is regulated and controlled, so that the splitting efficiency of carotenoid in vivo is reduced, and the accumulation of carotenoid in fish muscle tissue is promoted, thereby improving the color and luster of the muscle and improving the meat quality.
  2. 2. The method for increasing carotenoid content in fish muscle according to claim 1, wherein the bco2 gene is a nucleotide sequence shown in SEQ ID No. 1 or has at least 55% or more of homology with SEQ ID No. 1.
  3. 3. The method of claim 2, wherein the modulating the expression of the β -carotene cleavage oxygenase 2 (β -carotene oxygenase 2, bco 2) gene or a functional homolog thereof is reducing the transcription level of the β -carotene cleavage oxygenase 2 (β -carotene oxygenase 2, bco 2) gene or a functional homolog thereof by silencing, knocking down, knocking out or inhibiting, inhibiting mRNA stability, blocking protein translation or disrupting the genomic integrity of the coding sequence thereof.
  4. 4. The method of claim 3, wherein the means for inhibiting gene expression is selected from the group consisting of any one of double-stranded RNA (dsRNA), small interfering RNA (siRNA), micro RNA (miRNA), short hairpin RNA (shRNA), antisense oligonucleotide (ASO) and CRISPR/Cas gene editing system, or a combination thereof.
  5. 5. The method for increasing the carotenoid content in fish muscle according to any one of claims 1 to 4, (1) Designing and synthesizing a gene suppression molecule targeting the functional homologous sequence of SEQ ID NO. 1 or at least 55% sequence identity thereof; (2) The inhibitor molecule is introduced into the fish of interest to reduce the expression of bco2 or its homologous genes.
  6. 6. The method of claim 5, wherein the inhibitory molecule is a functional homologous sequence targeting SEQ ID No.1 or at least 55% sequence identity thereof, i.e. double stranded RNA (dsRNA).
  7. 7. A double-stranded RNA molecule for inhibiting the expression of fish beta-carotene cleavage oxygenase 2 gene is characterized in that the double-stranded RNA molecule is a nucleotide sequence shown as SEQ ID NO. 2.
  8. 8. Use of the double-stranded RNA molecule of claim 7, wherein the double-stranded RNA molecule is used in the preparation of a formulation for increasing fish muscle carotenoid content.
  9. 9. A regulating preparation for regulating the carotenoid content of fish muscle, which is characterized in that the regulating preparation contains the double-stranded RNA molecule of claim 7.

Description

Method for improving carotenoid content in fish muscle Technical Field The invention belongs to the technical fields of aquatic biology, molecular breeding and gene expression regulation and control, and in particular relates to a method for improving carotenoid content in fish muscle. Background Carotenoids are a class of fat-soluble pigments that are widely found in plants, algae and parts of microorganisms and have important physiological functions in animals, including as vitamin a precursors, scavenging active oxygen, enhancing immune function, and participating in reproduction and development, etc. Since most animals, including fish, cannot self-synthesize carotenoids, they must be obtained by ingestion. In aquatic animals, carotenoids are essential nutrients for maintaining normal physiological functions, and directly affect the body color and meat quality of fish, so that the commodity value of the carotenoids is determined. Fish with bright color and rich carotenoids are often favored by consumers, such as salmon, rainbow trout, etc., which rely on carotenoid accumulation to develop a characteristic color. However, there are significant differences in the ability of different fish species to deposit carotenoids in muscle, and many important farmed fish species have limited market competitiveness due to their higher carotenoid metabolic rates or enhanced degradation, resulting in white flesh. Therefore, improving the carotenoid content of fish muscles and improving the color quality has important significance in aquatic breeding and quality improvement. Studies have shown that carotenoid metabolism in vertebrates is closely related to the carotenoid-cleaving oxygenase family (carotenoid cleavage oxygenases, CCOs), where β -carotene oxygenase 2 (BCO 2) catalyzes the cleavage reaction of various carotenoids, which in partly terrestrial vertebrates is believed to be related to carotenoid content in skin, feathers or adipose tissue. It should be noted that bco2 and its homologous genes in fish differ significantly from other vertebrates in gene copy number, family composition. In addition to the typical bco2 gene, there is a bco2 like homologous gene with undefined functions in the fish genome, and the expression regulation and control characteristics and biological functions of the bco2 like homologous gene have not been systematically elucidated. Meanwhile, fish muscle tissue is not a typical metabolic or deposition tissue of carotenoid, and whether bco2 and homologous genes thereof directly participate in pigment metabolism regulation in fish muscle or not is still lack of systematic research and sufficient experimental evidence support. Disclosure of Invention The invention aims to provide a method for improving carotenoid content in fish muscle. In order to achieve the above purpose, the invention adopts the technical scheme that: A method for increasing carotenoid content in fish muscle, which reduces the cracking efficiency of carotenoid in vivo by regulating and controlling the expression of beta-carotene cracking oxygenase 2 (beta-carotene oxygenase 2, bco 2) gene or its functional homologous gene, thereby promoting the accumulation of carotenoid in fish muscle tissue, and realizing the improvement of muscle color and luster and the improvement of meat quality. The bco2 gene is a nucleotide sequence shown as SEQ ID NO. 1 or has at least 55% of homologous nucleotide sequence with SEQ ID NO. 1. The bco2 genes include, but are not limited to, bco2a, bco2b, bco2-like, and the like, to enhance muscle carotenoid deposition. The regulation of the expression of the beta-carotene cleavage oxygenase 2 (beta-carotene oxygenase 2, bco 2) gene or a functional homologue thereof is to reduce the transcription level of the beta-carotene cleavage oxygenase 2 (beta-carotene oxygenase 2, bco 2) gene or a functional homologue thereof, inhibit mRNA stability, block protein translation or destroy the genome integrity of the coding sequence thereof by silencing, knocking down, knocking out or inhibiting. When the inhibition mode is adopted for gene expression, the inhibition mode is selected from any one or combination of double-stranded RNA (dsRNA), small interfering RNA (siRNA), micro RNA (miRNA), short hairpin RNA (shRNA), antisense oligonucleotide (ASO) or CRISPR/Cas gene editing system. The method comprises the following steps: (1) Designing and synthesizing a gene suppression molecule targeting the functional homologous sequence of SEQ ID NO. 1 or at least 55% sequence identity thereof; (2) The inhibitor molecule is introduced into the fish of interest to reduce the expression of bco2 or its homologous genes. The inhibitory molecules are introduced into fish bodies in the step (2) through injection, dipping, feed feeding, nanoparticle delivery or carrier expression and other modes. And then, adopting qPCR, western blot, enzyme activity measurement and other methods to evaluate the gene expression inhibition eff