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CN-122012502-A - RNAi target sequence for channel catfish dnmt gene knockout, sex control method and application

CN122012502ACN 122012502 ACN122012502 ACN 122012502ACN-122012502-A

Abstract

The invention provides a channel catfish dnmt gene knockout RNAi target sequence, a sex regulation method and application, and belongs to the technical field of sex regulation. The invention designs RNAi sequence based on dnmt gene, and realizes gonadal differentiation intervention independent of exogenous hormone in the species by inhibiting dnmt gene expression in channel catfish. Experimental results show that the down regulation of dnmt1 expression can effectively induce genetic female individuals to generate partial reversion, and typical testis-like structures appear in ovarian tissues, so that the key effect of dnmt1 in maintaining female gonad development stability is proved, and the establishment of a stable and efficient all-male channel catfish breeding system by dnmt1 regulation and control combined genetic screening or gene editing technology is expected, and a new path is opened for all-male offspring seed production.

Inventors

  • ZHANG SHIYONG
  • CHEN XIAOHUI
  • LIU HONGYAN
  • WANG MINGHUA
  • ZHONG LIQIANG

Assignees

  • 江苏省淡水水产研究所

Dates

Publication Date
20260512
Application Date
20260228

Claims (9)

  1. 1. The channel catfish dnmt gene knocked out RNAi target sequence is characterized in that the RNAi target sequence is shown as SEQ ID NO. 4.
  2. 2. The channel catfish sex regulation method is characterized by comprising the following steps of: (1) RNAi plasmid design for lentiviral packaging Designing RNAi target sequences, sense strands and antisense strands aiming at channel catfish dnmt1 genes, preparing an LV3-shRNA vector based on the RNAi target sequences, the sense strands and the antisense strands and the LV3-pGLV-h1-GFP-puro vector, transforming DH5 alpha competent cells from the LV3-shRNA vector to obtain dnmt gene RNAi plasmids, and packaging the dnmt gene RNAi plasmids by slow viruses to obtain RNAi plasmids packaged by slow viruses; (2) Channel catfish sex regulation Injecting RNAi plasmid packaged by the lentivirus in the step (1) into yolk of the channel catfish embryo close to the animal pole, incubating and identifying to obtain the hermaphrodite channel catfish.
  3. 3. The method for sex control of channel catfish according to claim 2, wherein in the step (1), the RNAi target sequence is shown as SEQ ID NO.4, the Loop structure in the sense strand uses a "TTCAAGAGA" sequence to avoid formation of a termination signal, a "GATCC" sequence is added to the 5 'end of the sense strand, and an "AATTC" sequence is added to the 5' end of the antisense strand.
  4. 4. The method for regulating and controlling the sex of channel catfish according to claim 2, wherein the sense strand is shown as SEQ ID NO.5 and the antisense strand is shown as SEQ ID NO. 6.
  5. 5. The method for sex control of channel catfish according to claim 2, wherein in step (2), the lentiviral packaged RNAi plasmid has a titer of 10 7~8 and an injection amount of 50 nL.
  6. 6. An application of the channel catfish dnmt gene knockout RNAi target sequence in the construction of a hermaphrodite channel catfish model.
  7. 7. A hermaphroditic channel catfish model constructed by the channel catfish sex control method of any one of claims 2-5.
  8. 8. The hermaphrodite channel catfish model of claim 7, wherein the hermaphrodite channel catfish model is a reversion of channel catfish of female genes to hermaphrodite channel catfish with male traits.
  9. 9. Use of the hermaphrodite channel catfish model of any one of claims 7-8 in sex determination mechanism research.

Description

RNAi target sequence for channel catfish dnmt gene knockout, sex control method and application Technical Field The invention relates to the technical field of sex regulation, in particular to a channel catfish dnmt gene knockout RNAi target sequence, a sex regulation method and application. Background The channel catfish (Ictalurus punctatus) is used as a global important freshwater economic fish, has the advantages of fast growth, strong disease resistance, excellent meat quality and the like, and is widely applied to the aquaculture industry. In actual production, male individuals typically exhibit significantly better growth rates and feed conversion efficiencies than females, and achieve better meat yield and specification consistency over the same cultivation period. Therefore, the total androgenetic cultivation can not only greatly improve the yield per unit area and the economic benefit, but also optimize the utilization of feed resources, shorten the cultivation period and reduce the environmental load. However, the sex-determining mechanism of channel catfish is extremely complex, and the lack of typical heterogenic chromosomes (such as XX/XY or ZW/ZZ systems) in the genome thereof, sex differentiation is highly dependent on the interaction of genetic background with environmental factors (such as temperature, pH, population density, etc.), and belongs to a typical "multi-factor regulatory" sex-determining mode. This uncertainty results in a proportion of male and female in the natural population approaching 1:1, severely restricting mass production of all male offspring. Although the traditional exogenous hormone (such as methyltestosterone) induced sex reversal technology can realize sex control in partial fishes, the traditional exogenous hormone (such as methyltestosterone) induced sex reversal technology has unstable effect in channel catfish, has multiple problems of drug residue, ecological risks, hidden danger of food safety, consumer conflict and the like, and has been strictly limited or prohibited by multiple countries. Under the background, developing a new precise sex regulation strategy which does not depend on exogenous hormone and is based on an endogenous molecular mechanism becomes a core attack direction in the field of channel catfish breeding. In recent years, epigenetic regulation, particularly DNA methylation, has received increasing attention for use in vertebrate sex determination and gonadal differentiation. DNA methyltransferase 1 (DNMT 1) serves as a core enzyme for maintaining the DNA methylation pattern, and is responsible for accurately transmitting methylation information on a parent DNA strand to a daughter strand during cell division, thereby guaranteeing genome methylation homeostasis. However, the specific function of dnmt gene in the sex control of channel catfish is still blank. Although the traditional hormone-induced sex reversal method achieves effect in partial fishes, the problems of residual risk, ecological potential safety hazard, low public acceptance and the like exist, and a safer and more accurate molecular regulation means are needed to be developed. Disclosure of Invention The invention aims to provide an RNAi target sequence for channel catfish dnmt gene knockout, a sex regulation method and application, and discloses a core regulation effect of dnmt 1-mediated DNA methylation in channel catfish sex determination, and further provides a novel safe, controllable and convertible sex control strategy, which has important scientific value and industrial application prospect. In order to achieve the above object, the present invention provides the following technical solutions: The invention provides an RNAi target sequence for channel catfish dnmt gene knockout, which is shown as SEQ ID NO. 4. The invention also provides a channel catfish sex regulation method, which comprises the following steps: (1) RNAi plasmid design for lentiviral packaging Designing RNAi target sequences, sense strands and antisense strands aiming at channel catfish dnmt1 genes, preparing an LV3-shRNA vector based on the RNAi target sequences, the sense strands and the antisense strands and the LV3-pGLV-h1-GFP-puro vector, transforming DH5 alpha competent cells from the LV3-shRNA vector to obtain dnmt gene RNAi plasmids, and packaging the dnmt gene RNAi plasmids by slow viruses to obtain RNAi plasmids packaged by slow viruses; (2) Channel catfish sex regulation Injecting RNAi plasmid packaged by the lentivirus in the step (1) into yolk of the channel catfish embryo close to the animal pole, incubating and identifying to obtain the hermaphrodite channel catfish. Preferably, in the step (1), the RNAi target sequence is shown as SEQ ID NO.4, the Loop structure in the sense strand uses a "TTCAAGAGA" sequence to avoid formation of a termination signal, a "GATCC" sequence is added to the 5 'end of the sense strand, and an "AATTC" sequence is added to the 5' end of the antisense stra