Application of siRNA of cynoglossus semilaevis heat shock transcription factor in controlling cynoglossus semilaevis pseudo male fish

Abstract

The invention relates to application of siRNA of a cynoglossus semilaevis heat shock transcription factor in control of cynoglossus semilaevis pseudo male fish, and belongs to the field of molecular genetic breeding, siRNA is designed according to the cynoglossus semilaevis heat shock transcription factor, small RNA female fish has a good knock-down effect and can control generation of the pseudo male fish, the cynoglossus semilaevis heat shock transcription factor is hsf1, and the molecular genetic breeding of the cynoglossus semilaevis heat shock transcription factor is hsf2. The siRNA (small interfering Ribonucleic Acid) designed for the hsf2 or hsf5a is shown as SEQ ID NO. 1-6. The invention also provides a cynoglossus semilaevis pseudo male fish biological injection preparation. The preparation contains at least one of siRNA designed according to a cynoglossus semilaevis heat shock transcription factor hsf1, hsf2 or hsf5a. According to the invention, an effective RNA interference technology can be established at the early development stage of the cynoglossus semilaevis, the formation of pseudo male fishes can be effectively controlled, and gender control is realized.

Description

Technical Field

[0001] The invention belongs to the field of molecular genetic breeding, and specifically relates to the application of siRNA of a heat shock transcription factor of half-smooth tongue sole in controlling pseudo-male fish of half-smooth tongue sole. Background Art

[0002] Cynoglossus semilaevis is a type of flatfish belonging to the family Glossodactylidae and the genus Glossodactylus. It is an important marine aquaculture fish in Northeast Asia. Cynoglossus semilaevis is deeply loved by consumers for its delicious meat and smooth taste, and has become a precious species among marine aquaculture fish. However, there are obvious growth differences between male and female Cynoglossus semilaevis. Female fish can grow 2-4 times larger than male fish. Therefore, increasing the proportion of female fish in aquaculture is of great significance for increasing production. Cynoglossus semilaevis belongs to the ZW sex determination type, that is, in theory, the ZW genotype will develop into female fish, and the ZZ type will develop into male fish. However, in actual production, high temperature will cause a considerable number of ZW individuals to reverse their sex to "pseudo-male fish". Since pseudo-male fish grow similarly to male fish, this greatly limits the development of the tongue sole industry.

[0003] Although gene editing technology has been successfully applied in Cynoglossus semilaevis, difficulties such as low survival rate after microinjection and long period of obtaining homozygous bodies (sexual maturity period: 1 year for males and 2 years for females) limit its application. In comparison, in vivo RNA interference saves time and money, and is more suitable for studying gene function and achieving gene regulation, but how to efficiently screen effective small RNA is the key. Summary of the invention

[0004] In view of the above technical problems, the present invention provides an application of a small RNA of heat shock transcription factor of half-smooth tongue sole in controlling pseudo-male fish of half-smooth tongue sole. The inventors previously analyzed through multi-omics and found that genes of multiple heat shock transcription factors hsf and other genes are closely related to sex reversal (individuals with genotype ZW should develop into female fish, but develop into pseudo-male fish). Three of them (hsf1, hsf2, hsf5a) were used as objects to design multiple pairs of siRNAs, which were verified in vivo and had a good knockdown effect on ZW fish. This method can establish an effective RNA interference technology in the early development of half-smooth tongue sole, can effectively control the formation of pseudo-male fish, and achieve sex control.

[0005] The present invention is achieved through the following technical solutions:

[0006] The invention discloses an application of a heat shock transcription factor siRNA of half-smooth tongue sole in controlling pseudo-male fish of half-smooth tongue sole. The siRNA is designed according to the heat shock transcription factor of half-smooth tongue sole, and the hsf1, hsf2 or hsf5a gene of female half-smooth tongue sole is knocked down by the siRNA to control the production of pseudo-male fish. The heat shock transcription factor of half-smooth tongue sole is hsf1, hsf2 or hsf5a. The siRNA is shown in SEQ ID NO.1-6.

[0007] The present invention also provides a semilaevis pseudomale fish biological injection preparation, wherein the preparation comprises at least one of siRNAs designed based on the semilaevis heat shock transcription factors hsf1, hsf2 and hsf5a.

[0008] The beneficial effects of the present invention compared with the prior art are as follows:

[0009] The present invention can establish effective RNA interference technology in the early development of semilaevis semilaevis by siRNA injection, especially hsf knockdown can significantly reduce the male gene expression in ZW fish, and has important application value in controlling the formation of pseudo-male fish and increasing the proportion of female fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The effect diagram of hsf gene interference in vitro in gonadal cell lines, where A is hsf1, B is hsf2, and C is hsf5a;

[0011] Figure 2 The effect of hsf gene interference in gonads, where A is hsf1, B is hsf2, and C is hsf5a;

[0012] Figure 3 The expression levels of other genes after hsf gene was knocked down by siRNA in the ovary in vivo, where A is hsf1, B is hsf2, and C is hsf5a. DETAILED DESCRIPTION

[0013] The technical solution of the present invention is further studied below through examples, but the protection scope of the present invention is not limited in any form by the examples.

[0014] Example 1

[0015] According to the gene sequences of hsf1, hsf2, and hsf5a (gene accession numbers: 103393760, 103380605, and 103395174), two pairs of specific siRNAs for each of the hsf1, hsf2, and hsf5a genes were designed online (https: / / sidirect2.rnai.jp / ), targeting two different targets, respectively, and NC was used as a negative control. After preliminary screening of cell lines, the siRNA with the best interference effect was selected for in vivo experiments, as shown in Table 1. Figure 1 Among the designed siRNAs, hsf1-1, hsf2-1 and hsf5a-1 had the best interference effects.

[0016] In vivo RNAi experiments were conducted on 50-day-old Cynoglossus semilaevis (body length 1.966±0.444cm, body width 0.430±0.176cm, body weight 1.299±0.647g). A control group (NC) and three experimental groups (4 in each group) were set up. Each experimental group was injected with siRNAs designed for different heat shock factor genes, and the injection concentration was 0.025nmol / g. The gonads were taken 48 hours after injection, and RNA was extracted using the Trizol method. About 30mg of tissue was added to 500μL Trizol (Ambion, USA), and fully ground in a sample grinder. After multiple centrifugation, chloroform was added for extraction. After isopropanol precipitation and 75% alcohol washing, DEPC water was added to dissolve RNA. After reverse transcription, quantitative primers were designed for qPCR analysis to detect the expression level of hsf in the gonads (primers are shown in Table 2, and qPCR configuration system is shown in Table 3).

[0017] Table 1 Designed siRNA sequences

[0018]

[0019] Table 2 Detection primers used for quantitative PCR

[0020]

[0021] Table 3 Quantitative system (10 μL)

[0022] SYBRGreen 5μL cDNA template 1μL Forward and reverse primers (F, R) 0.2μL each <![CDATA[ddH2O]]> Make up to 10 μL .

[0023] Compared with NC, the expression levels of hsf1, hsf2, and hsf5a were significantly reduced after knockdown in ZW fish, but there was no significant difference in ZZ fish ( Figure 2 ).

[0024] According to the instructions of the CP Regent transfection kit, PBS (control group, NC) and siRNA (experimental groups hsf1-1, hsf2-1 and hsf5a-1) were injected into the gonads of 50-day-old semi-smooth tongue soles by microinjection (siRNA concentration was 0.025 nmol / g). 48 hours after injection, the gonads were taken to extract RNA, and the expression level was calculated by qPCR after reverse transcription. The results are shown in Figure 3 As shown, knockdown of hsf can reduce the expression of the male-determining gene dmrt1 and the spermatogenesis-related gene tesk1 in the ovary, indicating that it has application potential in inhibiting pseudo-masculinization of ZW fish.

Claims

1. The use of siRNA of heat shock transcription factor of Cynoglossus semilaevis in controlling pseudo-male fish of Cynoglossus semilaevis, characterized in that: The application is to design siRNA according to the heat shock transcription factor of half-smooth tongue sole, and use the siRNA to knock down the hsf1, hsf2 or hsf5a gene of female half-smooth tongue sole to control the production of pseudo-male fish; the heat shock transcription factor of half-smooth tongue sole is hsf1, hsf2 or hsf5a, and the nucleotide sequence of siRNA is shown in SEQ ID NO.1-6.

2. A biological injection preparation of Cynoglossus semilaevis, characterized in that: The preparation contains at least one siRNA designed based on the heat shock transcription factor hsf1, hsf2 or hsf5a of Semilaevis semilaevis.

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