Application of siRNA of heat shock transcription factor in control of pseudo-male cobb's cunioffera variegate

Abstract

The present application relates to the application of siRNA of Cynoglossus semilaevis heat shock transcription factor in controlling Cynoglossus semilaevis pseudo-male, and belongs to the field of molecular genetic breeding, wherein siRNA is designed according to Cynoglossus semilaevis heat shock transcription factor, the small RNA female has good knockdown effect, and can control the generation of pseudo-male, the Cynoglossus semilaevis heat shock transcription factor is hsf1, hsf2 or hsf5a, and the designed siRNA is shown as SEQ ID NO. 1-6. The present application also provides a Cynoglossus semilaevis pseudo-male biological injection preparation, wherein the preparation contains at least one of siRNA designed according to Cynoglossus semilaevis heat shock transcription factor hsf1, hsf2 or hsf5a. The present application can establish effective RNA interference technology in the early development of Cynoglossus semilaevis, effectively control the formation of pseudo-male, and realize gender control.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetic breeding, specifically relating to the application of a heat shock transcription factor siRNA of the tongue sole in controlling pseudo-male tongue sole. Background Technology

[0002] Half-smooth tongue sole (Cynoglossus semilaevis) is a type of flatfish belonging to the family Cynoglossidae and the genus Cynoglossus. It is an important marine aquaculture fish in Northeast Asia. Half-smooth tongue sole is highly prized by consumers for its delicious, smooth flesh, making it a valuable species in marine aquaculture. However, there is a significant difference in growth between males and females; females can grow 2-4 times larger than males. Therefore, increasing the proportion of females in aquaculture is crucial for improving yield. Half-smooth tongue sole exhibits ZW sex determination, meaning that theoretically, the ZW genotype will develop into females, and the ZZ genotype into males. However, in actual production, high temperatures can cause a significant number of ZW individuals to undergo sex reversal and become "pseudo-males." Because pseudo-males grow similarly to males, this greatly limits the development of the tongue sole industry.

[0003] Although gene editing technology has been successfully applied in tongue sole, difficulties such as low survival rate after microinjection and long cycle to obtain homozygotes (sexual maturity cycle: 1 year for males and 2 years for females) limit its application. In comparison, in vivo RNA interference is time-saving and economical, and is more suitable for studying gene function and realizing gene regulation. However, how to efficiently screen effective small RNAs is the key. Summary of the Invention

[0004] This invention addresses the aforementioned technical problems by providing an application of heat shock transcription factor small RNAs in controlling pseudomales in half-smooth tongue sole. The inventors previously discovered through multi-omics analysis that several heat shock transcription factors (hsf) are closely related to sex reversal (individuals with the ZW genotype should develop into females but instead develop into pseudomales). Using three of these factors (hsf1, hsf2, and hsf5a) as targets, multiple pairs of siRNAs were designed and validated in vivo, showing good knockdown effects on ZW fish. This method can establish an effective RNA interference technique in the early stages of half-smooth tongue sole development, effectively controlling pseudomale formation and achieving sex control.

[0005] This invention is achieved through the following technical solution:

[0006] Application of heat shock transcription factor siRNA in controlling pseudo-males in half-smooth tongue sole: siRNA was designed based on the heat shock transcription factor of half-smooth tongue sole, and the hsf1, hsf2, or hsf5a genes in female half-smooth tongue sole were knocked down using the siRNA to control the production of pseudo-males; 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 biological injection preparation of pseudo-male half-smooth tongue sole, the preparation comprising at least one of siRNAs designed based on the heat shock transcription factors hsf1, hsf2 and hsf5a of half-smooth tongue sole.

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

[0009] This invention establishes an effective RNA interference technique in the early development of the tongue sole using siRNA injection, particularly hsf knockdown, which significantly reduces the expression of male genes in ZW fish. This has significant application value in controlling pseudo-male formation and increasing the female ratio. Attached Figure Description

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

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

[0012] Figure 3 The expression levels of other genes after ovarian siRNA knockdown of the hsf gene in vivo are shown in the figures, where A represents hsf1, B represents hsf2, and C represents hsf5a. Detailed Implementation

[0013] The technical solution of the present invention will be further studied through the following embodiments, but the scope of protection of the present invention is not limited in any way by the embodiments.

[0014] Example 1

[0015] Based on the gene sequences of hsf1, hsf2, and hsf5a (accession numbers: 103393760, 103380605, 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 sites respectively. NC was used as a negative control. After preliminary screening with cell lines, the siRNAs with the best interference effect were selected for in vivo experiments, as shown in Table 1. Figure 1 Among the designed siRNAs, hsf1-1, hsf2-1, and hsf5a-1 showed the best interference effect.

[0016] For the in vivo RNAi experiment, 50-day-old half-smooth tongue sole (body length 1.966±0.444cm, body width 0.430±0.176cm, weight 1.299±0.647g) were selected. A control group (NC) and three experimental groups (n=4 per group) were set up. Each experimental group was injected with siRNAs designed from different heat shock factor genes at a concentration of 0.025 nmol / g. Forty-eight hours after injection, gonads were harvested, and RNA was extracted using the Trizol method. Approximately 30 mg of tissue was added to 500 μL of Trizol (Ambion, USA), thoroughly ground in a sample grinder, and extracted with chloroform after multiple centrifugations. After precipitation with isopropanol and washing with 75% ethanol, the RNA was dissolved in DEPC water, reverse transcribed, and quantitative primers were designed for qPCR analysis to detect the expression level of hsf in the gonads (primers are shown in Table 2, qPCR configuration system is shown in Table 3).

[0017] Table 1 Designed siRNA sequences

[0018]

[0019] Table 2 Primers used for quantitative PCR detection

[0020]

[0021] Table 3. Quantitative system (10 μL)

[0022] SYBRGreen 5μL cDNA template 1μL Forward and reverse primers (F, R) 0.2 μL 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, while no significant difference was observed 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 half-smooth tongue soles 50 days after hatching using a microsyringe (siRNA concentration: 0.025 nmol / g). 48 hours after injection, gonads were harvested to extract RNA, which was then reverse transcribed and expressed using qPCR. Results are as follows: Figure 3 As shown, knockdown of hsf can reduce the expression of male-determining gene dmrt1 and spermatogenesis-related gene tesk1 in the ovary, indicating its potential application in inhibiting pseudomastosis in ZW fish.

Claims

1. The application of siRNA of heat shock transcription factor in half-smooth tongue sole in controlling pseudo-male half-smooth tongue sole, characterized in that, The application involves designing siRNA based on the heat shock transcription factor of the tongue sole, and using the siRNA to knock down the hsf1, hsf2, or hsf5a genes in female tongue sole to control the production of pseudo-males; the heat shock transcription factor of the tongue sole is hsf1, hsf2, or hsf5a, and the nucleotide sequence of the siRNA is shown in SEQ ID NO.1-6.

2. A biological injection preparation for pseudo-male tongue sole, characterized in that, The formulation contains at least one siRNA designed based on the half-smooth tongue sole heat shock transcription factors hsf1, hsf2 or hsf5a, the nucleotide sequence of which is shown in SEQ ID NO.1-6.

Images