Application of Cynoglossus semilaevis histones and their variant small RNAs in sexual differentiation of Cynoglossus semilaevis

Abstract

The present invention relates to the application of semi-smooth tongue sole histone and its variant small RNA in the sex differentiation of semi-smooth tongue sole, belonging to the field of molecular genetic breeding, and the application method is to design siRNA according to semi-smooth tongue sole histone H3 and its variant H3.3-like, and the siRNA can interfere with the gene expression of histone and its variant, and interfere with the expression of semi-smooth tongue sole sex-related genes dmrt1, tesk1 and neurl3, so that the expression is significantly reduced; the present invention also provides a biological preparation including siRNA. The biological preparation can inhibit the sex differentiation of semi-smooth tongue sole and reduce the production of pseudo-male fish or male fish.

Description

Technical Field

[0001] The invention belongs to the field of molecular genetic breeding, and specifically relates to an application of a half-smooth tongue sole histone variant and a variant small RNA thereof in the sex differentiation of the 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. In China, 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, a considerable number of ZW individuals will reverse their sex to become "pseudo-male fish". Since pseudo-male fish grow similarly to male fish, this greatly limits the development of the tongue sole industry.

[0003] Genes play an important role in the process of sexual differentiation, and regulating gene expression is an important means of achieving sex control. 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 male fish and 2 years for female fish) limit its application. In comparison, in vivo RNA interference is time-saving and economical, and is more suitable for studying gene function and achieving gene regulation, but how to efficiently screen effective small RNAs is the key. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a half-smooth tongue sole histone variant and its variant small RNA for use in sex differentiation. The inventors previously analyzed transcriptome, proteome and ubiquitination group, and found that multiple histones are closely related to male differentiation and spermatogenesis. Two of the histones, H3 and H3.3-like, were used as targets to design multiple pairs of siRNAs. First, siRNAs with good knockdown effects were screened through the half-smooth tongue sole testicular cell line, and then verified in male fish in vivo to obtain better knockdown effects. Establishing effective RNA interference technology in the early development of half-smooth tongue sole can effectively shorten the research cycle and obtain relevant phenotypes as soon as possible.

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

[0006] The invention discloses an application of half-smooth tongue sole histones and their variants in the sex differentiation of half-smooth tongue sole. The application method comprises designing siRNA according to half-smooth tongue sole histones and their variants, wherein the siRNA can interfere with the gene expression of the histones and their variants, and interfere with the expression of half-smooth tongue sole sex-related genes dmrt1, tesk1 and neurl3, thereby significantly reducing the expression; the half-smooth tongue sole histones are H3 or its variant H3.3-like.

[0007] Application of siRNA of half-smooth tongue sole histone and its variants in sex differentiation of half-smooth tongue sole, wherein the siRNA can interfere with the expression of half-smooth tongue sole sex-related genes dmrt1, tesk1 and neurl3, and the nucleotide sequence of the siRNA is such as SEQ ID NO.7-8 or SEQ ID NO.9-10.

[0008] The present invention also provides a biological preparation, which comprises any one group of the siRNAs, and can inhibit the sex differentiation of Cynoglossus semilaevis, specifically reduce the generation of pseudo-male fish or male fish.

[0009] The beneficial effects of the present invention compared with the prior art are as follows: the technology can effectively inhibit the genes of male differentiation and spermatogenesis, thereby controlling the transformation of female fish into pseudo-male fish or inhibiting the production of sperm in male fish, which is beneficial to increasing the proportion of female fish in the breeding group or improving the growth of male fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The expression levels of histones in testicular cell lines after transfection with different siRNAs (different letters indicate significant differences, p<0.05);

[0011] Figure 2 The expression of histones after siRNA injection in testis (* indicates significant difference, p<0.05);

[0012] Figure 3 The expression levels of other genes after siRNA injection into the testis (different letters indicate significant differences, p<0.05). DETAILED DESCRIPTION

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

[0014] Example 1

[0015] Based on the sequences and previous multi-omics results, we found that four histones H1.1-like, H1.2-like, H3, and H3.3-like had differences at the protein level, and this difference may be closely related to post-translational modifications. Therefore, we designed specific siRNAs for H1.1-like, H1.2-like, H3, and H3.3-like (the gene sequence numbers in NCBI are 103395672, 103379258, 103379247, and 103394976, respectively), and used NC as a negative control, see Table 1.

[0016] The cell line interference steps are as follows: after the semi-smooth tongue sole testis cell line successfully established in the laboratory was revived, it was cultured in a 24°C incubator with L-15 medium containing a final concentration of 10% fetal bovine serum (FBS), 5ng / ml bFGF, 5ng / ml EGF, 27.5μmol / L beta-mercaptoethanol and 5% triple antibody. When the cells are full of culture bottles, they are subcultured. When the density reaches 80% to 90%, they are plated in 12-well cell culture plates. The CP Regent transfection kit was used to perform siRNA interference transfection experiments on the plated semi-smooth tongue sole testis cell line. The siRNA group and NC group experiments were repeated three times. In the RNAi experiment in the cell line, according to the instructions of the CP Regent transfection kit, the transfection time is recommended to be 48-72 hours. Our preliminary test showed that the knockdown effect of 72 hours was the best. The total RNA of semi-smooth tongue sole was extracted using the RNA purification tissue kit (Tiangen Bio). Reverse transcription was performed using PrimeScript with gDNAEraser TM cDNA was prepared using the RT kit (TaKaRa, Japan). The reverse transcription kit was used in two steps according to the instructions. The first step was to remove genomic DNA. The second step was reverse transcription, and the final product was stored at -20°C. The qPCR reaction conditions were: 95°C 30s, 95°C 5s, 60°C 30s, and 40 cycles. The experimental data were calculated using the 2-ΔΔct method for gene expression. SPSS26.0 was used to perform multiple comparison analysis on the data, and a p value less than 0.05 was considered to be significantly different between the two groups. In the multiple comparisons, a and b are groups with different degrees of differential expression. The expression levels of H3 and H3.3-like were detected by qPCR. The detection primers are shown in Table 2, and the siRNA with the best knockdown effect was screened for in vivo experiments.

[0017] For the in vivo RNAi experiment, 50-day-old Cynoglossus semilaevis (body length 1.966±0.444 cm, body width 0.430±0.176 cm, body weight 1.299±0.647 g) were selected, and the control group (NC) and four experimental groups (4 in each group) were injected with 0.025 nmol / g concentration. 72 hours after injection, gonads were taken for gene expression analysis of H3, H3.3-like and other sex-related genes (including foxl2, figla, dmrt1, cyp19a, sox-9, tesk1 and neurl3) (primers are shown in Table 2).

[0018] Results: Compared with the control group, the expression levels of H1.1, H1.2-like-1, H1.2-like-2, H3, and H3.3-like in testicular cells were significantly reduced after knockdown ( Figure 1 ); According to the effect of siRNA in Cynoglossus semilaevis cells, siRNA was selected and injected into the gonads of 90-day-old female and male fish. Compared with the NC group, the expression levels of H3 and H3.3-like genes in male fish were significantly reduced ( Figure 2 After knocking down H3 and H3.3-like, the expression levels of spermatogenesis-related genes tesk1 and neurl3 were significantly reduced ( Figure 3 ).

[0019] Table 1 Designed siRNA sequences

[0020]

[0021] Table 2 Detection primers used for quantitative PCR

[0022]

Claims

1. Application of siRNA in sex differentiation of Cynoglossus semilaevis, characterized in that: The application method is to design siRNA based on the semi-smooth tongue sole histone and its variants, and the siRNA can interfere with the gene expression of the histone and its variants, and interfere with the sex-related genes of the semi-smooth tongue sole dmrt1、tesk1 and neurl3 The expression of the half-smooth tongue sole histone is H3, the variant of the half-smooth tongue sole histone is H3.3-like, and the nucleotide sequence of the siRNA is shown in SEQ ID NO.7-8 or SEQ ID NO.9-10.

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