Two variable spliceosomes of cynoglossus semilaevis adcyap1b gene and application thereof

Abstract

The invention relates to two variable spliceosomes of a cynoglossus semilaevis adcyap1b gene and application of the two variable spliceosomes, and belongs to the technical field of functional gene screening and application in the aquatic biotechnology, the two variable spliceosomes are adcyap1b-tv1 and adcyap1b-tv2, coding region CDS sequences of the adcyap1b-tv1 and the adcyap1b-tv2 are SEQ ID NO: 1 and SEQ ID NO: 2, and protein sequences of the adcyap1b-tv2 are SEQ ID NO: 3 and SEQ ID NO: 4. The two variable spliceosomes of the adcyap1b gene are applied to regulation and control of hormone secretion of the pituitary of the cynoglossus semilaevis. By adding the recombinant protein of which the two variable spliceosomes are adcyap1b-tv1 and adcyap1b-tv2 into pituitary cells, the expression of a growth hormone gene gh, a prolactin gene prl, a growth lactin gene sl, a proogonin gene pomc and an adrenocorticotropic hormone release hormone gene crh of the cynoglossus semilaevis is regulated and controlled, so that the growth and development of the cynoglossus semilaevis are influenced.

Description

Technical Field

[0001] This invention belongs to the field of functional gene screening and application technology in aquatic biotechnology, specifically involving two alternative splice variants of the adcyap1b gene of the half-smooth tongue sole and their applications. Background Technology

[0002] The protein PACAP (pituitary adenylate cyclase activator peptide), encoded by Adcyap1, is a multifunctional neuropeptide first isolated from the bovine hypothalamus. Studies in mammals such as rats, cattle, and sheep have shown that after PACAP binds to its receptor, it can activate the ERK and cAMP / PKA signaling pathways, affecting the secretion of various hormones (such as growth hormone GH, prolactin PRL, gonadotropin-releasing hormone GnRH, follicle-stimulating hormone FSH, luteinizing hormone LH, adrenocorticotropic hormone ACTH, thyroid-stimulating hormone TSH, and steroid hormones) in the hypothalamic-pituitary-adrenal (HPI) axis, thereby playing a key role in hormonal regulation.

[0003] The regulatory role of PACAP on growth hormone (GH) varies by species. For example, in mammals and birds, pituitary growth hormone secretion mainly depends on ghrh, a member of the glucagon family, which promotes GH secretion by stimulating the cAMP / PKA and MAPK signaling pathways. In contrast, fish such as goldfish and killifish primarily rely on PACAP, a pituitary stimulator, to stimulate the secretion of pituitary hormones like GH. As amphibians occupying an intermediate evolutionary position, these two neuropeptides work synergistically to stimulate the secretion of pituitary hormones such as GH. From a gene structure and evolutionary analysis perspective, the adcyap1 and ghrh genes in mammals have different evolutionary trajectories, while in submammals and fish, these two genes evolved from the same gene precursor.

[0004] Half-smooth tongue sole ( Cynoglossus semilaevis The tongue sole, a valuable and economically important marine fish species unique to my country, exhibits typical female heterogamous chromosomes (ZW / ZZ). Females typically grow 2-4 times faster than males, displaying a typical female-dominated sex size variation. Therefore, does the adcyap1 gene play a dominant role in pituitary hormone secretion in the tongue sole? In-depth analysis of this gene will help us understand the molecular mechanisms regulating the interaction between sex and growth in the tongue sole. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention provides the application of two alternative splice variants of the adcyap1 gene in regulating pituitary hormone secretion in the half-smooth tongue sole.

[0006] This invention is achieved through the following technical solution: One objective of this invention is to provide two alternative splice variants of the adcyap1b gene, namely adcyap1b-tv1 and adcyap1b-tv2, wherein the coding region CDS sequences of adcyap1b-tv1 and adcyap1b-tv2 are SEQ ID NO:1 and SEQ ID NO:2, and the protein sequences are SEQ ID NO:3 and SEQ ID NO:4.

[0007] The second objective of this invention is to provide the application of two alternative splice variants of the adcyap1b gene in regulating pituitary hormone secretion in the half-smooth tongue sole.

[0008] Furthermore, the application method involves recombinantly expressing two alternative splice proteins of adcyap1b using a eukaryotic expression system, and then regulating the growth hormone gene (growthhormone) of the half-smooth tongue sole by adding the recombinant protein to pituitary cells. gh ), prolactin gene ( prl ), somatolactin gene ( sl ), proopiomelanocortin ( pomc ) and corticotropin-releasing hormone gene ( crh The expression of ).

[0009] The beneficial effects of this invention compared to the prior art are as follows: This invention discovered that the adcyap1b gene produces two transcripts and proteins, adcyap1b-tv1 and adcyap1b-tv2, during expression. Further research revealed the important roles of adcyap1b-tv1 and adcyap1b-tv2 in regulating pituitary hormone secretion in the half-smooth tongue sole. Attached Figure Description

[0010] Figure 1 Phylogenetic tree construction of the glucagon family (ADCYAP1 / VIP / GHRH) and gene expression heatmap of the half-smooth tongue sole family. A is the phylogenetic tree of the glucagon family (ADCYAP1 / VIP / GHRH), and B is the heatmap of glucagon family gene expression in different tissues of the half-smooth tongue sole. Figure 2 Half-smooth tongue sole adcyap1b Variable shear identification and shear body expression pattern analysis, A is half-smooth tongue sole adcyap1b Identification of gene alternative splicing: B is *Half-smooth Tongue Sole*. adcyap1b Expression patterns of the two splice bodies in male and female brain tissues at different developmental stages; Figure 3 The following data were used to analyze the eukaryotic protein expression of the two splice variants of ADCYAP1B from the tongue sole: A shows the gradient elution results of intracellular purification of cell samples after transfection of HEK293 cells with pcDNA3.1-adcyap1b_tv1 plasmid; B shows the gradient elution results of purification of culture supernatant after transfection of HEK293 cells with pcDNA3.1-adcyap1b_tv2 plasmid; and C shows the SDS-PAGE electrophoresis results of purified protein samples of ADCYAP1B_tv1 and ADCYAP1B_tv2. Figure 4 Functional analysis of two splice proteins, ADCYAP1B, from the tongue sole. A, B, C, D, and E show the results of incubation of tongue sole pituitary cells with the splice proteins. gh , prl , sl , pomc and crh The relative expression level of genes. Detailed Implementation

[0011] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0012] Example 1: Phylogenetic tree construction and gene expression heatmap analysis of the glucagon family (ADCYAP1 / VIP / GHRH) in half-smooth tongue sole. To comprehensively identify members of the glucagon family, to which ADCYAP1 belongs, four glucagon family members were first identified from *Coccus semilazi* through genome-wide screening. These were then compared with those found in *Coccus semilazi*. Cynoglossus semilaevis ), Blue medaka ( Oryzias latipes Nile tilapia ( Oreochromis niloticus ), Redfin Pufferfish ( Takifugu rubripes ), zebrafish ( Danio rerio ), spotted catfish ( Ictalurus punctatus ),people( Homo sapiens ) and mice ( Mus musculusPhylogenetic trees of glucagon family genes were constructed using MEGA 11.0 software with homologous protein sequences and the neighbor-joining method, and the reliability of the branches was assessed using the bootstrap test. The results showed that these sequences mainly clustered into three major branches: adenylate cyclase-activating peptide 1 (ADCYAP1), vasoactive intestinal peptide (VIP), and growth hormone-releasing hormone (GHRH). The ADCYAP1 branch was further divided into two sub-branchs: ADCYAP1b and ADCYAP1a. Both ADCYAP1a and ADCYAP1b of *Sinocyclocheilus spp.* are most closely related to ADCYAP1b of *Nile tilapia* and *Psilocybe maculatus*. In the VIP branch, the *Sinocyclocheilus spp.* VIP sequence clustered closely with the VIP sequences of *Pueraria lobata* and *Psilocybe maculatus*. In the GHRH branch, the *Sinocyclocheilus spp.* GHRH sequence formed an evolutionary cluster with the GHRH sequences of *Psilocybe maculatus* and *Nile tilapia*. This phylogenetic tree clearly reveals the evolutionary patterns of the ADCYAP1, VIP, and GHRH families in bony fishes and mammals.

[0013] To further investigate the expression patterns of glucagon family members in the half-smooth tongue sole, transcriptome data from two-year-old male and female half-smooth tongue soles published in our laboratory were used to create heatmaps of gene expression in different sexes and tissues. The results showed that... adcyap1b The gene showed extremely high expression levels in both male and female brain tissues, but almost no expression in other tissues. adcyap1a The genes are mainly expressed in the male and female brains, pituitary glands, and male gonads. vip Genes are primarily expressed in the brain tissues of both males and females. ghrh It is expressed at extremely low levels in brain tissue and is almost undetectable in other tissues.

[0014] Example 2: Identification of Alternative Splicing of the adcyap1b Gene in Half-smooth Tongue Sole and Analysis of Splice Variation Expression To investigate the expression pattern of the adcyap1b gene in half-smooth tongue sole at different stages, cDNA from brain tissue of 1.5-year-old half-smooth tongue sole was used as a template. PCR was performed using the coding region primers cdsF and cdsR. The PCR reaction system was 50 μL (KODMix 25 μL, primers 1.5 μL each, ddH2O 20 μL, template cDNA 2 μL), and the reaction conditions were: 98℃ for 10 s; 58℃-62℃ for 5 s; 68℃ for 20 s, 40 cycles; 72℃ for 7 min. The results showed that PCR amplification produced two clear, distinct bands with different molecular weights. Sequencing and sequence analysis revealed that these two bands were 546 bp and 441 bp in size, respectively. The difference was mainly due to exon 4 skipping, indicating that the adcyap1b gene of *C. half-smooth tongue sole* exhibits alternative splicing events. This gene can be transcribed and translated into two alternative splice proteins, ADCYAP1B_tv1 and ADCYAP1B_tv2. The nucleotide sequences of the two alternative splice proteins, ADCYAP1B_tv1 and ADCYAP1B_tv2, are shown in SEQ ID NO. 1-2, and the amino acid sequences are shown in SEQ ID NO. 3-4.

[0015] Two pairs of specific quantitative PCR primers (tv1F, tv1R, tv2F, and tv2R) were designed for the two alternative splice variants. Using cDNA from the brain tissues of male and female half-smooth tongue sole at different stages as templates and β-actin gene as an internal reference gene, quantitative PCR was performed. The reaction system and conditions are as follows: 10 µL reaction system: 5 µL SYBR Taq, 0.2 µL primer F (forward primer), 0.2 µL primer R (reverse primer), 1 µL cDNA, 3.6 µL H2O.

[0016] The reaction conditions were as follows: 95℃, 30 s; 95℃, 3 s, 60℃, 33 s, 40 cycles; 95℃, 15 s; 60℃, 60 s; 95℃, 15 s.

[0017] The relative expression levels of genes were calculated using the 2-ΔΔCt method, and bar charts were generated using GraphPad 8.0.2 software. The LSD multiple comparison method of analysis of variance was used to analyze the differences between groups. The results showed that the relative expression level of adcyap1b-tv1 in brain tissue was higher than that of adcyap1b-tv2. The expression levels of both were higher in female brain tissue than in male brain tissue, and both reached their highest values ​​at 2 years of age.

[0018] The nucleotide sequence of the coding region CDS of adcyap1b-tv1 is (SEQ ID NO:1): Atggagcgcaagataaacatggccagttcgagtaaagccactttaatcttgctcatctatggaatcttaatgcactacagcgtattctgcacacctattggactaagttaccccaagatcagacttgaaaacgacgccttcgatgaggatgggaattcattaaatgacatgggctttgatagcgaccagattgctatacgcagcccagcatccttaaacgacgacgcctactccctgtactaccccccggagaagagaccagaaaggcatgctgaggaagaattagatagagccttgagggagatcctgggtcagttaacagcgagacattatctgcattctctgatgacagttcgtgcaggtgaagacaacagcatggaggacgaatcagagcctctatctaagagacattcagatggcatcttcaccgacagctacagccgctacagaaagcagatggccgtgcagaaatacctggctgcggttctgggacgaaggtacagacagagagtaaggaacaaaggacgccggcttgcctatttgtag; Nucleotide sequence of the coding region CDS of adcyap1b-tv2 (SEQ ID NO:2): Atggagcgcaagataaacatggccagttcgagtaaagccactttaatcttgctcatctatggaatcttaatgcactacagcgtattctgcacacctattggactaagttaccccaagatcagacttgaaaacgacgccttcgatgaggatgggaattcattaaatgacatgggctttgatagcgaccagattgctatacgcagcccagcatccttaaacgacgacgcctactccctgtactaccccccggagaagagtgaagacaacagcatggaggacgaatcagagcctctatctaagagacattcagatggcatcttcaccgacagctacagccgctacagaaagcagatggccgtgcagaaatacctggctgcggttctgggacgaaggtacagacagagagtaaggaacaaaggacgccggcttgcctatttgtag; The amino acids of the coding region CDS of adcyap1b-tv1 are (SEQ ID NO:3): MERKINMASSSKATLILLIYGILMHYSVFCTPIGLSYPKIRLENDAFDEDGNSLNDMGFDSDQIAIRSPASLNDDAYSLYYPPEKRPERHAEEELDRALREILGQLTARHYLHSLMTVRAGEDNSMEDESEPLSKRHSDGIFTDSYSRYRKQMAVQKYLAAVLGRRYRQRVRNKGRRLAYL; The amino acid sequence of the coding region CDS of adcyap1b-tv2 (SEQ ID NO:4): MERKINMASSSKATLILLIYGILMHYSVFCTPIGLSYPKIRLENDAFDEDGNSLNDMGFDSDQIAIRSPASLNDDAYSLYYPPEKSEDNSMEDESEPLSKRHSDGIFTDSYSRYRKQMAVQKYLAAVLGRRYRQRVRNKGRRLAYL。

[0019] Primers used in this part: cdsF: ATGGAGCGCAAGATAAACATG (SEQ ID NO.5), cdsR: CTACAAATAGGCAAGCCGGCG (SEQ ID NO.6); tv1F:GCATTCTCTGATGACAGTTCGT (SEQ ID NO.7), tv1R:ACCGCAGCCAGGTATTTCT (SEQ ID NO.8); tv2F: GAGAAGAGTGAAGACAACAGCA (SEQ ID NO.9), tv2R: CTACAAATAGGCAAGCCG (SEQ ID NO.10); actinF:TTCCAGCCTTCCTTCCTT (SEQ ID NO.11), actinR: TACCTCCAGACAGCACAG (SEQ ID NO. 12).

[0020] Example 3: Preparation and identification of recombinant protein from the adcyap1b gene alternative splice of the half-smooth tongue sole. To further analyze the function of adcyap1b in *Cocculus semismoothienos*, the expression and purification of recombinant eukaryotic proteins with two alternative splice variants of adcyap1b were studied using HEK 293T mammalian cells. After codon optimization of the coding sequences of adcyap1b_tv1 and adcyap1b_tv2, an exogenous signal peptide (MYRMQLLSCIALSLALVTNS, SEQ ID NO.13) and a Flag+His purification tag were added to the N-terminus and C-terminus, respectively. Then, using EcoRI and HindIII double restriction sites, the proteins were subcloned into the eukaryotic expression vector pcDNA3.1(-), successfully constructing the eukaryotic recombinant expression vectors pcDNA3.1-adcyap1b_tv1 and pcDNA3.1-adcyap1b_tv2. The two recombinant vectors were transfected into HEK 293T cells using liposome transfection reagent. Supernatants and cell lysates were collected 48-72 hours after transfection, and the recombinant proteins were identified by Western blotting using a Flag-tagged antibody. Two hundred µg each of intracellularly expressed recombinant ADCYAP1B_tv1 and secreted recombinant ADCYAP1B_tv2 proteins were successfully obtained using the His purification method. The purified protein samples were desalted and deimidazole-free, and stored in DMEM medium.

[0021] Example 5: Functional study of recombinant protein from the adcyap1b gene alternative splice in half-smooth tongue sole. To investigate the effects of two alternative splice variants of adcyap1b recombinant proteins on the regulation of pituitary hormone secretion, an incubation experiment was conducted between the recombinant proteins and pituitary cells, as detailed below: Using laboratory-cultured female pituitary cells from the tenth generation of *Cocculus semismoothii*, two groups were established: an ADCYAP1B-tv1 recombinant protein treatment group, an ADCYAP1B-tv2 recombinant protein treatment group, and a control group without any treatment. Two flasks (25cm x 1000ml) were used in each group. 2 Cells. In the recombinant protein treatment group, 0.5 mL of recombinant protein at a concentration of 0.05 μg / μL was added to each flask of cells, i.e., 25 μg per flask. After incubation at 24°C for 48 hours, RNA was extracted using the Trizol method, reverse transcribed into cDNA, and then detected by quantitative real-time PCR for pituitary genes. gh , prl , sl , pomc , crh The expression level was measured, and β-actin was used as the internal reference gene. The primers for these genes are as follows: ghF: ATCCACGCAGCCGGTTATAG (SEQ ID NO.14), ghR: CTCATGCTTGTTGTCGGGGA (SEQ ID NO. 15); prlF: ACAGTGGAGGCAACGACA (SEQ ID NO.16), prlR: CAACGCAGGACTTTCAGG (SEQ ID NO. 17); slF: CTGGACTGTAAGGACGAGC (SEQ ID NO.18), slR: CTGAGATGCGGGAGATGA (SEQ ID NO. 19); pomcF: GTCCTCTGTGGCTATTGGT (SEQ ID NO.20), pomcR: ATGGCACTGCTCTTGGAG (SEQ ID NO. 21); crhF: CCGTCCTCCTCCCTGTAT (SEQ ID NO. 22), crhR: GCCCTGATGTTCCCAAAT (SEQ ID NO. 23); actinF:TTCCAGCCTTCCTTCCTT (SEQ ID NO.11), actinR: TACCTCCAGACAGCACAG (SEQ ID NO. 12).

[0022] The reaction system and conditions for quantitative PCR are as follows: 10 µL reaction system: 5 µL SYBR Taq, 0.2 µL primer F, 0.2 µL primer R, 1 µL cDNA, 3.6 µL H2O.

[0023] The reaction conditions were as follows: 95℃, 30 s; 95℃, 3 s, 60℃, 33 s, 40 cycles; 95℃, 15 s; 60℃, 60 s; 95℃, 15 s.

[0024] The relative expression levels of genes were calculated using the 2-ΔΔCt method, and bar charts were generated using GraphPad 8.0.2 software. The T-test was used to analyze the differences between the treatment and control groups. The results showed that both ADCYAP1B-tv1 and ADCYAP1B-tv2 significantly promoted [gene expression]. gh , pomc and crh The expression level, and ADCYAP1B-tv1 on gh and pomc The promoting effect was significantly stronger than that of ADCYAP1B-tv2. And for... prl and sl Significant gene-promoting effects were observed only in the ADCYAP1B-tv1 recombinant protein treatment group.

[0025] in, gh It is a core hormone that regulates cell proliferation and promotes fish growth and development, and also participates in metabolic balance and reproductive processes. (Fish) prl They play an important role in regulating osmotic pressure, reproductive activity, and growth and development. (This is unique to fish.) sl It plays multiple functions, including gonadal development, reproductive regulation, and body color formation. pomc The encoded precursor protein can be cleaved to produce peptides such as adrenocorticotropic hormone and melanocyte-stimulating hormone, which can regulate the feeding behavior, body color formation, growth and reproductive behavior of fish. crh It is a key initiator of the hypothalamic-pituitary-renal axis in fish, and under external stressors such as high temperature, it can regulate and reverse behaviors by releasing cortisol. crh They also participate in the feeding and development processes of fish.

[0026] Based on the results of this experiment, which showed the regulatory effects of the two recombinant proteins of ADCYAP1B on these hormones, it can be concluded that the two splice variants of ADCYAP1B in *Sinonovacula semismoothica* can promote the regulation of these hormones. gh , pomc andcrh The secretion of this substance plays a role in growth and development, feeding behavior, and sex reversal, and ADCYAP1B-tv1 has a stronger regulatory effect. In addition, ADCYAP1B-tv1 can also promote… prl and sl The secretion of hormones regulates osmotic pressure and reproductive behavior. These results provide an important foundation for a deeper understanding of the molecular mechanisms by which ADCYAP1 regulates pituitary hormone secretion in fish.

Claims

1. Two alternative splice variants of the adcyap1b gene, characterized in that, The two alternative splice variants are adcyap1b-tv1 and adcyap1b-tv2, and the coding region CDS sequences of adcyap1b-tv1 and adcyap1b-tv2 are SEQ ID NO:1 and SEQ ID NO:2, and the protein sequences are SEQ ID NO:3 and SEQ ID NO:

4.

2. The application of the two alternative splice variants of the adcyap1b gene as described in claim 1 in regulating pituitary hormone secretion in the half-smooth tongue sole.

3. The application according to claim 2, characterized in that, The application involves using a eukaryotic expression system to recombinantly express two alternative splice variants, adcyap1b-tv1 and adcyap1b-tv2, to obtain recombinant proteins with the two alternative splice variants adcyap1b-tv1 and adcyap1b-tv2. These recombinant proteins are then added to pituitary cells to regulate the growth hormone gene in the half-smooth tongue sole. gh prolactin gene prl , growth prolactin gene sl , Acortin gene pomc and adrenocorticotropic hormone-releasing hormone gene crh The expression.

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