Clone And Expression Analysis Of Goose FSH Gene

FSH (Follicle-stimulating hormone) gene is an important candidate gene in animal reproductive traits, and also plays a key role in the regulation of egg laying in poultry. In this study, RT-PCR and RACE technology are used to acquire the FSHa gene CDS sequence, and the full length of cDNA of FSHβ gene, expression analysis of goose FSHβ mRNA at different physiological stages by qRT-PCR, and slso use the gene fusion technology to explore FSH gene recombinant expression in COS-7 cells, the expression changes of related gene in FSH signaling pathway. The study aims to provide some reference for a comprehensive analysis of the structure and function of the goose FSH gene, meanwhile, also provide a theoretical basis to improve and enhance the goose laying performance.1. Clone and sequence analysis of FSH gene in gooseIn this study, we have cloned FSHa, FSHβ CDS sequence, from goose pituitary gland by reverse transcription-polymerase chain reaction(RT-PCR) and also use rapid amplification of cDNA end(RACE) method to cloned 5’UTR and 3’UTR sequence of FSHβ. The cloned goose FSHa gene CDS contained 363bp open reading frame; FSHβ cDNA contained a 16-bp 5’UTR, a 396-bp open reading frame, meanwhile,3’UTR existed two different spliced transcripts, fragment size 518bp and 780bp, respectively. The amino acid sequence of FSHa, FSHβ analyzed by bioinformatics software, we found that the protein of FSHa, FSHβ are hydrophilic and secreted protein, also had cleavage sites for signal peptide.2. Expression Analysis of Goose FSHβ mRNA at different physiological stagesThis experiment was conducted to reveal expression character of goose FSHβ mRNA at breeding cycle. The experiment adopted the RT-PCR and qRT-PCR to detect expression of geese FSHβ mRNA in different tissues at different reproductive stage (pre-laying, laying period (pre-ovulation stage, Oviposit period), nest period) of Zhejiang white geese and Yangzhou white geese as the object of study. The result of RT-PCR showed that the expression of FSHβ mRNA have high levels in the tissues that related with reproduction, such as pituitary, hypothalamus, ovarian, fallopian tubes compared with other tissues. The qRT-PCR indicated that the expression of FSHβ mRNA in the pituitary and fallopian tubes is higher than hypothalamus and ovarian during 3 different reproductive stages (pre-laying period, laying period and nest period). And the expression of FSHβ mRNA of the four tissues have the same change trend, first increased and then decreased. During laying period, the expression of FSHβ mRNA significantly greater than pre-laying period in the pituitary, ovarian and fallopian tube (P< 0.05), is higher significantly than nest period in the ovarian (P <0.01), while the expression of FSHβ mRNA in the hypothalamus have no significant difference during three reproductive cycles (P>0.05); During laying period, the expression of FSHβ mRNA have highly difference between Zhejiang white goose and Yangzhou goose only in the ovarian.3. Fusion gene expression of FSH in COS-7 cellsIn this study, we build four recombinant FSH by gene fusion technology, transfected these plasmid into COS-7 cells, respectively detect the secretion amount of FSH protein in cell culture supernatants at 12h,24h,36h,48h by radioimmunoassay. Cells which transfected pVITRO2-FSHβand pVITRO2-FSHβ-CTP, the results showed that the CTP sequence can increase the secretion amount of FSH protein. Cells which transfected pVITRO2-FSHβ-CTP-a and pVITRO2-FSHaβ-CTP, the secretion amount of FSH protein was opposite trend. qRT-PCR was used to detect the expression chang of FSH signaling pathway genes, FSHβ, GnRH, GH and BMP gene, after transfection, respectively in 6h,12h,24h,36h, 48h, the results showed that every gene has expression trend.