Molecular Cloning Of Goose Hsd3B1, Sequence Analysis, And Primary Functional Research In Ovary Follicle Of Goose

3beta-hydroxysteroid dehydrogenase/A5-A4isomerase1(HSD3B1) is one of the most critical enzymes in steroidogenesis pathway. It is a promiscuous enzyme receiving many different substrates:P5,17-OH-pregnenolone (17-OH-P5), dehyroepiandrosterone (DHEA), and androst-4-diene-17-dione (4-AD) changing them into P4,17-OH-P4, androstenedione (A4) and testosterone. The inactive or slightly active steroid is turned into an active one by HSD3B1.The steroid hormone in follicular yolk is important for the development of follicle and oocyte,however, there is little known about the steroid concentrations in follicles from small one to large one(every one millimeter). In this experiment, the coding domain sequence (CDS) of HSD3B1in Sichuan white goose was cloned by RT-PCR, and then was analyzed through bioinfomation.. The expression pattern of the HSD3B1was detected using qRT-PCR method in different size of follicle in goose. The steroid hormone were also measured in small follicles from3-4mm to9-10mm by every one millimeter difference and hierarchical follicles (F5、F4、F3、F2、Fl) Also, we analyzed the relationship between HSD3B1mRNA expression and steroid hormone concentration.The results are shown as follows:(1) The HSD3B1CDS of Sichuan white goose contained1134bp nucleotide,which encode377amino acid. The sequence of goose HSD3B1shows a higher homology with birds than with mammalian or others. The HSD3B1CDS has a conservative conserved domain sequence named3Beta-HSD, which contains284amino acid.(2) By competitive radioimmunoassay, E2, P, T was assayed.E2has a maximum level in4-5mm follicle. The level is keeping slightly changed from5-10mm,then followed a second ridge of the concentrations curve.Stepping up in F5(4.572±1.147)(P<0.05),the concentration drop out then.The level of P4vary low in small follicles (3-10mm).But when follicles turn into F5,there is a noticeable upgrade,there is a reduction in F4,then the level enhanced last to the time before ovulation. Testosterone in small follicle level changes slightly, only a little breakdown to F5. And when grow up F4, there is a noticeabe up,then, there is a down and up until F1, except a lower point in the F2.(3) The results of tissue differential expression showed that HSD3B1mRNA had the highest expression level in ovarian, then followed hypothalamus, uterine tube, liver and hypophysis.(4) The level of HSD3B1mRNA expression raise to a peak in5-6mm follicles, there is a slight raise after the level reach it’s lowest point in6-7mm follicles. In7-8mm follicles,the level of HSD3B1mRNA expression reach (0.00272±0.00285) before breakdown.When follicles grow up to9-10mm,this expression level is lowest in the whole follicle development period.Then the expression level increase rapidly until the follicle grow into F2(0.00873±0.00489)(P<0.05). In F1,there is a sligntly breakdown.