The Cytological Observation On Fertilization And The Cloning And Expression Of Rhodanese In Urechis Unicinctus

This paper consists two parts, the first part is the cytological observation on fertilization in Urechis unicinctus, and the other is the cloning and expression of rhodanese.Fertilization is the fusion of gametes to produce a new organism. In animals, the process involves the fusion of an ovum with a sperm, which eventually leads to the development of an embryo. In the first part of this paper, we observed the sperm penetration into the oocyte and the changing of the nuclear phase in Urechis unicinctus using histology technique and fluorescence microscopy.The results showed that the mature oocyte of U. unicinctus was egg-shaped and remained at the prophase of the first maturation division with uncracked germinal vesicle. Sperms quickly attached to the surface of the oocyte after mixing of germ cells at (21±1)℃water temperature, after 5 minutes of insemination, the sperm penetrated into the oocyte at the site close to vegetal pole and the fertilization membrane started to lift. Later on the sperm nucleus expanded first time and the fertilization membrane completed after 25 minutes. The first and second maturation divisions finished after about 10-50 minutes of insemination, simultaneously the first and the second polar body were released respectively. About 50-70 min later, sperm and egg nucleus expanded respectively and developed into the pronuclei, and they moved to the center of the egg and finally formed an associated nucleus. The first cleavage finished after 70-90 minutes and divided into two daughter cells. The fertilization process lasted about 70 minutes, slightly longer than most of the marine invertebrates. In this study, with the observation of the process of fertilization, we can not only enrich the basic information of the biological fertilization, but also provide theoretically foundation for artificial breeding and the future cultivation of new varieties for U. unicinctus.The second part of this paper is that we cloned the full-length cDNA sequence of rhodanese in U. unicinctus, and analyzed tissue expression with the response of sulfide. Rhodanese, also known as sulfurtransferase, is the last menber of the detoxification enzyme system of mitochondrial sulfide oxidation, which transfers a persulfide to a sulfite, producing thiosulfate.The full-length of U. unicinctus rhodanese cDNA was cloned by homology cloning and 3'- and 5'-RACE as a 2318 bp consisting of an open reading frame of 870 bp encoding a putative protein of 289 amino acids. The deduced amino acid sequence contained conserved amino acid domains(CEVGVT) and conserved active Cys 230.There are differences in the expression of the tissues in U. unicinctus by real-time RT-PCR. The results showed the highest rhodanese mRNA expression was in the anal sacs, followed by the hindgut and mid-gut, finally, coelomic fluid cells and the body wall with a relatively low level of expression. In hindgut of U. unicinctus after the future sulfide exposure (50μM and 150μM), rhodanese mRNA expression was not increased significantly compare to control within 72 h. The investigation of rhodanese can deepen the understanding of sulfide metabolic molecular mechanism and provides more evidence to explore U. unicinctus as a model organism in sulfide metabolic research.RACE techniques. Subsequently, we demonstrated some bioinformatics analysis data of U. unicinctus rhodanese, and then we investigated its mRNA expression in different tissues and in hindgut after sulfide exposure.The full-length of U. unicinctus rhodanese cDNA was cloned by homology cloning and 3'- and 5'-RACE as a 2318 bp consisting of an open reading frame of 870 bp encoding a putative protein of 289 amino acids. The deduced amino acid sequence contained conserved amino acid domains(CEVGVT) and conserved active Cys 230. The sequence prediction results showed that U. unicinctus rhodanese was located in the mitochondria, as a hydrophilic protein without a signal peptide or transmembrane region. Glycosylation modification prediction showed that U. unicinctus rhodanese was characterized by the presence of one N-glycosylation sites.