Cloning ZP3 gene and egg chorion microstructure of tilapia

• Main Authors: Han-Hieh Kuo, 郭瀚介
• Other Authors: Jui-Sen Yang
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/39281202799147245562

碩士 === 國立臺灣海洋大學 === 海洋生物研究所 === 94 === The ZP3 is a female specific protein on the surface of eggs for binding the sperm in reproduction. The purpose of this thesis is to gate ZP3 gene sequnce by using genatic clonning method, and by using scanning electron microscopy to observe microstructure of egg surface of the tilapia including Oreochromis niloticus, O. aureus and Oreochromis sp.. We tried to identified three kinds of tilapia by cloning ZP3 gene and analyzing similarity of gene and protein sequence and structure and observing the microstructure of egg surface and micropyle. The shape of tilapia eggs is oval. The egg size of O. niloticus is 2.24× 1.59 mm in diameter with irregular wavy feature in surface, and with a funnel mocropyle of ca. 4.3 μm in diameter. The egg size of O. aureus is 2.31×1.68 mm in diameter. The micropyle is funnel and it’s diameter is ca. 3.75 μm. There are many short pillar on the egg surface. Oreochromis sp. is 1.76×1.40 mm in diameter. The micropyle is funnel and it’s diameter is ca. 5 μm. There are many granular substances on the egg surface. A 578 bp nucleotide segment of ZP3 gene of O. niloticus was cloned and translated to the 195 amino acids. A 576 bp nucleotide segment of ZP3 gene of O. aureus was cloned and translated to the 195 amino acids. A 578 bp nucleotide segment of ZP3 gene of Oreochromis sp. was cloned and translated to the 195 amino acids. The ZP3 nucleotides of tilapias were 45~47 % similar to other fishes as Carassius auratus gibelio, Cyprinus carpio, Pimephales promelas, Danio rerio, Oryzias latipes, Sparus aurata and Oncorhynchus mykiss. The amino acid translated from the selected ZP3 nucleotides of three tilapias were 21~23 % similar to other fishes. The ZP3 nucleotides and amino acids cloned from three tilapias had more then 99 % similarity. O. niloticus, O. aureus and Oreochromis sp. all have two glycosylation sites and three casein kinase II phosphorylation sites. O. niloticus amd Oreochromis sp. have two protein kinase C phosphorylation sites, and O. aureus has one. The amino acid of glycosylation site is NVSS in many fishes, but NISE and NRST in tilapia. The hydrophobic amino acids as alanine, valine, leucine, proline, phenylalanine, tryptophan, methionine and isoleucine were found in the ZP3 segments of the three tilapias. The cloned ZP3 of three tilapias have 70 (36%) hydrophobic amino acids. Cysteines compose secondary and third tertiary structures of ZP protein and the chorion strcture of the fish egg with disulfide bund. Three tilapias all have eight cysteines in ZP3 protein. The microstructure of egg surface and ZP3 gene sequence analysis will be usful in identification of tilapia species.