Semester

Summer

Date of Graduation

2012

Document Type

Thesis

Degree Type

MS

College

Davis College of Agriculture, Natural Resources and Design

Department

Wildlife and Fisheries Resources

Committee Chair

Jianbo Yao.

Abstract

Zygote arrest 1 (Zar1) is a maternal-effect gene that is essential for early embryonic development. Recently, a novel gene called Zar1-like (Zar1l) was discovered. Functional studies showed that Zar1l plays an important role in regulating oocyte-to-embryo transition in the mouse. The objectives of this study were to characterize the rainbow trout Zar1 and Zar1l genes and evaluate the potential roles of these genes in controlling egg quality in rainbow trout. Through database mining, we identified the cDNAs encoding rainbow trout Zar1 and Zar1l. The Zar1cDNA codes for a protein of 333 aa ,and the Zar1l cDNA encodes a protein of 323 aa. The sequences at the C terminus of the two proteins are highly conserved and contain a conserved Zinc-binding domain. Analysis of tissue distribution by RT-PCR showed Zar1 is predominantly expressed in ovary and testis with minor expression in other somatic tissues, while Zar1l is exclusively expressed in ovary. The expression patterns of Zar1 and Zar1l genes during early embryonic development (0h, 7.5h, 10.5h, 19.5h, 2d and 7d post fertilization) were determined by quantitative real-time PCR analysis. Both genes are highly expressed in unfertilized oocytes (0h), but they show different expression patterns. While the expression of Zar1 gene decreases from 0h to 10.5h post fertilization, and then increases in 2d embryos, the Zar1l gene shows continuous reduction of expression during early embryonic development (from 0h to 2d embryos). The expression patterns of Zar1 and Zar1l genes during ovarian development (pre, early, middle, and late vitellogenesis) were also determined by quantitative real-time PCR analysis. Both genes showed high expression in pre-vitellogenesis and reduced expression in early and middle vitellogenesis. In late vitellogenesis, expression of Zar1 gene increases again, but Zar1l gene expression continues to decrease. To determine the role of Zar1 and Zar1l genes in controlling egg quality, we analyzed the expression of both genes in eggs of different qualities (Day1, Day7, and Day14 post-ovulation). Both genes showed reduced expression in eggs of low quality. Finally, we performed a yeast-two hybridization screening to identify proteins that interact with Zar1l protein. Five proteins showing interactions with Zar1l protein were identified which include Di-N-acetylchitobiase, Serine/threonine/tyrosine-interacting protein (STYX), Ariadne-2 homolog, GH20 HexA HexB-like, and C-type mannose-binding lectin.

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