A Study Of Screening Of The Proteins Interacting With Zebrafish Spr2-Like2 Via Yeast Two-hybrid System.

Zebrafish (Brachydanio rerio), as a new model animal wildly used on embryo development study of vertebrate, became popular and useful. In recent ten years, the study of vertebrate embryo development using this model has developed rapidly. There are several advantages of zebrafish to be a model animal. Firstly, they are small and easy to be raised. The female can lay more than 200 eggs once a week and the embryos need only 3 months to be hatched. Secondly, the zygotes of zebrafish are big and limpid, so it is quite convenient to observe the whole developmental processes of zebrafish embryo and analyse the feasible endpoints in embryotoxicological testing. Finally, the development of zebrafish embryo is quite rapid, only need 3 days from impregnated to incubated. Recently a new method which microinject Morpholino into the embryo of zebrafish to inhibit special genes activity has effectively to be used. As a result, we decided to use zebrafish as a model to study an important protein named zebrafish Spr2.spr2 is a restrictly expressed gene in both mesoderm and ectoderm during the development of zebrofish embryo. Spl-like proteins and Kruppel-like factors (KLFs) are highly related zinc-finger proteins that are important components of the eukaryotic cellular transcriptional machinery. By regulating the expression of a large number of genes that have GC-rich promoters, Spl-like/KLF transcription regulators may take part in virtually all facets of cellular function, including cell proliferation, apoptosis, differentiation, and neoplastic transformation. Individual members of the Spl-like/KLF family can function as activators or repressors depending on which promoter they bind and the coregulators with which they interact.spr2 is expressed in both hypoblast and epiblast cells during late blastulation/early gastrulation, and in some mesodermal andneural tissues at later stages. The spr2 knockdown and overexpression data shows that spr2 expression is dependent on Fgf, Nodal and Wnt signals, and it is implicated in mesoderm induction during early development in zebrafish embryos. Because of the importance of mesoderm development during organgenesis, We use yeast two hybrid system to search for proteins interacting with spr2 from the early embryos of zebrafish cDNA library and explore the mechanism of spr2 function in Fgfs signal pathway.In this study, the yeast two-hybrids system is used to searched for proteins interacting with Spr2. From the early embryos of zebrafish cDNA library, 78 positive clonies from 6.3×106 transformants were found by yeast mating. 11 kinds of proteins are identified after sequencing the plasmids of the positive clonies such as Paired box gene 6a, mSin3A-associated protein 130. We suggest that spr2 can participate in Fgfs signal pathway by the interaction with fgf8.