Cloning And Expression Analysis Of Embryo Development Related Gene P26 In Artemia

Artemia is a kind of small crustacean, which distributes widely over the world. They are remarkably resistant to adverse environments such as long-term anoxia, desiccation, temperature extremes, hypersaline, and irradiation. P26 is a small heat shock protein that functions as a molecular chaperone to protect embryos by preventing irreversible protein damage during embryonic development. The expression of the p26 gene is embryo-specific, it expresses only in cysts and diapause embryos. In this study, primers were designed based on the mRNA sequence of p26 gene in A. sinica available in the GenBank database, and total RNA of A. sinica was amplified by RT-PCR. A fragment of 542 bp was amplified, and the quantity of (A+T) is higher than (C+G). The fragment was identified as p26 of A.sinica after analyzing by Blastn. The putative protein p26 contained 174 amino acid and a-crystallin domain. Phylogenetic trees were constructed based on the sequences of the putative protein of p26 and other sHSPs (small heat shock proteins) by software ClustalX 1.83 and MEGA 3.0. P26 of A. sinica and sHSPs of invertebrate were clustered in a clade, and sHSPs of vertebrate were clustered in the other clade. DNA sequences were generated by eight bisexual Artemia strains living in different regions of the world. Phylogenetic trees based on p26 gene sequence indicated that bisexual Artemia strains were consisted of four groups : Group 1: East Asia bisexual group; Group 2: Middle Asia bisexual group; Group 3: North America bisexual group and Group 4: South America bisexual groups. To analyze the relationship between the expression level of p26 gene and embryonic development,expression of the p26 gene at different developmental stages of A. sinica and A.parthenogenetica was quantified using Real-time Quantitative Polymerase Chain Reaction (RQ-PCR) at the first time. The results indicated that perfect standard curves were obtained by RQ-PCR, so object gene can been detected in a broad linear range. Good amplification profiles dissociation curves were get in any sample, indicating that RQ-PCR is convenient,rapid,highly efficient and feasible. p26 were expressed in A. sinica and A.parthenogenetica during embryonic development, and expression levels of the p26 gene in A. sinica were higher than in A.parthenogenetica at other developmental stages except from 0 h. At the developmental stage of 0 h, both A. sinica and A.parthenogenetica expressed the highest level of p26. As development progressed, expression levels of the p26 gene reduced significantly. There was a small quantity of p26 gene expression at the developmental stages of 16 h and 24 h. We come to conclusion of p26 is involved in protecting the embryo from adverse environments during embryonic development.