Cloning And Analysis Of β-actin Gene In Brachionus Plicatilis

Rotifer is a kind of multicellular invertebrate with pseudocoelom. Its tiny body size fits the caliber of juvenile fish just right. Also, it is nutritious and easy for artificial feeding, which makes it the perfect primary diet for fish fries. Among the various species of rotifers, Brachionus plicatilis inhabits mainly in brackish water, and it is the sole rotifer species that can be raised artificially in seawater. Only a few of existed studies have referred to the molecular biology field of rotifer. So in the present study, a relatively conserved gene, β-actin, is chosen as the subject. To acquire the full-length cDNA sequence of Brachionus plicatilis β-actin, degenerate primers are designed according to the alignment results of β-actin homologs. Then the promoter sequence is achieved by genome walking, and its structure and function are predicted and analyzed. These results lay the foundation for further vector construction and transgene techniques in rotifer.In this study, the whole DNA sequence of Brachionus plicatilis β-actin gene is identified to be 2987 bp, with no intron in the coding region. Its cDNA sequence is 1377 nt, consisting a 5’UTR of 76 nt, a 3’UTR of 170 nt, and an ORF of 1131 nt, which is predicted to encode 376 amino acid residues. The promoter sequence of Brachionus plicatilis β-actin is 1706 bp. By Blastp analysis, Brachionus plicatilis β-actin ORF is compared to its homologs in Dugesia japonica, Glohodera rostochiensis, Urechis unicinctus and shellfishes. The results show that these β-actin genes share high similarities, even above 75%, regardless of their evolutionary relationship. It can be inferred that β-actin keep a high gene stability during the Jong evolution progress. Thus it may be the preferred gene to study molecular genetics in lower animals.The first step of this study is cloning the center cDNA sequence of Brachionus plicatilis β-actin. Then its promoter sequence can be fuse to external genes, such as genes encoding antimicrobial peptides or growth hormones, to construct vectors in vitro. These vectors can be further applied to various transgene techniques like electroporation and microinjection, in order to explore the transgene field in rotifer. Taking advantage of the low breeding cost, the short life cycle and high reproductive efficiency, rotifers can be the bioreactor for expressing many kinds of target products, which will serve many fields like clinical pharmacy, and will open another avenue for transgene bioreactor research and application.