Isolation and functional analysis of the promoter of the amphioxus Hsp70a gene

Abstract

Amphioxus is a promising laboratorial model animal for studying the evolutionary and developmental mechanisms that appeared during the invertebrate-chordate to vertebrate transition. However, the main drawback for the use of amphioxus as a model organism is the lack of well-developed technical approaches. Conditional gene expression, as performed with thermal control, is a very useful strategy in gene function studies. To make this method possible in amphioxus studies, here we report the isolation and characterization of an amphioxus Hsp70 gene (Hsp70a) and its promoter in Chinese amphioxus (Branchiostoma belcheri). Hsp70a showed very low expression at normal temperatures but was robustly induced in animals upon heat shock. The basal cis-acting elements (CAAT and TATA), as well as four heat shock elements (HSEs), were found within the regulatory region (−1031 to −11 upstream from the start codon), but surprisingly most of the elements were located in the 5′UTR region (−252 to −10). Reporter constructs, including sequences from both the transcription start site (TSS) and ATG were tested for transient expression in EPC cells and microinjected zebrafish embryos. Results suggested that the 5′UTR region, which includes a TATA box at −92bp, a CAAT box at −152bp, and three HSE elements (−212 to −106), represents the core hsp promoter sequence of the B. belcheri Hsp70a gene. Therefore in this study we identified an effectively thermo-inducible promoter in amphioxus that could be used for the establishment of a conditional gene expression system in which the target gene can be regulated in a temporal- or tissue-specific way in amphioxus.