Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.
Gene. 2012 Nov 15;510(1):39-46. doi: 10.1016/j.gene.2012.08.031. Epub 2012 Aug 28.
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.
文昌鱼是研究无脊椎动物-脊索动物过渡时期出现的进化和发育机制的有前途的实验室模型动物。然而，文昌鱼作为模型生物的主要缺点是缺乏完善的技术方法。热控制下的条件基因表达是基因功能研究中非常有用的策略。为了使这种方法在文昌鱼研究中成为可能，我们在这里报告了中国文昌鱼（Branchiostoma belcheri）中一种文昌鱼 Hsp70 基因（Hsp70a）及其启动子的分离和特性。Hsp70a 在正常温度下表达水平很低，但在热休克后动物中强烈诱导。在调控区（起始密码子上游-1031 到-11）中发现了基本顺式作用元件（CAAT 和 TATA）以及四个热休克元件（HSE），但令人惊讶的是，大多数元件位于 5'UTR 区（-252 到-10）。包括转录起始位点（TSS）和 ATG 在内的报告基因构建体在 EPC 细胞和微注射斑马鱼胚胎中进行了瞬时表达测试。结果表明，5'UTR 区包含-92bp 处的 TATA 盒、-152bp 处的 CAAT 盒和三个 HSE 元件（-212 到-106），代表 B. belcheri Hsp70a 基因的核心 hsp 启动子序列。因此，在这项研究中，我们在文昌鱼中鉴定了一种有效的热诱导启动子，可用于建立条件基因表达系统，其中靶基因可以在时间或组织特异性方式在文昌鱼中进行调控。
