The Expression Patterns Of BMPR-â… ,BMPR-â…¡,Smad1,Smad4 And Smad7 In Amphioxus Tail Regeneration Process And Prokaryotic Expression,Polyclonal Antibody Preparation, Immunohistochemical Localization Of BMP2/4,BMPR-â…  And BMPR-â…¡

Regeneration is a process in which some organisms can restore the lost tissues or organs by re-growth. Regeneration is an evolutionary trait and the regenerative abilities among different species are various. For better understanding regeneration, it is necessary to study it in an evolutionary perspective and especially to study it in important evolutionary animals. The cephalochordate amphioxus, the most basal living chordate, has long been considered to be a living fossil to bridge the invertebrate and vertebrate. Recently it has been reported that amphioxus can regenerate its tail in a vertebrate-like manner. However, the studies in amphioxus regeneration, especially in its molecular mechanism, are very limited.It has been found that BMP signaling pathway plays an important role in limb and tail regeneration in some vertebrate species. BMP (Bone morphogenetic protein), an important member of transforming growth factors β family, is a kind of multi-function secreted glycoprotein without collagen except BMP-1. The pH of BMP is weak acidity and its relative molecular mass is about 30 kDa. BMP signaling pathway is implicated in various biological processes such as cell proliferation, cell differentiation, embryo development, and so on. Although BMP signal pathway is involved in regeneration process in vertebrate, it is obscure whether it plays roles in regeneration in amphioxus.In this paper amphioxus tail regeneration was focused. BMP signal related genes including BMP2/4, BMPR-Ⅰ, BMPR-Ⅱ, Smad1, Smad4 and Smad7 were cloned, which act as ligand, recipient and regulation factors respectively in the signaling pathway. The roles of the BMP signaling pathway in amphioxus tail regeneration were explored by using multiple techniques such as gene cloning, in situ hybridization, the prokaryotic expression and so on.First of all, the total RNAs extracted from amphioxus at different regeneration stages were used for the reverse transcription and the complete BMPR-1, BMPR-II, Smadl, Smad4 and Smad7 gene sequences were amplificated by RT-PCR method. These amplified sequences were connected to the pGEM-T easy vector containing T7 or SP6 promoter, and RNA probes (digoxigenin labeled) were synthesized using the method of in vitro transcription. The spatial and temporal expression patterns of BMPR-I, BMPR-II, Smadl, Smad4 and Smad7 at different stages of tail regeneration in amphioxus are studied by in situ hybridization. It was revealed that these genes are mainly expressed at early regeneration stage (from 1 to 14 days after amputation) close to the incision region, especially in the notochord part at the highest expression level. With the regeneration undergoing, their expression levels gradually decreased and even no expression signals can be detected at late regeneration stage (14 days after amputation). These suggested that BMPR-Ⅰ, BMPR-Ⅱ, Smadl, Smad4 and Smad7 play roles in the early regeneration stages in amphioxus.In order to further verify whether the BMP signaling pathway is involved in the amphioxus tail regeneration process at the protein level, the complete BMP2/4, BMPR-I and BMPR-II cDNA sequences containing enzyme cutting sites were cloned into pET-32a(+) vector simultaneously to construct the prokaryotic expression vectors. Through induction of IPTG, BMP2/4, BMPR-Ⅰ and BMPR-Ⅱ fusion proteins were successfully expressed and mainly accumulated in inclusion bodys, and subsequently purified through Nickel column chromatography method. New Zealand Rabbits were immunized by injecting the purified proteins subcutaneously. Antiserum against BMP2/4, BMPR-Ⅰ and BMPR-Ⅱ were collected and their titres were determined respectively. Using these antibodies, immunohistochemical tests were performed on amphioxus at early regenerative stages. The immunohistochemical staining demonstrated that BMP2/4, BMPR-I and BMPR-II were located in the vicinity of amputation plane at early regeneration stages, matching to the expression patterns of their mRNAs.In short, it was shown that BMP signaling pathways related genes express during the process of tail regeneration in amphioxus, and their specific expression patterns suggest their roles in the regeneration process. This paper provide an insight into roles of BMP signaling pathway in regeneration. In addition, the antibodies prepared will definitely provide tools for further probe the functions of BMP signaling pathways in regeneration.