| Because of its special physical and chemical properties, Lead has been used in the industry widely, and some of them deposited in the environment. However, Lead is not the necessary element for organisms, and it is more harmful for its toxicity to nerve, blood, gastrointestinal tract, reproductive system, circulatory system and so on.On the basis of our result that high level of Lead can inhibit the expression of trypsin (ela2l) in zebrafish (Danio rerio), I treated zebrafish embryos with 0.1?0.2 and 0.3 g/L Pb(NO3)2-Real-time PCR and Whole mount in situ hybridization results show that the expression of other trypsins (such as try and ctrl) in zebrafish treated by Lead were all declined, compared with the control.The results of o-dianisidine staining showed that 0.2g/L Pb(NO3)2 reduced the level of heme in the larves of zebrafish. And the RT-PCR and real-time PCR demonstrate that the reduction of heme has two effects,0.2g/L Pb(NO3)2 on the one hand reduced the expression of some genes related with heme synthesis, on the other hand elevated the expression of ho-1 which controled the degradation of heme. That is to say, Lead can both depress the synthesis of heme and promote heme degradation. Recent report has confirmed that heme regulates trypsins in zebrafish through Bachlb/Nrf2a-MafK pathway. Hence, it is obvious that Lead can depress the expression of trypsins by reducing the level of heme, mediated with the heme-Bach/Nrf-Maf pathway.However, when zebrafish embryos were treated with hemin, a heme analogue, after treated with different concentration of Pb(NO3)2, the results of Real-time PCR showed that hemin can not rescue the decline of trypsins caused by Lead, suggesting that there may be other pathway of Lead toxicity.bach (The basic leucine zipper transcription factor) is a member of the basic leucine zipper family. It plays an important role in oxidative response and gene regulation as a transcriptional repressor. Nrf (Nuclear factor erythroid 2/p45-related factor) is a member of the CNC basic leucine zipper family. It plays an important role in the regulation of oxidative response genes and Phase ? metabolism in vertebrate. Zebrafish embryos were treated with different concentration of Pb(NO3)2 to examine the responds of bach?nrf and maf to Lead, the results showed that the transcription of bachla was dramatically up-regulated, while the transcription of nrf1a?nrf1b?nrf2a? nrf2b and nrf3 were down-regulated, and the transcription of mafs were dramaticlly changed, suggesting that Lead might depress trypsins through Bach/Nrf-Maf pathway directly.bachl and bach2 are homologes of bach in many species. Recently bach1a and bachlb were found as paraloges of bachl in zebrafish, medaka, tilapia, fugu and platyfish through the sequence search and comparison in Ensemble.In addition, bach2a and bach2b were found as paraloges of bach2 in zebrafish, medaka and tilapia. The open reading frames of four zebrafish bach paraloges were cloned in this experiment. The length of open reading frame of bach1a, bach1b, bach2a and bach2b are 2031,1899, 2286 and 2472bp, respectively, and their predicted protein sequence are 676,632,761 and 823 amino acids, respectively. Then the phylogenetic tree was built by Neighbor-Joining method with the proteins of Bach from animals. The tree showed that the two functional domains of Bach are highly conserved among animal genomes, and their molecular evolution are consistent with the species divergence. The spatial-temporal expressions of bachla, bachlb, bach2a and bach2b were also studied in this paper by whole mount in situ hybridization and RT-PCR. The results showed that all of bach1a, bach1b, bach2a and bach2b began to express from the one cell stage, suggesting that these four genes are maternally inherited. They express ubiquitously in the early stage of development of zebrafish embryos. Only bach1b has specific expression in internal organs and neuromasts at the late stage of development. Strikingly, bach1a, bach1b, bach2a and bach2b responded differently to the different concentration of Pb(NO3)2. bachla was up-regulated, bachlb had no significant difference, while bach2a and bach2b were down-regulated, suggesting that bach1a and bach1b were functional divergence in the zebrafish genome, as well as bach2a and bach2b. |
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