| The proteins of musculoaponeurotic fibrosarcoma oncogene (Maf) family control many life processes in cell as important transcription factors. They are usually divided into large Maf and small Maf. Small Maf members have not the transactivation domain, which Large Maf members have. Then small Maf proteins need to form heterodimers with other basic and leucine zipper (bZIP) proteins, in order to regulate gene expression via MAF recognition element (MARE) site. In zebrafish, small Maf family includes Mafk, Mafg1, Mafg2, Maft, mafgl and mafg2are two paraloges of mafg in fish, the similarity between MafGl and MafG2is88%. The spatiotemporal expressions of such small maf genes and their effects on other gene transcription in zebrafish were doned in this thesis, it is benifit to study on evolution of maf paralogs and their functional divergences in fish.The open reading frames of four small maf were cloned in this experiment. The results of sequencing shows that, the length of open reading frame of mafk is465bp, coded154amino acids. The length of open reading frame of mafgl is489bp, coded162amino acids. The length of open reading frame of mafg2is516bp, coded171amino acids. The length of open reading frame of maft is477bp, coded158amino acids. The multiple sequence alignment analysis and phylogenetic tree showed that the evolutionary between small Maf is conserved. The syntenic analyses among different species of small maf show that mafk and maff are more conservative than mafg.The overlay and replace of four Maf proteins were confirmed. It was reported that four small maf express widely in various organizations of zebrafish, signals can be detected at later embryonic development. However, the spatial expression patterns of small maf are not published yet during zebrafish embro development. So ten different periods of zebrafish embryonic development (1cell,2cell,3.5hpf,6hpf,12hpf,24hpf,36hpf,48hpf,72hpf,96hpf) were chosed in our experiment, the results of semiquantitative PCR and whole-mount in situ hybridization show that the expression quantities of four small maf are different during zebrafish embryonic development. The mafg2expresses the most, mafk and maft are more, mafgl is the least. Moreover, the expressional localization of four small maf mainly concentrated in the head. The signs in the torso and tail during the late stage of embryonic development of mafk and maft were detected, except mafk and mafg1. In addition, the express pattern between two paralogs of mafg is strikingly different in zebrafish.Previous studies have shown that heme is the upstream regulatory factors of Maf-Nrf2/Bach1pathway. The deficiency of heme lead to down regulation of zymogens in zebrzfish, but the study on regulation mechanism is rare. In order to investigate whether heme controls the transcriptional level of zymogens through Maf-Nrf2/Bach1pathway or not, vectors of small maf and upstream region of zymogens were co-transfected into293cell or EPC cell in our experiment. The results from dual-luciferase system showed that the expression level of zymogens (try) was regulated by different amount of Hemin in293cells, significantly increased at the concentration of10μM and20μM of Hemin. The expression levels of try were also regulated by the expressional amount of small maf and nrf2a. The expression level of try was up-regulated at250ng-transfected dose of nrf2a, while significantly up-regulated at500ng-transfected dose of nrf2a. The down-regulation of the try is dosage-dependent on overexpress of small maf. High dose of mafk (250ng), led to significant down-regulate of try, can no longer be restored by Nrf2a, while low dose of mafk (50ng), also led to significant down-regulate of try, can be up-regulated by Nrf2a in this case. In EPC cell, the down-regulation of the try is also dosage-dependent on overexpress of small maf. However, the expression level of try was decreased with Hemin increasing, contrast with the results in293cells. |
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