| 其他摘要 | Amphibians that are the most primitive terrestrial vertebrates have wide distribution throughout the world. Their skins are naked and smooth. In order to adapt a wide range of habitats and ecological conditions, they have developed a variety of skin effective defense system. As an important component of the body's innate defense system of the amphibians, antimicrobial peptides (AMPs) are abundant in the skin secretions. We selected Bombina microdeladigitora and Hyla annectans in Yunnan province to study the diversity, function and structure of AMPs in amphibian skin secretions. In this study, 64 novel cDNAs encoding 44 Maximins and 30 Maximin Hs were cloned from skin cDNA library of B. microdeladigitora, of which 32 Maximins and 20 Maximin Hs were novel and others were same as AMPs cloned from other Bombina species. Besides skin, we also found AMPs genes were abundantly expressed in brain of B. microdeladigitora and B. maxima. Twenty one novel cDNAs encoding 16 Maximins and 10 Maximin Hs and 39 novel cDNAs encoding 27 Maximins and 20 Maximin Hs were cloned in brain of B. microdeladigitora and B. maxima, respectively. Among the AMPs, all Maximins are cations and tens of Maximin Hs are anions including Maximin H5 identified before. The analysis of base transition/transversion showed RMaximin<1 while RMaximin H >1. These results indicated that the diversity of Maximins caused mainly by base transition, while Maximin Hs by transversion. The interspecies evolutionary distance analysis showed that B. maxima are close to B. microdeladigitora, while they are both far away from B. variegata. The intraspecific evolutionary distance is differing from domains. The distances between the mature peptides are larger than that between signal peptides and acidic spacer peptides. Our results also showed that the evolutionary rate was faster in Maximins than in Maximin Hs. Furthermore, there is a positive selection on Maximins and Maximin Hs intra- and inter-species (ω>1), while no positive selection is found in signal peptides and acidic spacer peptides. These results suggested that the rapid evolution of Darwin’s positive selection on Maximins and Maximin Hs is the primary cause of diversity in AMPs. This is consistent with the most important defensive functions of AMPs that is essential for amphibian survival in different conditions. Functional study found that AMPs in B. microdeladigitora not only have activites of antibacteria and anti-fungi, but also have a strong anti-oxidative function. In addition, the expression of AMP genes in brain tissue gives us information that AMPs may also play a role in neural transmission. A large number of cDNAs encoding bradykinin-related peptides were cloned from B. microdeladigitora. These sequences include 1-4 Bombinakinin tandem repeats or 1-4 Bombinakinin tandem repeats with a Bombinakinin-GAP, while previous reports show that 1-8 Bombinakinin tandem repeats or 1-8 Bombinakinin tandems repeats with a Bombinakinin-GAP in B. maxima. We also gained three cDNAs encode Bombinakinin tandems from the brain of B. maxima, two have 6 Bombinakinin tandems with or without a Bombinakinin-GAP, the other one has 2 Bombinakinin tandems. By comparing the cDNA sequences between Bombinakinin and Bombinakinin with a Bombinakinin-GAP, we found that the missing sequence TGCGGGTA lead to frameshift mutation, which brought the termination of coding sequence forward and caused the loss of Bombinakinin-GAP. Two serine protease inhibitors (named BMSI 1 and BMSI 2, respectively) were identified from the skin secretions of the toad, B. microdeladigitora. The cDNAs encoding BMSIs were cloned from the cDNA library prepared from the toad skin. The deduced complete amino acid sequences of BMSIs indicate that mature BMSI 1 and BMSI 2 are composed of 60 amino acids including 10 half-cystines to form 5 disulfide bridges. A FASTA search in the databanks revealed that BMSIs exhibit sequence similarity with other serine protease inhibitors from amphibians of the genus Bombina. BMSI 1 potently inhibited trypsin and thrombin with a K(i) value of 0.02 μM and 0.15 μM, respectively. Sequence analysis revealed that all serine protease inhibitors from five amphibians of the genus Bombina share highly conserved primary structures. By random screening of cDNA library of the brain of B. maxima, a complete Somatostatin（SST）sequence was obtained, then two variant sequences SST-L (Leu11-SST-14) and SST-R (Arg14-SST-14) were gained by means of gene clone from B. maxima and B. microdeladigitora. The two mutational proteins have similar function with SST that inhibit proliferation of some tumor cell lines and release of cytokines, and have analgesic activity. Moreover, the cDNA sequences of POMC and proenkephalin homologous with B. orientalis were screened in brains of B. maxima and B. microdeladigitora. Two types of active peptides called annin were found in skin of Hyla annectans,. One type of annin cDNAs encodes peptides with 15-17 amino acid residues. Their signal peptides are higher homologous to those from Hylidae, while the acidic spacer peptides and mature peptides are significantly difference. They share homologous with AMPs from other Hylidae species but have neither antibacterial nor anti-oxidative activity. MTT assay showed they could promote cancer cell and endothelial cell (HUVEC) growth at a high concentration. Another type of cDNAs encode pentapeptide with analgesic activity which might be caused by the antagonism of bradykinin. |
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