| In recent years, the wide use of pesticides and spread of honeybee diseases causes serious damage to the ecosystem and bee breeding habitat, which is closely related to honeybee Colony Collapse Disorder(CCD). In America and parts of Europe, the loss of honeybees(Apis mellifera Linnaeus) is more than 30%, inducing incalculable direct colony and subsequent crop losses. The cause of CCD is not clear and types of diseases and usage of pesticides become the speculated factorsof CCD by the scholars. Apis mellifera Linnaeus and Apis cerana Fabricius, both belonging to the same genus, have close genetic relationship, with A. mellifera Ligustica and A. c. cerana in China as the representative species. The colonies of A. mellifera Ligustica kept in our country outnumber A. c. cerana Fabricius and they are even breed in the same region in some parts of the nation, but the CCD do not occur in A. c. cerana. The underlying mechanisms are unclear, thus more study of autoimmunity system should be enhanced in A. c. cerana.MsrBs are involved in removing free radicals, avoiding antioxidant trauma in organism and defending exogenous toxic substance. Through regulating free iron ions, iron-metabolism proteins play a key role in inhibiting free radical production, eliminating existent reactive oxygen species and preventing the infection of the pathogenic microorganism. In our study, we selected A. c. cerana as the experiment material to explore the biological functions of MsrB and iron-metabolism proteins genes and their relationships with immunity of A. c. cerana. The major results and conclusions in this thesis arepresented as follows: Molecular property and functional analysis of MsrB gene in A. c. ceranaA novel MsrB gene in A. c. cerana was cloned based on the conserved regions of MsrBs from A. mellifera Fabricius by RT-PCR and RACE-PCR, and we named the putative gene as AccMsrB. The sequence analysis indicated that the full-length c DNA of AccMsrB was 757 base pair(bp), including a 138 bp 5′untranslated region(UTR), a 2054 bp 3′UTR and a 414 bp complete open reading frame(ORF). The ORF encoded a polypeptide of 137 amino acids with a predicted molecular mass(Ms) of 15.5 kDa and a theoretical isoelectric point(pI) of 7.77. AccMsrB shares higher sequence with other insect species, especially from Apidae family, and contains the conserved features of the cytosolic MsrB superfamily. Using the TFSEARCH database, several important transcription factors required for regulating various environmental stresses(HSF and Dfd) were identified in the 5’-flanking region of AccMsrB. These results suggested that AccMsrB might be involved in development and environmental stress responses.The qRT-PCR and immunohistochemical staining were performed to examine the developmental regulation and tissue distribution of AccMsrB. The results indicated that Acc MsrB was expressed in the whole period of metamorphosis and the highest expression level was detected in the transition stage of development such as first instars larvae, pre-pupae and 15-day old adult. AccMsrB had a significantly different expression levels in varying types of tissue, with highest in the epidermis and second highest in muscle. Immunohistochemical staining revealed that AccMsrBexiting in body wall and the surrounding tissue of the eye. All of these results implyed that AccMsrB may play a protective role in the transitions of metamorphosis development, defending environmental oxidative damage and cleaning free radicals.The relative expressions of AccMsrB under various biotic and abiotic stimulis were detected by qRT-PCR. The results showed that the transcript levels of AccMsrB can be markedly accumulated by treatment of various abiotic stimulis such as un-fitted temperature, UV, H2O2, pesticides and CdCl2. Whereas the transcription of this gene was gradually inhibited by phoxim treatment. And the assay of oxidation statuses(H2O2 concentration) indicated that this induced expression of Acc Msr B might be directly related to the accumulations of H2O2. Taking into account these results, we speculate that AccMsrB may be essential in protection against oxidative damage and metamorphosis development.(2) Molecular characterization and functional analysis of iron metabolism protein genes in A. c. ceranaBy the above mentioned methods,a group of iron metabolism protein genes in A. c. cerana were isolated and named as AccsTf, Acc FTH and AccFTL(GenBank ID: JF330111.1, JF330112.1, and JF340051.1, respectively). The full-length 2346 bp, 1028 bp and 1018 bp c DNA sequence of AccsTf, AccsFTH and AccsFTL included a 74 bp, 219 bp and 274 bp 5′UTR, and a 133 bp, 134 bp and 90 bp 3′UTR, and a 2139 bp, 675 bp and 654 bp complete ORF thatencoded peptides with 712, 219 and 218 amino acids, respectively, with computed theoretical pI of 7.07, 6.25 and 6.52 and Mr of 78.6 kDa、25.5 kDa and 25.2 kDa. Multiple sequence alignments revealed higher sequence identity ofAccsTf, AccFTH, Acc FTL with other insects.By analyzing the gene sequence related to iron metabolism, in the 5’-flanking region of Acc FTH, an iron response element(IRE) which can form a stem-loop structure, involving in iron regulatory proteins(IRPs), could regulate the uptake and release of iron. And the promoter sequences of iron metabolism protein genes contained several important transcription factors associated with immune response, including HSF、AP-1、Nrf2 and NFκB.We also found that there wasiron binding site composed by some conserved amino acids in N terminal of protein; iron oxide center composed by conserved amino acids including cysteine in heavy chain of ferritin; and conserved glycosylation site(N-E-F) and cysteine. These putative transcription factor binding sites imply that AccTf, Acc FTH, AccFTL are transferrin and ferritin andmight involved in not only environmental stress responses and iron metabolism in A. c. cerana.The qRT-PCR was performed to determine temporal and spatial expression profiles of Acc Tf and AccFTH.We noticed that a significant increase of AccTf and AccFTH expression in L4 and PP, which always correlated with the transition of diet and development stages. The highest expression level was detected in the fat body compared to other tissue; while ferrintin was expressed in various types of tissue, but highest in midgut, indicating the similar expression pattern for this genome relative to other organisms. The expressions profiles of AccTf and AccFTH under various experiment conditions were detected by qRT-PCR. We found that the transcript levels of AccTfcould be markedlyup-regulated when treated by unbeneficial temprature, different oxidative stress, and pesticides. However, the transcription of AccFTH showed up-regulation just in some treated conditions and suppression during 4 oC and 42 oC and pesticide treatment. These results demonstrated that transferrin and ferritin were functionally important in defending most disadvantage factors that caused stresses. After treated with FeCl3, the iron concentration in beesincreased andthe transcript levels of Acc Tf and AccFTH were markedlyup-regulated, suggesting that these two genes was induced by free ironic ions and functioned in iron storage and transportation as to prevent the generation of oxygen free radical and protect the body from oxidative damage.In conclusion, our results showed that AccMsrB, AccTf, Acc FTH and AccFTL genes had common features from their own gene families conferring them with ability of removing endogenous free oxidative radicals and defending exogenous oxidative stress. Transferrin and ferritin co-worked to fulfill iron transportation and storage tasks and regulate iron concentration in body, thus to enhance antioxidant capability of bees. |
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