| Varroa destructor has become improtant pests in the world, causing large economic damage on beekeeping. As mite native hosts, Apis cerana cerana evolved several strategies against mites, including grooming and hygienic behavior. These behaviors allow for co-existence between Chinese honeybees and mites. In this study, for the sake of exploring genes involved in the process of Chinese honeybee resitance to mites, the transcriptomes of Apis cerana cerana colonies during V. destructor infection were analyzed in compared to healthy colonies by using the Illumina Solexa sequencing method. Two candidate genes, named mab-21 and Calcineurin B that were differentially expressed, were selected for sequence cloning and further for analysis of mRNA expression pattern during different developmental stage. The main results are as follows:1. By high throughput RNA sequencing technology, A total of 31 739 all unigenes were obtained by De novo assembly, and the average length is 6 067 bp. 29 792 unigenes were annotated after blast with Nr, GO and KEGG database. 513 unigenes were differentially expressed in Chinese honeybees colonies after V. destructor infection, of which 502 were up-regulated and 11 down-regulated.2. The mab-21 gene is one of the differentially expressed genes. The mab-21 gene in A. cerana cerana was cloned by RT-PCR and named as Accmab21(GenBank accession no. KR000001).The full length of open reading frame(ORF) is 1 098 bp, and encodes 395 amino acids with the molecular weight of 41.63 kD and the theoretical isoelectric point of 8.53. The phylogenetic analysis showed that mab-21 of A. cerana cerana, A. mellifera, A. florea and Bombus impatiens clustered in the same clade.3. A quantitative analysis of mab-21 expression patternin the head part of honeybee workers at different developmental stages(newly emerged bee, nurse, guarder and forager) after infection of V. destructor mites was conducted using real-time RT-PCR. The mab-21 transcript was clearly detected in different developmental stages and was significantly higher in nurses than in other stages(P<0.05). After V. destructor infection, the expression level of mab-21 in the nurses and guarders significantly decreased in compared to the healthy control(P<0.05), but no obvious difference was detected in the newly emerged bees and foragers. These results suggest that mab-21 may be involved in the process of A. cerana cerana resistance to mites.4. Calcineurin B, the regulatory subunit of Calcineurin, is another candicate genes. Calcineurin is a protein phosphatase regulated by Ca2+/calmodulin. Full length cDNA sequence of Calcineurin B in A. cerana cerana was obtained by RT-PCR. Sequence analysis indicated that its open reading frame(ORF) is 513 bp encoding 170 amino acids. The molecular weight and theoretical isoelectric point of predicted protien is 19.36 kD and 4.33. Multiple sequences alignment showed that Calcineurin B from A. cerana cerana has the highest amino acid sequence identity with that of A. mellifera(99.4%), while has higher amino acid sequence identity with that of B. impatiens(97.1%), Tribolium castaneum(97.6%), Drosophila melanogaster(94.7%), Bombyx mori(96.5%). Thephylogenetic analysis indicated that A. cerana cerana, A. mellifera, A. dorsata and A. florea have a close relationship of Calcineurin B evolution. 5. The expression pattern of Calcineurin B were also quantitatively analyzed. The Calcineurin B transcript was clearly detected in different developmental stages of the workers of A. cerana cerana. The expression levels are ordered as follows: guarder>forager>nurse>newly emerged bee(P<0.05). After V. destructor invasion, the expression of Calcineurin B in the newly emerged bee and nurse significantly increased as compared to thehealthy control(P<0.05), but decreased in foragers. no obvious difference was detected in guarder. |
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