Identification And Vertification Of Genes Involved With Varroa Destructor Resistance In The Eastern Honeybee (Apis Cerana)

Varroa destructor became the greatest threat to apiculture almost throughout the world, and a more direct path toward mite resistance is to breed resistant bees by marker-assisted markers and genetic engineering technology. So it is important to identify genes involved with resistance to V. destructor and understand the genetic mechanisms underlying the resistance of honeybee to Varroa mites. In this study, the transcriptomes and proteomes of four Apis cerana colonies were analyzed using the Illumina Solexa sequencing method and iTRAQ technology respectively. Two colonies were highly affected by mites whereas the others displayed strong resistance to V. destructor. We determined differences in gene expression and protein expression in the susceptible colonies and the resistant colonies unchallenged and challenged by V. destructor. The objective of this study was to identify possible genes involved with resistance to V. destructor parasitism, which may provide insights into the genetic mechanisms underlying the resistance of honeybee to Varroa mites and genes for breeding. The main results were as follows:1. The A. cerana colonies were challenged by combs from Apis melleferina highly infected by mites varroa mite, and the following measures are employed to decrease the genetic background between the various colonies:a) the challenged resistant colony(C+) and the challenged sensitive colony(M+) accepted combs with similar mite infection degree and similar number of capped cells; the unchallenged resistant colony(C) and the challenged sensitive colony(M) accepted combs with no mite infection and similar number of capped cells. b) full sisters were selected by10microsatellite makers. c) the sensitive colony showed poorer resistance, whereas resistant one showed its highest resistance within24h after challenged, so the samples were collected at24h challenged by mites.2. A total of91,172all-unigenes were obtained by de novo sequencing. There were23,790unigenes identified with25COG categories.85,577unigenes were categorized into biological process of GO, and the most participated in cellular process(14,797) and metabolic process(10,317);48,194were in celluar component, and the most were in cell(10,323) and cell part(10,323);29,301were in molecular function, and the most were in binding (11,370) and catalytic activity(11,042). pathway analysis revealed that there are39,625unigenes associated with257pathways, and4793were annotated to metabolic pathway. 3.40,255unigenes were differentially expressed in C+VS M+, and19,702were down-regulated in C+while20,553up-regulated. GO enrichment analysis suggested that C+and M+responded differently to mite challenge companied with changing of transcript factors. Several significantly enriched pathways involved in virus and bacterial infection and biosynthesis of antibiotic substance, so it suggested that there were different natural resistances between the resistant and the sensitive.21,252unigenes were differentially expressed in C VS M, and962were down-regulated in C while20,290up-regulated. GO and pathway enrichment analysis suggested that there were differences in immune response, muscle development, learning, memory and smell sensitivity in the two colony.36,691unigenes were differentially expressed in C+VS M+, and17,799were down-regulated in C+while18,892up-regulated. GO enrichment analysis suggested that C+responded to mite challenge companied with changing of transcript factors. Several significantly enriched pathways involved in virus and bacterial infection and biosynthesis of antibiotic substance, and it suggested that C+activated natural resistances when infected by bacteria.24,508unigenes were differentially expressed in M+VS M, and21,479were down-regulated in M+while3029up-regulated. GO enrichment analysis suggested that M+changed olfactory function companied with changing of transcript factors. Several significantly enriched pathways involved in virus and bacterial infection, and only one in biosynthesis of antibiotic substance, which suggested that the natural resistance of the resistant was low.4.270up-regulated and8down-regulated DEGss had differences greater than15-fold, and the folowing DEGss were associated with varroa resistance, including troponin, calcium-transporting ATPase, obp, transcript factors, genes related to immunity, apidermin and synapsin.5. Quantitative protemic analysis by iTRAQ using the A. cerana transcriptome as reference. There were1532proteins identified. Most of proteins(61.02%) were categorized into cell and cell part of celluar component. Binding contained the most proteins (45.30%) in molecular function. Most proteins participated in basic biological process such as cellular process(20.01%) and metabolic process(19.68%). pathway analysis revealed that there are1503proteins associated with239pathways, and the most were annotated to metabolic pathway, the result was same as transcriptome.6.72proteins were differentially expressed in C+VS M+, and31were down-regulated in C+while41up-regulated. GO enrichment analysis suggested that C+and M+responded differently to mite challenge companied with changing of transcript factors. There was only one significantly enriched pathway(Sphingolipid metabolism).154proteins were differentially expressed in C VS M, and82were down-regulated in C while72up-regulated. GO enrichment analysis suggested that there were differences in vitality. Metabolic pathway was the most significantly enriched among17.202unigenes were differentially expressed in C+VS M+, and81were down-regulated in C+while121up-regulated. GO enrichment analysis suggested that C+responded to mite challenge companied with changing of transcript factors. Parkinson’s disease was the most significantly enriched pathway among14.161unigenes were differentially expressed in M+VS M, and94were down-regulated in M+while67up-regulated. GO enrichment analysis suggested that the basic life of M+affected by mites. Parkinson’s disease was the most significantly enriched pathway among25.7. According to transcriptomic analysis and Venn analysis, endocuticle structural glycoprotein SgAbd-2-like and obp13were associated with Varro resistance.8. The corrections of differential expressed proteins and corresponding transcripts was low (r C+VS M+=0.0737, r C VS M=0.3269, r C+VS C=0.0774, r M+VS M=0.0066).9. A total of15DEGs involved in Varro resistance were verified by qRT-PCR, and the result showed that the expression regularity was consistent with transcriptome data, illustrating the transcriptome sequencing results were reliable. The coding sequences of obpl7, obp18and Mklb-1were408bp,399bp and4887bp respectively. Sequence homology, amino acid structure, protein secondary and tertiary structure of obp4, obp17, obp18and mklb-1were analyzed. The ORFs and amino acid constitutions of the4genes in A. cerana had high homology to A. mellifera. The three obps have signal peptides, and no signal peptides were predicted for mklb-1. All of the four genes had have no transmembrane helixes, and were a proteins.