【作者】 殷玲；

【导师】 陈国宏； 梁勤；

【作者基本信息】 扬州大学 ， 特种经济动物饲养， 2013， 博士

【摘要】 狄斯瓦螨(Varroa destructor)是对蜜蜂威胁最大的病虫害,几乎对全世界范围内的养蜂业都造成了巨大的损失。本研究基于课题组前期研究所获得的宝贵的蜂螨敏感型群体,采用Illumina Solexa测序技术及iTRAQ技术初步筛选出蜂螨抗性相关的候选基因,通过荧光定量PCR技术验证候选差异基因,并对相关基因编码区序列测定分析。本研究为进一步研究蜜蜂抗螨分子机理奠定基础,同时筛选出来的差异基因可为蜜蜂抗螨育种研究提供基因资源。主要研究结果如下：1.本研究选择狄斯瓦螨感染的西方蜜蜂巢脾对抗性群体及敏感性东方蜜蜂群体进行胁迫,采用以下措施降低各群体间的遗传背景：a)受蜂螨胁迫的抗性群体(C+)和受胁迫的敏感性群体(M+)所接受的西方蜜蜂巢脾受狄斯瓦螨蜂螨感染程度相当且具有数量相当的工蜂和雄蜂的封盖蜂房；抗性对照组(C)及敏感性对照组(M)使用未受瓦螨感染的西方蜜蜂巢脾。b)采用10对微卫星引物选择全同胞姐妹用于下一步实验。c)对蜂群受瓦螨胁迫的情况进行连续观察,发现敏感性群体抗螨能力较差,而抗性群体则在遭到蜂螨胁迫后24h时抗螨行为达到高峰,因此确定胁迫24h后进行样本采集。2.采用Illumina Solexa转录组测序技术对蜂螨胁迫及未遭蜂螨胁迫的敏感性和抗性东方蜜蜂群体进行哺育蜂头部转录组测序,从头组装获得了91,172个unigenes,对这些unigenes进行功能注释。COG分类中,共有23,790条unigene归类到25类基因功能。GO分类注释了85,577条unigene的生物学途径,其中参与细胞生理过程(14,797)和代谢过程(10,17)的最多；注释了48,194条unigene的细胞学组件,其中细胞及细胞组分最多,都为10,323条；描述了29,301条unigene的分子功能,其中结合功能(11,370)和催化活性(11,042)的最多。39,625条在KEGG数据库中注释到257条通路,定位到代谢通路的unigene数量最多(4793)。3.C+VS M+共产生了40,255条差异unigene,其中19,702条在C+下调表达,20,553条在C+上调表达。GO显著富集分析表明蜂螨抗性及敏感性东方蜜蜂在受到蜂螨胁迫时体内发生了不同的应答反应,而这些应答反应很可能伴随转录因子的变化。多条显著富集的pathway涉及病菌感染及抗菌物质的生物合成,说明敏感性蜂群和抗性蜂群对病菌的抵抗力存在差异。C VS M共有21,252条差异unigene,其中962条在C下调表达,20,290条在C上调表达。GO显著富集分析说明东方蜜蜂蜂螨抗性群体和敏感群体间存在免疫反应、肌肉发育、学习和记忆方面的差异。有71个DEGs被富集到了嗅觉转导通路中,说明可能两个群体间的嗅觉系统存在差异。C+VS C共有36,691条差异unigene,其中17,799条在C+下调表达,18,892条在C+上调表达。GO显著富集分析表明在蜂螨胁迫的情况下,东方蜜蜂产生了一定的应答反应,伴随的可能为转录因子的表达差异。多条显著富集的pathway涉及病菌感染及抗菌物质的生物合成,说明抗性群体在受到蜂螨胁迫时也同时受到了细菌的感染,同时通过自身免疫积极抵抗细菌。M+VS M共有24,508条差异unigene,其中21,479条在M+下调表达,3029条在M+上调表达。GO显著富集分析说明蜂螨敏感性蜂群在受到蜂螨胁迫时,其嗅觉功能发生了变化,可能形成了短期记忆,并伴随转录因子的表达差异。多条显著富集的pathway涉及细菌及病毒感染,只有O聚糖生物合成(0.49%)与免疫相关,说明敏感性群体对于细菌病毒的抵抗力低于抗性群体。4. Venn分析关注到的差异基因中,在C+中呈现极高表达的DEGss (Log2Ratio≥15)上调的270个,下调的8个,结合所有差异基因的GO及pathway显著富集分析结果进行进一步的分析,发现以下基因与抗螨相关：气味结合蛋白、肌钙蛋白、钙离子运输ATPase、转录因子、免疫相关基因、表皮蛋白、突触蛋白。5.以东方蜜蜂转录组数据作为参考序列,对四样本哺育蜂头部蛋白质进行iTRAQ定量分析,共鉴定到1532个蛋白质。GO分析显示与细胞组分相关的表达蛋白最多的是细胞和细胞组分相关蛋白,共占61.02%。结合功能包含的蛋白数量最多(45.30%)。大部分蛋白参与到基本的生物学过程,如细胞进程(20.01%)及代谢进程(19.68%)。pathway分析发现共有1503条蛋白注释到239个通路中,注释到新陈代谢通路的蛋白最多,这与转录组数据一致。6. C+VSM+共产生了72个差异蛋白,其中31个在C+下调表达,41个在C+上调表达。GO显著富集分析说明蜂螨抗性及敏感性东方蜜蜂在受到蜂螨胁迫时体内发生了不同的应答反应,这与转录组数据也是相符合的。差异蛋白显著富集的pathway为鞘脂类代谢。C VS M共有154个差异蛋白,其中82个在C下调表达,72个在C上调表达。GO显著富集分析说明蜂螨抗性东方蜜蜂和蜂螨敏感性东方蜜蜂的生命活力存在差异。差异蛋白显著富集于17个pathway,最为显著的是代谢途径。C+VS C共有202个差异蛋白,其中81个在C+下调表达,121个在C+上调表达。GO显著富集分析说明东方蜜蜂在受到蜂螨胁迫时体内发生了一系列的代谢产能反应及应答反应。差异蛋白显著富集于14个pathway,最为显著的为震颤性麻痹病。M+VS M共有161条差异unigene,其中94个在M+下调表达,67个在M+上调表达。GO显著富集分析说明蜂螨敏感性东方蜜蜂在受到蜂螨胁迫时,其生命活动受到了影响,并且体内发生了一定的应答反应。差异表达蛋白显著富集于25个pathway,最为显著的为震颤性麻痹病。7.韦恩分析及与转录组关联分析显示endocuticle structural glycoprotein SgAbd-2-like及obp13与抗螨相关。8.将基于参考基因得到的蛋白质鉴定结果和转录组结果进行关联,C VSM组中关联性(r)为0.3269；rM+VS M为0.0066；C+VS M+为0.0737；C+VS C为0.0774。结果表明两者间相关性较低。9.对嗅觉通路中的3个差异基因及其它与抗螨性状相关的差异基因共15个DEGss进行了qRT-PCR验证,结果显示其表达规律与转录组数据一致,说明转录组测序结果是可靠的。对obp17, obp18及mklb-1编码区进行了克隆测序,获得的obp17,obp18及mklb-1编码区序列长度分别为408bp,399bp,4887bp。将东方蜜蜂obp4, obp17, obpl8及mklb-1的氨基酸序列进行生物信息学分析,结果表明东方蜜蜂四个蛋白与西方蜜蜂高度同源,Obps都有信号肽,mklb-1没有信号肽,四个蛋白都没有跨膜区,都属于α型蛋白。更多还原

【Abstract】 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.更多还原