摘要
【目的】通过RNA seq技术对纯培养的蜜蜂球囊菌Ascosphaera apis孢子和球囊菌侵染的蜜蜂幼虫肠道组织进行测序,de novo组装球囊菌的参考转录组,并对其进行功能与代谢通路注释,进而基于该转录组数据开发球囊菌的SSR分子标记。【方法】首先通过差速离心获得活化的球囊菌孢子,配制含1×107孢子/m L的饲料饲喂4,5和6日龄的意大利蜜蜂Apis mellifera ligustica幼虫和中华蜜蜂Apis cerana cerana幼虫,通过Illumina Hi SeqTM2500平台同时对上述蜜蜂幼虫肠道及纯化球囊菌孢子进行深度测序,原始数据过滤后通过Trinity软件组装得到unigenes,进而通过BLASTX比对NCBI Nr,Swiss-Prot,KOG和KEGG数据库对unigenes进行功能与代谢通路注释。利用MISA软件对所有unigenes进行SSR搜索,并利用Primer Premier 5软件设计SSR特异性引物,通过PCR对不同来源的球囊菌SSR位点进行扩增。【结果】本研究共获得146 135 308条高质量reads,de novo组装得到42 609个unigenes。BLASTX比对结果显示,29 316个unigenes在上述公共数据库中具有功能和代谢通路注释。注释到法夫酵母Xanthophyllomyces dendrorhous上的unigenes最多,达6 050个。KEGG注释结果显示,unigenes可注释到117个代谢通路,其中富集在核糖体(ribosome)上的unigenes数量最多(529)。所有unigenes中共预测到7 968个SSRs,通过PCR开发出5个球囊菌SSR分子标记。【结论】本研究成功组装球囊菌的参考转录组,并进行了功能与代谢通路注释,可为在分子水平深入研究球囊菌提供重要的参考信息。基于该转录组信息开发的5个SSR分子标记可推动菌株鉴定、基因图谱构建及基因定位等研究。
【Aim 】This study aims to deeply sequence the purified spores of Ascosphaera apis and honeybee larval gut infected with A. apis using RNA seq technology,to de novo assemble a reference transcriptome of A. apis,to further conduct functional and metabolic pathway annotation,and finally to develop SSR markers for A. apis. 【Methods】A. apis spores purified after differential centrifugation werefed to the 4-,5-and 6-day-old larvae of Apis mellifera ligustica and A. cerana cerana at a concentration of1 × 107 spore / m L. The purified spores of A. apis and the infected honeybee larval guts were sequenced at Illumina Hi SeqTM2500 platform. After filtration of raw reads,unigenes were de novo assembled with Trinity software and annotated through BLASTX against the NCBI Nr,Swiss-Prot,KOG and KEGG databases. The MISA software was used to search SSRs in all unigenes,and Primer Premier 5 software was used to design specific primers for SSR loci. PCR was carried out to amplify SSR loci in A. apis from different regions in China. 【Results 】 In this study,a total of 146 135 308 high-quality reads were produced from RNA seq,and 42 609 unigenes were de novo assembled. BLASTX results indicated that29 316 unigenes have function or metabolic pathway annotations in the aforementioned public databases.And the most unigenes（ 6 050） were annotated to Xanthophyllomyces dendrorhous. The results of KEGG pathway annotation revealed that all unigenes were annotated to 117 metabolic pathways,among which ribosome（ 529 unigenes） was the most enriched one. In total,7 968 SSRs were predicted from all unigenes,and five SSR markers were developed by PCR amplification. 【Conclusion】In this study,a reference transcriptome of A. apis was successfully de novo assembled and annotated,providing significant information for further studying A. apis at the molecular level. Five SSRs developed in this study can facilitate the research on A. apis including strain identification, gene map construction and gene localization.
出处
《昆虫学报》
CAS
CSCD
北大核心
2017年第1期34-44,共11页
Acta Entomologica Sinica
基金
现代农业产业技术体系建设专项资金(CARS-45-KXJ7)
福建农林大学科技发展资金(KF2015123)
福建省教育厅科技项目(JA10104)
福建省大学生创新创业训练计划项目(201610389053)
