黑须污蝇不同发育阶段转录组及嗅觉相关基因分析

doi:10.16380/j.kcxb.2019.06.004

摘要/Abstract

摘要：

【目的】建立黑须污蝇Wohlfahrtia magnifica转录组数据库，挖掘黑须污蝇基因数据，为更深入地研究双峰驼阴道蝇蛆病提供理论依据。【方法】采用高通量测序平台Illumina Hiseq^TM4000对黑须污蝇幼虫、蛹和成虫进行转录组测序，并进行生物信息学分析。利用黑须污蝇幼虫、蛹和成虫转录组数据，通过基因差异表达分析以及GO功能显著性富集分析的方法筛选出嗅觉相关基因，并通过荧光定量PCR技术对黑须污蝇幼虫、蛹和成虫中OBP99b, OBP56a和OBP99a 3个嗅觉相关基因表达水平进行验证。【结果】结果显示，每个黑须污蝇样本的转录组数据量在4.96 Gb以上，G+C含量在35.35%以上，Q20含量在97%以上。与NCBI_nr, GO, KEGG, Pfam, Swiss-Prot和eggNOG数据库进行对比，共注释到73 303条unigenes。其中eggNOG数据库注释到的unigenes最多，为35 066条，分布在23个蛋白功能中，其中的翻译修饰、蛋白质周转的unigenes所占比例较大；GO数据库注释到的unigenes数为29 193条，功能包括分子功能、细胞组分、生物学过程三大类50个分支，其中参与生物学过程和氧化还原过程的unigenes比例较大；KEGG数据库注释到的unigenes数为15 068条，其中参与信号转导过程的unigenes比例较大。上述数据表明该转录组测序结果较好，为后续研究奠定了基础。依据黑须污蝇幼虫、蛹、成虫转录组数据筛选到30个嗅觉相关基因，其中包含9个气味结合蛋白(OBP)基因，进一步基因注释发现OBP99b, OBP56a和OBP99a基因在黑须污蝇不同发育阶段表达差异显著(|log₂FC|>1, P<0.05)，这在荧光定量PCR分析结果中得到了验证。【结论】本研究获得了黑须污蝇幼虫、蛹和成虫转录组数据，筛选出黑须污蝇各发育阶段嗅觉相关基因，并验证了OBP99b, OBP56a和OBP99a基因在黑须污蝇幼虫、蛹和成虫3个发育阶段差异表达，为防治双峰驼阴道蝇蛆病提供了新思路。

关键词: , 黑须污蝇, 转录组, 高通量测序, 生物信息学分析, 荧光定量PCR

Abstract: 【Aim】 To establish the transcriptome database of the spotted flesh fly, Wohlfahrtia magnifica, and to explore its genetic data, so as to provide a foundation for further studying the bactrian vaginal fly plague. 【Methods】 The transcriptomes of the larva, pupa and adult of W. magnifica were sequenced by a high-throughput sequencing platform (Illumina Hiseq^TM4000) and subjected to bioinformatics analysis. Olfaction-related genes were screened by differential gene expression analysis and GO function significant enrichment analysis based on the transcriptome data of the larva, pupa and adult of W. magnifica. The expression levels of three olfaction-related genes (OBP99b, OBP56a and OBP99a) in the larva, pupa and adult of W.magnifica were verified by qPCR. 【Results】 The results showed that each sample of W. magnifica generated the transcriptome data of above 4.96 Gb, in which the G+C content was above 35.35%, and the Q20 content was above 97%. A total of 73 303 unigenes could be annotated in NCBI_nr, GO, KEGG, Pfam, Swiss-Prot and eggNOG databases. Among them, the largest number of unigenes (35 066) were annotated in the eggNOG database, and involved in 23 protein functions, among which the unigenes involved in the translation modification and protein turnover accounted for a larger proportion. A total of 29 193 unigenes were annotated in the GO database, and involved in 50 branches of molecular function, cellular component and biological process, with a higher proportion of unigenes involved in biological process and oxidation-reduction process. There were 15 068 unigenes annotated in the KEGG database, with higher proportion of unigenes involved in the signal transduction process. The above data indicated that the transcriptome sequencing result was good, laying a foundation for the follow-up experiment. Based on the transcriptome data of the larva, pupa and adult of W. magnifica, 30 olfaction-related genes including 9 odor-binding protein (OBP) genes were screened. Further gene annotation revealed that OBP99b, OBP56a, and OBP99a showed differential expression (|log₂FC|>1, P<0.05) in different developmental stages of W. magnifica, which was verified by the results of fluorescence quantitative PCR. 【Conclusion】 In this study, the transcriptome data of larva, pupa and adult of W. magnifica were obtained, the olfaction-related genes at different developmental stages of W. magnifica were screened, and the differential expression of OBP99b, OBP56a and OBP99a in the three developmental stages of larva, pupa and adult of W. magnifica was verified. These results provide a new idea for preventing and treating the vaginosis of the bactrian alpaca.

Key words: Wohlfahrtia magnifica, transcriptome, high-throughput sequencing, bioinformatics analysis, fluorescence quantitative PCR

王超, 包花尔, 乌云高娃, 崔阿拉腾乌拉, 巴音吉日嘎拉, 额尔敦木图. 黑须污蝇不同发育阶段转录组及嗅觉相关基因分析[J]. 昆虫学报, 2019, 62(6): 685-693.

WANG Chao, BAO Hua-Er, WUYUN Gaowa, CUI Altenwula, BAYIN Jiragara, ERDEMTU. Analysis of the transcriptomes and olfaction-related genes of Wohlfahrtia magnifica (Diptera: Sarcophagidae) at different developmental stages[J]. Acta Entomologica Sinica, 2019, 62(6): 685-693.

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黑须污蝇不同发育阶段转录组及嗅觉相关基因分析

Analysis of the transcriptomes and olfaction-related genes of Wohlfahrtia magnifica (Diptera: Sarcophagidae) at different developmental stages

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