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意大利蜜蜂细胞内吞相关基因与全长转录本鉴定
Identification of endocytosis-associated genes, and full-length transcripts, in Apis mellifera ligustica
范小雪,王思懿,朱乐冉,荆 欣,郭思佳,牛庆生,蒋海宾,徐细建,陈大福,付中民,徐国钧,郭 睿
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DOI:10.7679/j.issn.2095-1353.2023.076
作者单位:福建农林大学动物科学学院(蜂学学院),福州 350002;福建农林大学蜂疗研究所,福州 350002
中文关键词:意大利蜜蜂;细胞内吞;纳米孔测序;全长转录本;可变剪切
英文关键词:Apis mellifera ligustica; endocytosis; Nanopore sequencing; full-length transcript; alternative splicing
中文摘要:

【目的】 利用已获得的纳米孔(Nanopore)长读段测序数据对意大利蜜蜂Apis mellifera ligustica的细胞内吞相关基因和全长转录本进行鉴定、分析和验证,为深入开展相关研究提供参考和依据。【方法】通过Blast工具将意大利蜜蜂全长转录本比对NrKEGG数据库以筛选出细胞内吞相关基因和全长转录本。采用gffcompare软件将全长转录本与西方蜜蜂Apis mellifera参考基因组(Amel_HAv3.1)上注释的转录本进行比较以优化已注释基因的结构。使用Astalavista软件鉴定细胞内吞相关基因的可变剪接(Alternative splicing,AS)事件类型,利用IGV浏览器对剪切体结构进行可视化,通过PCR验证AS事件的真实性。利用TAPIS pipeline进行可变多聚腺苷酸化(Alternative polyadenylation,APA)位点预测和分析,并通过MEME软件鉴定APA位点上游的基序(motif)。【结果】 共鉴定到意大利蜜蜂细胞内吞相关的170个基因与991条转录本。共优化了89个细胞内吞相关基因的结构,其中5端延长的基因有36个,3端延长的基因有35个,5端和3′端同时延长的基因有18个。鉴定到细胞内吞相关的28个基因的666AS事件,其中最丰富的AS类型是3端可变剪接(Alternative 3' splice site,A3SS)。PCR结果证实了涉及整合素β-nu样异构体基因、RasGTP结合蛋白基因和rab11家族相互作用蛋白4基因的2种类型的4AS事件真实性。共鉴定到120个细胞内吞相关的基因含有1个及以上的APA位点,并在APA位点上游鉴定到多个motif,一致性序列为NBMNHHHBMNSNNCBNVSNNNNNNNNNNNVNNNN  【结论】 鉴定到意大利蜜蜂细胞内吞相关的170个基因及991条全长转录本,发现细胞内吞相关基因发生大量的AS事件并含有丰富的APA位点,优化了西方蜜蜂参考基因组上注释的细胞内吞相关基因的结构。

英文摘要:

[Objectives]  To identify, analyze and confirm the genes and full-length transcripts associated with endocytosis in Apis mellifera ligustica using previously obtained Nanopore long-read sequencing data, thereby providing a foundation for further functional study of these genes. [Methods]  All full-length transcripts available for Apis mellifera ligustica were Blast-searched in the Nr and KEGG databases to screen out endocytosis-related genes and full-length transcripts. Structures of annotated genes were optimized by using gffcompare software to compare endocytosis-associated full-length transcripts with annotated transcripts in the A. mellifera reference genome (Amel_HAv3.1). Types of AS events in genes related to endocytosis were identified using Astalavista software. Visualization of isoforms’ structure was performed with an IGV browser. The authenticity of AS events was verified using PCR. Prediction and investigation of APA sites were done using a TAPIS pipeline, followed by identification of motifs upstream of APA sites with MEME software. [Results]  In total, 170 genes and 991 full-length transcripts relevant to endocytosis were identified. Structures of 89 annotated genes were optimized, among these, 5' ends of 36 genes were prolonged, 3' ends of 35 genes were prolonged, and both 5' ends and 3' ends of 15 genes were prolonged. In addition, 666 AS events in 28 genes were identified, the most common of which was an alternative 3' splice site. PCR results validated the authenticity of four randomly selected AS events involving an integrin beta-nu-like isoform, ras-like GTP-binding protein and Ras-like GTP-binding protein. Moreover, 120 genes were identified containing one or more APA sites in endocytosis, the most abundant of which were genes containing three APA sites (14, 11.67%). Several motifs upstream of APA sites were observed, with the consensus sequence: NBMNHHHBMNSNNCBNVSNNNNNNNNNNNVNNNN. [Conclusion] 170 genes and 991 full-length transcripts associated with endocytosis in A. m. ligustica were identified. A number of AS events in endocytosis-related genes that also contained abundant APA sites were detected, and structures of endocytosis- related genes annotated in the reference genome of A. mellifera were optimized.

 

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