中华蜜蜂幼虫肠道参考转录组的de novo组装及SSR分子标记鉴定

doi:10.3864/j.issn.0578-1752.2017.06.016

摘要/Abstract

摘要： 【目的】利用RNA seq技术对中华蜜蜂（Apis cerana cerana，简称中蜂）幼虫肠道参考转录组进行de novo组装，并进行功能及代谢通路注释，进而利用该转录组数据进行中蜂幼虫的SSR分子标记鉴定。【方法】实验室条件下饲养中蜂幼虫，将纯化的蜜蜂球囊菌（Ascosphaera apis，简称球囊菌）孢子饲喂3日龄幼虫，剖取4、5和6日龄幼虫肠道，液氮速冻。将健康幼虫肠道与感染球囊菌的幼虫肠道同时进行Illumina测序。通过对raw reads的过滤得到clean reads，利用Trinity软件组装得到unigenes。通过BLASTx（E-value＜10−5）比对NCBI Nr、Swiss-Prot、KOG和KEGG数据库，对unigenes进行功能和代谢通路注释。利用MISA软件对所有unigenes进行SSR搜索，并利用Primer Premier 5软件设计特异性SSR引物，通过常规PCR对来源于北京、辽宁兴城和四川成都的中蜂幼虫肠道样本进行SSR位点鉴定。【结果】中蜂幼虫肠道的RNA seq共得到35 670 000条reads，de novo组装得到43 557个unigenes，平均长度为898 nt。共有18 225个unigenes可被注释到上述公共蛋白数据库，单独注释到NCBI Nr、Swiss-Prot、KOG和KEGG数据库的unigenes数分别为3 899、443、37和10个。KOG注释结果显示，11 442条unigenes分布于25个基因家族，其中注释到RNA加工和修饰家族的基因数最多，达1 249个。9 679个unigenes的GO分类结果显示，在生物学进程分类中，注释到细胞进程的基因最多，达4 201个，在分子功能和细胞组分类中，注释到结合与细胞的基因数最多，分别为4 935和2 900个。4 517个unigenes可注释到KEGG数据库中的216个代谢通路，注释到核糖体的基因数最多，达385个。利用MISA软件，可在7 763个unigenes搜索到13 448个SSR位点，随机选取20对SSR引物对国内3个不同来源的中蜂幼虫肠道样本的SSR位点进行扩增，有6对引物可鉴定出SSR分子标记。【结论】成功组装并注释了中蜂幼虫肠道参考转录组，可为中蜂及其幼虫的分子生物学及组学研究提供重要的参考信息，也可用于补充、丰富和检验东方蜜蜂的参考基因组，基于此转录组数据开发出6个中蜂的SSR分子标记，可应用于中蜂的基因图谱构建、基因多样性分析、基因定位等研究，也说明利用转录组数据开发非模式生物SSRs的方法可行。

关键词: RNA seq, 参考转录组, 中华蜜蜂, unigene, SSR

Abstract: 【Objective】 The objective of this study is to de novo assemble a reference transcriptome for Apis cerana cerana larval gut, perform gene function and pathway annotation for this transcriptome, and to identify specific SSR molecular markers for A. c. cerana larvae. 【Method】 3-day-old instar A. c. cerana larvae were fed with the purified Ascosphaera apis spores, the guts of 4-, 5- or 6-day-old honeybee larvae were sampled and used as sequencing material for RNA seq. After filtration, clean reads were obtained, and unigenes were assembled using Trinity software. BLASTX tool (E-value＜10⁻⁵) was used to search the unigenes against NCBI Nr, Swiss-Prot protein, KOG as well as KEGG databases to perform gene function and pathway annotation. MISA software was used to search microsatellite markers in the larval gut’s transcriptome. The specific primers of all SSRs were designed using Primer Premier 5 program and several pairs were used to amplify SSR loci in A. c. cerana larvae samples from 3 different regions (Beijing, Xingcheng, and Chengdu) in China by method of PCR. 【Result】 In this study, RNA seq produced 35 670 000 high quality reads, which were assembled into 43 557 unigenes with a mean length of 898 nt. 18 225 unigenes were annotated in the public protein databases. a total of 11 442 unigenes had a KOG classification and they distributed in 25 KOG categories, among them, RNA processing and modification was the largest group (1 249). 9 679 unigenes could be classified into three gene ontology (GO) categories, in which the mostly enriched ones were cellular process (4 201 unigenes), cell (2 900 unigenes) and binding (4 935 unigenes). 4 517 unigenes were annotated to 216 KEGG pathways, among them, ribosome (385 unigenes) was the largest. Finally, 13 448 SSRs were found in 7 763 unigenes, and 6 out 20 SSR loci could be successfully amplified in A. c. cerana larvae samples from 3 different regions in China using PCR. 【Conclusion】 This study assembled and annotated a reference transcriptome for A. c. cerana larval gut, which will provide a key information not only to studies on eastern honeybee and its larvae such as molecular biology and omics, but also to improve and validate the genome of A. cerana. SSR markers developed here could be applied to future investigation of A. c. cerana including gene map construction, genetic diversity analysis as well as gene location. Meanwhile, this study suggested that developing molecular markers using transcriptome data of non-model organism is a rapid and efficient method.

Key words: RNA seq; de novo assembly, Apis cerana cerana, unigenes, SSR

徐细建，郭睿，骆群，熊翠玲，梁勤，张串联，郑燕珍，张曌楠，黄枳腱，张璐，李汶东，陈大福. 中华蜜蜂幼虫肠道参考转录组的de novo组装及SSR分子标记鉴定[J]. 中国农业科学, 2017, 50(6): 1157-1166.

XU XiJian, GUO Rui, LUO Qun, XIONG CuiLing, LIANG Qin, ZHANG ChuanLian, ZHENG YanZhen, ZHANG ZhaoNan, HUANG ZhiJian, ZHANG Lu, LI WenDong, CHEN DaFu. De novo Transcriptome Assembly for Apis cerana cerana Larval Gut and Identification of SSR Molecular Markers[J]. Scientia Agricultura Sinica, 2017, 50(6): 1157-1166.

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