不同发育阶段斯氏副柔线虫比较转录组学分析

doi:10.3864/j.issn.0578-1752.2017.23.017

摘要/Abstract

摘要： 【目的】探明不同发育阶段骆驼斯氏副柔线虫的转录组差异，了解不同发育阶段虫体在功能分类和代谢通路等方面的生物学特征，挖掘生长发育相关功能基因，丰富寄生性线虫转录组学信息。【方法】采用Illumina HiSeq2000TM高通量测序技术对斯氏副柔线虫虫卵、第三期幼虫和雌虫进行转录组测序，构建3个样本的cDNA文库，评估建库质量；利用Trinity软件对所得序列进行De novo组装及组装效率评估；之后对获得的有效序列进行功能注释及相关生物信息学分析。【结果】测序及组装后虫卵、第三期幼虫和雌虫分别获得47 717、76 342和54 624个Unigenes，在虫卵和第三期幼虫比对中共有33 579个差异表达基因，其中表达上调的基因有20 477个，表达下调的基因有13 102个；在雌虫和第三期幼虫比对中共有32 199个差异表达基因，其中表达上调的基因有9 293个，表达下调的基因有22 906个。将成对比较的差异表达基因分别进行GO功能分类，其中虫卵和第三期幼虫比对中有6 617、3 891和8 755个Unigenes，雌虫和第三期幼虫比对中有7 043、3 686和10 177个Unigenes分别注释到生物过程、细胞组成和分子功能三大类中；通过KEGG pathway数据库分析，虫卵和第三期幼虫比对中有6 521个差异表达基因参与到251条通路中，显著富集MAPK信号通路、Wnt信号通路和氧化磷酸化等通路，雌虫和第三期幼虫比对中有6 528个参与到251条通路中，显著富集新陈代谢通路、DNA复制和细胞周期等通路。差异表达基因功能聚类分析中，雌虫和虫卵在调控生长速率和生殖发育等方面高表达；第三期幼虫在防御机制和糖类代谢等方面高表达；且3个发育阶段均在胚胎发育，胚后发育中富集高表达，其中在胚胎发育和胚后发育中分别有242和202个相关功能基因在3个发育阶段都表达，这些基因可能在胚胎（后）发育过程中起核心作用。此外，获得了9种异时性基因（如：LIN-28、LIN-14和RHEB-1等）、48个核激素受体（NHRs），包括NHR-49，NHR-48，NHR-40，NHR-1等以及36个锌金属蛋白酶（NAS），包括NAS-36、NAS-33和NAS-14等，并对其在斯氏副柔线虫虫卵、第三期幼虫和雌虫中的富集程度进行分析，发现这些基因在维持线虫正常生长发育过程中发挥关键作用，【结论】利用RNA-seq技术对3个发育阶段的斯氏副柔线虫进行测序和生物信息学分析，研究了不同发育阶段虫体的差异表达基因在GO功能分类、KEGG代谢通路和基因功能聚类等方面的生物学特性，筛选出多种异时性基因和发育相关重要基因，为后续开展斯氏副柔线虫功能基因组学研究，虫体与宿主互作、致病机制、免疫逃避等研究提供了理论依据。

关键词: 斯氏副柔线虫, 转录组, 差异表达基因, 发育相关基因

Abstract: 【Objective】 The objective of this study was to identify the differentially expressed genes(DEG) and describe biological characteristics involved in functional classifications and metabolic pathways at different developmental stages of Parabronema skrjabini infecting camel, which is necessary to better understand functional genes involved in growth and development and enrich the transcriptome data of parasitical nematodes.【Method】Eggs, the third-stage larvae(L3s) and females of Parabronema skrjabini were sequenced by Illumina HiSeq2000^TM sequencing platform and constructed their cDNA libraries after quality filtering. De novo assembling and assembly efficiency assessment were carried out using Trinity, a short-read assembly program. Then all the effective sequential data obtained were assigned to the relevant databases to perform functional annotation and bioinformatic analysis.【Result】The results showed that 47 717, 76 342 and 54 624 unigenes were obtained respectively in eggs，the third-stage larvae and female stages. 33 579 differentially expressed genes(DEGs) were identified by comparing the unigenes obtained from eggs and L3s stages, of which 20 477 were up-regulated and 13 102 were down-regulated. There were 32 199 differentially expressed genes between L3s and female stages, of these genes, 9 293 were up-regulated genes and 22 906 were down-regulated genes. The differentially expressed genes of two pairwise comparisons were respectively enriched in Gene Ontology. 6 617, 3 891 and 8 755 differentially expressed genes comparing eggs and L3s stages were annotated into database of biological process, cellular component and molecular function respectively, while the number by comparing L3s and female stages were 7 043, 3 686 and 10 177 respectively. In KEGG pathways identification, 6 521 differentially expressed genes comparing eggs and L3s stages were assigned to 251 KEGG pathways, and clustered significantly in MARK, Wnt signaling pathways and oxidative phosphorylation. In comparison of L3s and female stages, 6 528 differentially expressed genes were enriched in metabolic pathways, DNA replication and cell cycle. Functional cluster analysis indicated that the regulation related differential genes of growth rate and the reproductive and genital development were highly expressed in eggs and females stages, then the genes of the defense and carbohydrate metabolism were enriched in exclusive L3s stage. Differential genes of embryonic and post-embryonic development were highly expressed in all three stages, and 196 function genes from embryonic development and 166 function genes from post-embryonic development were co-expressed in all three stages, implied that these genes played a crucial role in (post-)embryonic development. Moreover, we identified 9 heterochronic genes, such as LIN-28, LIN-14 and RHEB-1, 48 nuclear hormone receptors (NHRs) genes, including NHR-49, NHR-48, NHR-40 and NHR-1, and 36 zinc metalloproteinase(NAS) genes, including NAS-36, NAS-33 and NAS-14. Then the analysis of enrichment capacity in three stages showed these genes were necessary to regulate the different development stages of Parabronema skrjabini. 【Conclusion】The transcriptomic research of Parabronema skrjabini at three developmental stages using RNA-seq revealed biological characteristics of differentially expressed genes involved in development- related GO functional classification, KEGG pathway and functional cluster and identified many kinds of heterochronic genes and developmental genes, which provides a foundation and reference for further investigation of the whole genome sequence analysis, interaction between Parabronema skrjabini and host, pathogenic mechanism and immune evasion.

Key words: Parabronema skrjabini, transcriptome, differentially expressed genes, development-related genes

王文龙，冯陈晨，红梅，岳建伟，呼和巴特尔，刘春霞. 不同发育阶段斯氏副柔线虫比较转录组学分析[J]. 中国农业科学, 2017, 50(23): 4644-4655.

WANG WenLong, FENG ChenChen, HONG Mei, YUE JianWei, Huhebateer, LIU ChunXia. The Comparative Transcriptome Analysis of Parabronema skrjabini at Different Developmental Stages[J]. Scientia Agricultura Sinica, 2017, 50(23): 4644-4655.

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