幼虫、青年和成年日本医蛭不同生长阶段差异表达基因比较分析

Comparison on differentially expressed genes in larval,juvenile and adult Hirudo nipponia at different growth stages

【目的】筛选出调控日本医蛭生长发育的相关基因,揭示其生长发育、繁殖和抗凝分子机制,为后期开展日本医蛭养殖、生长发育及相关功能基因研究提供科学依据。【方法】以幼虫期(孵化10 d以内,HNL)、青年虫期(孵化后2个月左右,HNY)和成年虫期(孵化后2年左右,HNA)的日本医蛭为研究对象,利用Illumina HiSeq 4000平台对构建的cDNA文库进行转录组测序,按错误发现率(FDR)<0.05和|log2FC|>1的原则筛选差异表达基因(DEGs),并进行GO功能注释分析和KEGG信号通路富集分析。【结果】使用3个时期的样本构建24个cDNA文库,共产生541124257条原始序列(Raw reads),经Fastp质量控制及RNA-Seq组装,共获得20430个mRNAs转录本,并注释出88个新的mRNAs转录本。通过对日本医蛭不同生长阶段进行两两对比,共获得1348个差异表达基因,其中,在幼虫期—青年虫期(HNL vs HNY)中获得238个差异表达基因,在幼虫期—成虫期(HNL vs HNA)中获得976个差异表达基因,在青年虫期—成虫期(HNY vs HNA)中获得763个差异表达基因。幼虫期—成虫期的差异表达基因主要富集蛋白水解、肌球蛋白复合体、细胞骨架、几丁质代谢过程及对伤害的反应等GO功能条目上,以及苯丙氨酸/酪氨酸/色氨酸生物合成、丙烯酰胺转移酶等KEGG信号通路上。说明日本医蛭幼虫在生长过程中大量摄食并通过消化、代谢和合成物质为自身发育提供能量,而成年日本医蛭发育成熟后与其生长发育相关的代谢过程下调,且已适应环境,对于环境刺激的敏感度也有所降低。【结论】在幼虫期—成虫期(HNL vs HNA)获得的976个差异表达基因中,上调差异表达基因主要与肌肉发育相关,而与性腺发育或产卵调节及神经发育相关的差异表达基因具有明显周期特异性,其表达变化均与日本医蛭的生长发育有关。水蛭素和抗凝相关基因在青年期高表达,因此开展水蛭抗凝血能力研究时宜选用青年日本医蛭。

【Objective】In order to identify the genes that regulate the growth and development of the Hirudo nipponia,and to reveal the molecular mechanisms of growth and development,reproduction and anticoagulation,so as to provide a theoretical basis for the breeding,growth and development,and studies on related functional genes of H. nipponia.【Method】H. nipponia were studied at the larval stage(within 10 d of hatching,HNL),young stage(about 2 months after hatching,HNY)and adult stage(about 2 years after hatching,HNA). Using the Illumina HiSeq 4000 platform to transcriptome sequencing of cDNA library. Differentially expressed genes(DEGs)were screened by false discovery rate(FDR)<0.05 and |log2FC|>1,and then to perform GO functional annotation and KEGG signal pathway enrichment analysis.【Result】A total of 541124257 raw reads were generated from 24 cDNA libraries using samples from three stages.There were 20430 mRNAs transcripts were obtained by Fastp quality control and RNA-Seq assembly and 88 new mRNAs transcripts were annotated. A total of 1348 DEGs were obtained by two-by-two comparison of different growth stages of the H. nipponia,including 238 DEGs in HNL vs HNY,976 DEGs in HNL vs HNA,and 763 DEGs in HNY vs HNA.DEGs of HNL vs HNA were mainly enriched in GO functional items such as proteolysis,myosin complex,cytoskeleton,chitin metabolism and response to injury,phenylalanine/tyrosine/tryptophan biosynthesis,acrylamide transferase and other KEGG signal pathways. These changes revealed that larvae fed a lot during growth to provide energy for their own development through digestion,metabolism and synthetic of substances. After adult H. nipponia grew and matured,metabolic processes related to their growth and development were down-regulated. Moreover,adult H. nipponia have adapted to the environment,and their sensitivity to environmental stimuli was also reduced.【Conclusion】Among the 976 DEGs obtained from HNL vs HNA,the up-regulated DEGs are mainly related to muscle development,whereas the DEGs related to gonadal development,ovarian regulation and neurodevelopment are obviously periodic,and the expression changes of all these genes are related to the growth and development of H. nipponia. Hirudin and anticoagulation-related genes are highly expressed in young stages,so it is suitable to use young H. nipponia when performing studies on the anticoagulant ability of leeches.