刺参弧菌抗性相关基因筛选与表达研究

In recent years，the frequent occurrence of various diseases had caused serious economic losses and become a great challenge in the sustainable development of the sea cucumber (Apostichopus japonicus) industry. Epidemiologic studies showed that vibrio was the most important pathogen for sea cucumber disease. Thus, the genetic mechanism analysis of disease resistance for vibrio in sea cucmber will fancinate the selective breeding of disease resistant sea cucumber and support the healthy aquaculture and high production for sea cucumber industry. In this study, gene expression profiles of coelomic fluid from sea cucumber seeds with different anti-vibrio splendidus ability will be constructed using high throughput transcriptome sequencing and compared with each other in order to find disease resistance related genes using bioinformatics analysis. Then disease resistance related genes would be selected through analyzing the relationship between gene differential expression and its association with disease resistance ability. The result would promote marker-assisted selection technlogy and propel variety breeding in sea cucumber.

近年来，病害问题已成为制约仿刺参产业健康发展的瓶颈因子，流行病学研究结果表明弧菌是仿刺参病害的重要致病原，解析仿刺参弧菌抗性相关分子机理可为实施抗病育种筛选和培育抗病新品系，保障仿刺参健康养殖和高效产出提供科学依据。本项目拟从转录基因组学角度出发，以仿刺参家系为实验材料，以其非特异性免疫载体- - 体腔液为研究对象，采用高通量转录组测序技术，构建灿烂弧菌感染前后抗病与易感刺参苗种体腔液的基因表达谱，利用生物信息学分析方法，筛选参与响应细菌侵染过程的差异表达基因，验证基因表达与抗病力的相关性，解析刺参抗病分子机理，以期为发展仿刺参分子标记辅助育种技术、培育优良的仿刺参抗病品种提供理论依据和技术支撑。

为解析仿刺参弧菌抗性相关分子机理，本研究在建立了灿烂弧菌定性定量检测技术的基础上，构建了仿刺参体腔液细胞转录组文库，完成了仿刺参体腔液转录组学分析和不同抗病力仿刺参体腔液基因表达谱比较分析，筛选到仿刺参参与抗灿烂弧菌侵染的抗病基因及易感基因，并完成了相应基因差异表达与仿刺参抗病力的相关性分析。相应研究结果可为认知仿刺参抗病机理、培育仿刺参抗病品种提供理论依据和技术支撑。建立了灿烂弧菌的原位杂交技术、实时定量PCR检测技术和多种致病菌的多重PCR检测技术，可以完成灿烂弧菌的定性定量检测；刺参体腔液转录组共获得3.2亿条reads，拼接后获得89891个unigene，从转录组中筛选到8009个SSR位点和149745个SNP位点；获得了灿烂弧菌侵染前后、不同抗病力仿刺参体腔液的基因表达谱，按照P<0.01以及foldchange在2倍以上的显著性差异表达基因，筛选出抗病候选基因358个（其中13个上调基因，345个下调基因）和发病候选基因102个（86个上调基因及16个下调基因）；根据基因的注释信息及参与的信号通路，共筛选了30个潜在的抗性基因和19个易感基因。信号通路分析表明这些差异基因参与到5个免疫信号通路，包括溶酶体途径、内吞作用途径、趋化因子途径、丝分裂原蛋白激酶信号通路途径和表皮生长因子信号通路途径；利用实时定量PCR方法验证了所筛选的24个抗病基因和15个易感基因差异表达与抗病力相关性，结果表明，定量检测结果与高通量分析结果的一致率为94.87%。