Nrf2转录因子调控福寿螺夏眠期抗氧化防御的作用机理研究

Apple snails are world worst invasive pests. The adjustment of antioxidant defenses during their estivation is crucial for population outbreak of apple snails. However, there are few reports on molecular mechanisms of antioxidant defenses regulation. Previous studies showed that Nrf2 regulated several antioxidant enzymes, and it was considered as the most important transcriptional regulator. We have cloned the full-length of Nrf2 transcriptional regulator sequences, obtained the expression pattern of the hemolymph of apple snails, and established the cell culturing technique. In this study, we will build RNA interfered apple snail population on Nrf2 transcriptional regulator. We will detect mortality rate during estivation of the Nrf2 transcriptional regulator interfered population and activity and gene expression of related antioxidant enzymes, to explicit the regulation of Nrf2 transcriptional regulator on antioxidant enzymes. We will analyze the pathways of Nrf2 transcriptional regulator on antioxidant defenses based on RNA-seq. We will also elucidate the regulation effect of ROS on Nrf2 transcriptional regulator based on the correlational analyses between ROS and expression and transformation of Nrf2 transcriptional regulator. We all comprehensively analyze the network of Nrf2 transcriptional regulator effecting on antioxidant defenses of apples snails during estivation. Our expected findings will uncover the underlying mechanisms involved in Nrf2 regulating on antioxidant defenses of apples snails during estivation, and provide theoretic instruction on effective prevention and control strategy of apples snails.

福寿螺是世界恶性入侵生物，其夏眠期的抗氧化防御调整对种群暴发至关重要，但相关抗氧化防御调控分子机理的研究鲜有报道。研究显示，Nrf2转录因子作用于多种抗氧化酶，是参与抗氧化防御通路最核心的调控分子。项目组前期克隆了福寿螺Nrf2基因全长，获得了福寿螺夏眠不同时期血淋巴组织表达谱，并构建了细胞培养技术。在此基础上，本研究将建立Nrf2干扰的福寿螺种群，通过检测干扰种群夏眠期的死亡率、相关抗氧化酶活性及基因表达量，探明Nrf2转录因子对夏眠福寿螺相关抗氧化酶的调控作用；通过RNA-Seq分析Nrf2转录因子调控抗氧化防御的可能途径，并分析活性氧与Nrf2转录因子表达量及在胞内转移的相关性，明确活性氧对Nrf2的调节效应，综合分析Nrf2转录因子在夏眠福寿螺抗氧化防御中的作用网络。所得结果不仅可以阐明Nrf2转录因子调控夏眠福寿螺抗氧化防御的内在机制，还可为制定有效的福寿螺防控策略提供理论指导。

福寿螺是世界恶性入侵水生生物，其夏眠期间的抗氧化防御调整对种群扩散与暴发至关重要，但相关抗氧化防御调控分子机理的研究鲜有报道。本研究通过干旱胁迫诱导小管福寿螺夏眠，发现夏眠45天后死亡率为10%。夏眠期福寿螺腹足肌细胞失水，空隙变大，圆形肌细胞数量增多，觉醒20 min时纤维状梭形肌细胞增多，形状更细长，圆形肌细胞减少；肝胰腺在夏眠期管腔变窄，上皮细胞缩小且排列整齐，觉醒20 min时上皮细胞排列不规则、断裂，管腔狭窄。在福寿螺夏眠-觉醒周期中抗氧化酶SOD和CAT活性呈先下降后上升再下降的趋势，GST活性变化平缓。通过夏眠-觉醒周期中福寿螺腹足和肝胰腺的转录组分析，探明了差异表达基因的变化趋势，并通过GO和KEGG富集探明了差异表达基因所在的关键通路。明确了血红素加氧酶1（HO-1）是福寿螺夏眠-觉醒周期中Nrf2调控的关键抗氧化防御基因。克隆获得了小管福寿螺的Nrf2序列并进行原核表达，其CDS全长2253 bp，编码750个氨基酸，在肺和腹足中表达量较高；设计获得了福寿螺Nrf2基因的siRNA序列，RNAi干扰效率为61.77%，通过干扰Nrf2基因的转录组分析，探明了Nrf2调控的关键抗氧化防御、信号转导与代谢通路。进一步克隆获得了斑点福寿螺和隐秘福寿螺的Nrf2序列，发现三种福寿螺的Nrf2序列无差异；克隆获得了三种福寿螺的HO-1基因，分析预测了Nrf2转录因子与三种螺HO-1的结合位点，并进行Nrf2对HO-1的转录调控差异的分析。研究较深入探明了福寿螺Nrf2转录因子在调控夏眠时期抗氧化防御的作用及途径，揭示了Nrf2转录因子对夏眠福寿螺抗氧化防御的作用网络及机理，为科学的福寿螺防控奠定了理论基础。发表论文9篇，其中SCI论文7篇，中科院1区及TOP期刊论文4篇，授权发明专利2项。