重金属镉影响亚洲玉米螟性信息素调控生殖行为的分子机理研究

Cadmium (Cd) is a heavy metal that is seriously harmful to animals and human health. The animal reproductive system is very sensitive to cadmium. Cadmium and other heavy metals have been reported on large animal reproductive systems such as livestock, poultry and mice. However, it is unclear that the molecular mechanism of heavy metal cadmium influenced the reproductive behaviors regulated by the sex pheromones. The Asian corn borer, Ostrinia furnacalis, is a model animal for the studies of insect pheromones. The insect is easy to raise, its reproductive behavior is highly modeled, the pheromone composition is clear, and the chemical communication behaviors are very sensitive to the external stimulus response. In this study based on the Asian corn borer as the research object, its full-length mRNA transcripts will be obtained with the third-generation full-length transcriptome (PacBio) data, and the reference gene sequence of pheromone activating neuropeptide (PBAN) and its receptor (PBANR) related to sex pheromone biosynthesis will be obtained by gene annotation and will be cloned. RNAi technique will be used to verify the function of the cloned sequence, and qRT-PCR will be used to determine the expression of PBAN and PBANR in different tissues and organs of the Asian corn borer. The effects of the heavy metal cadmium on the biosynthesis and release of sex pheromone regulated by PBAN and PBANR and its behavioral responses will be investigated by correlation analysis of digital gene expression tag profiling and RNA-Seq. It will be elucidated the mechanism of effects of heavy metal cadmium stress on the reproductive behavior regulated by the sex pheromones in the Asian corn borers, and to provide a reference for studying the molecular mechanism of heavy metals affecting reproductive behavior and survival adaptability in other animals.

重金属镉对动物健康危害严重，动物生殖系统对镉非常敏感。有关镉对动物生殖系统的研究已有报道，但尚不清楚镉影响由性信息素调控动物生殖行为的分子机理。亚洲玉米螟是研究性信息素的模式昆虫，易于饲养，其生殖行为模式化程度高，信息素组份明确，化学通讯行为对外界刺激反应的灵敏度高。本项目以亚洲玉米螟为研究对象，借助第三代全长转录组（PacBio）测序获得其mRNA全长转录本，利用基因注释获得该虫信息素激活神经肽(PBAN)及其受体(PBANR)的参考序列，并进行克隆；运用RNAi技术对克隆所得序列进行功能验证，通过qRT-PCR技术测定PBAN及PBANR在不同处理中的表达量；进而采用RNA-Seq数字基因表达谱关联分析研究镉影响PBAN及PBANR调控其信息素生物合成与释放的分子机理，从而阐明镉胁迫条件下亚洲玉米螟生殖行为调控的内在机制，可为镉影响其他动物生殖行为变化和生存适应能力的分子机制提供参考。

镉(Cd)是一种对动物健康危害严重的重金属，在镉连续胁迫下亚洲玉米螟Ostrinia furnacalis 的性信息素通讯行为受到了抑制。为探明其分子机理，通过PacBio测序及数据校正，获得亚洲玉米螟39,045个转录本及其信息素生物合成激活神经肽(OfurPBAN)及其受体(OfurPBANR)基因。其中OfurPBAN基因编码性信息素生物合成激活肽、滞育激素和α、β、r-SGNP等3种神经肽，是一类以FXPRL-NH2结尾的酰胺肽家族；而OfurPBANR的开放阅读框为1089bp的核苷酸序列，编码了362个氨基酸，具有7个跨膜结构，在信息素腺中表达最高。.在土壤-作物茎杆-蛀茎螟虫的系统中，作物茎杆的镉含量与土壤镉含量、蛀茎螟虫体内镉残留量与作物茎杆镉含量均呈正相关关系，虫体内残留镉含量随茎杆镉含量增加而增加。受Cd胁迫昆虫的交配行为和繁殖力受到抑制，亚洲玉米螟的性信息素滴度显著降低。重金属镉低浓度（5mg/kg）与对照（无重金属）处理下，其体内PBAN的表达与对照组差异不显著，而在高浓度（20mg/kg）胁迫下PBANR出现显著性差异，表明高浓度Cd能刺激OfurPBANR的过表达。.为探析镉影响亚洲玉米螟PBAN及PBANR的分子机理，通过基因表达谱分析以及差异基因筛选，发现Cd处理组和对照组共产生123个差异基因，仅1个下调，其余均上调，其中有3个P450基因、2个锌脂蛋白基因、3个氨基肽酶N，以及锌金属蛋白酶、蛋白激酶Ca结合蛋白、戊二酰-CoA脱氢酶、酚氧化酶、NADPH氧化酶、线粒体钙单向转运体蛋白和表皮蛋白等；值得关注的是，[O.furnacalis]_4857和[O.furnacalis]_2541这2个基因参与了非饱和脂肪酸的合成通路和脂肪酸的代谢通路，因脂肪酸的生物合成途径是调控蛾类昆虫性信息素合成的重要途径。因此，推测这2个基因可能参与这一途径，从而影响玉米螟的性信息滴度。另一方面，金属硫蛋白（metallothionein, MT）也可能参与镉解毒相关的代谢过程。利用迭代BLAST搜索管道，建立300多个昆虫的MT序列，表明昆虫MT结构保守，序列较短，同一物种不同MT之间同源性低，但MT含量是由单个祖先MT的谱系特定串联重复形成的。.本项目在执行期间，发表学术论文8篇、会议论文7篇，培养了1名博士研究生和2名硕士研究生。

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