基于microRNA调控的东亚飞蝗繁殖与免疫权衡策略研究

Effective immune defense and reproductive success are two interrelated important parts of individual's lifetime fitness. Although studies shown that an individual in limited resources can trade off the costs of immunity with reproduction, but the mechanism is poorly understood. microRNAs are important regulatory factors involved in many physiological processes in animals, there have evidences suggest that some microRNA can regulate the JH levels, thereby affects the immune activity of the insects. Therefore, microRNAs may be involved in trade-offs between reproduction and immunity, which worth to do further more investigation. Locusta migratoris menilensis was an ideal organism which to study the trade-offs between reproduction and immunity in this project. transcriptome sequencing and Small RNA Sequencing technology, those reproduction-related ESTs and microRNAs which response to juvenile hormone analog (JHA) treatment, as well as immune-related ESTs and microRNAs which response to immune stress are obtained. The sequencing data and bioinformatics methods to predict the gene regulatory microRNAs involved in reproduction or/and immune, two cell lines of 293T and S2 are used to verify the interaction between microRNAs and target genes, the impacts of important microRNAs on reproduction and immune will be identified and analysis in order to evaluate the potential value of microRNA regulation on locust pest management. This project not only in theory clarify the intrinsic regulatory mechanism of trade-offs between the locust reproductive invests and immune activity, but also lay the foundation for the use of the microRNA on pest management.

对病原物的免疫抵抗和繁殖成功是个体适合度互为关联的两个重要组成部分。虽有研究表明，动物在有限资源情况下可对免疫与繁殖进行权衡，但对其机制知之甚少。microRNA是动物很多生理过程中重要的调控因子，已发现某些microRNA可以调控JH等激素水平，进而影响到昆虫的免疫活性。因此，microRNA可能参与了昆虫繁殖与免疫间的权衡，值得深入研究。本项目以东亚飞蝗为对象，通过转录组测序和小RNA测序获得繁殖与免疫相关的ESTs及microRNA，利用测序数据和生物信息学方法预测东亚飞蝗繁殖与免疫相关基因的调控microRNA，在细胞水平上验证microRNA和靶基因的相互作用关系，再在昆虫活体中鉴定和分析重要microRNA对蝗虫繁殖与免疫的影响，评价microRNA调控在蝗虫控制中的潜力。该项目不仅在理论上阐明蝗虫繁殖投入与免疫活性间权衡的内在调控机理，还为利用microRNA控制害虫奠定基础

1）采用第二代测序技术，对东亚飞蝗雌成虫的卵巢与脂肪体混合建库，采用Illumina HiseqTM 2000进行双末端测序，Trinity软件进行无参拼接，得到8G的转录组数据，获得54 740条功能基因作为后续DGE数据分析的参考基因。其中，共有21 044 (38.44%) 条功能基因通过BLAST比对到已知基因。基于GO注释， 175个功能基因被归类为免疫系统过程，709个功能基因被归类为繁殖，650个功能基因被归类为生殖过程；按照KEGG注释， 5496个功能基因被分配到31个不同的KEGG通路，其中191个功能基因被归类为与免疫系统功能相关联，210个功能基因被归类为在能量代谢中发挥作用；2）分别用线纹折麻蝇（Blaesoxipha lapidosa）寄生、藤黄微球菌（Micrococcus luteus）感染、葡聚糖微珠（dextran beads，模拟寄生物）注射诱导处理东亚飞蝗羽化第一天雌虫，诱导处理后4小时和6天分别提取脂肪体和卵巢等组织样本，用于DGE分析，获得12个DGE。DGE基因表达水平的估算采用RSEM对bowtie的比对结果进行统计，以RPKM值分析得到差异表达基因。从每一个DGE分析中，每个DGE- SEQ可得到超过9.3 × 106的原始序列标签，而后每个DGE –SEQ得到超过9.1 × 106的清洁序列标签被映射到参考序列；3）用每个实验样本的RPKM值做聚类分析来识别不同群体间的DGE差异，并对9个对比组中所有差异表达的功能基因进行了注释。利用BLAST检索了NR数据库中免疫和生殖相关的基因，并将功能基因分别归类到免疫相关，或繁殖相关，免疫相关的功能基因再分为18类，并找出了其中10个差异表达最显著的功能基因；4）与免疫相关的差异表达功能基因进行了识别，包括pacifastin-related peptide precursor，allergen，AGAP-PA，multidrug resistance-associated protein，chitinase-like protein，caspase-1，immunoglobulin，trypsin，putative defense protein，tolloid-like protein，I-type lysozyme，carboxylesterase和regenecti