靶向隐花色素cry基因的miRNAs对全周期型禾谷缢管蚜生殖方式转换的调控作用

The bird cherry-oat aphid, Rhopalosiphum padi (L.) (Hemiptera: Aphididae), is one of the most important wheat pests worldwide. It has holocyclic (cyclical parthenogenesis) and anholocyclic (obligate parthenogenesis) life cycles, The cyclical parthenogenesis lineages could switch from parthenogenesis to sexual reproduction under short photoperiod inducing condition, whilst the obligate parthenogenesis lineages can never produce sexual generation even in inducing condition. We found that short photoperiod played a decisive role in the switch of reproductive mode, but the molecular mechanism was unknown. Previous research showed that the cryptochrome (cry) genes and miRNAs play an important role in the insect photoperiodism. We found that the RNA interference of cry1 or cry2 gene could affect the number of parthenogenetic female, gynoparae, male and oviparae female of offspring in holocyclic R. padi, and five miRNAs of the pest targeted the cry genes and were differentially expressed during the process of reproductive mode switch in holocyclic R. padi. In this project, the roles of cry genes in the process of reproductive mode switch will be determined. Dual-luciferase reporter assay system will be used to detect the interaction between microRNAs and cryptochrome genes. The miRNA mimics and inhibitor will be used to study the effects of miRNAs on the mRNA and protein levels of cry genes, as well as on the process of sexual generation production. The in vivo distribution of cry genes and miRNAs will be examined in holocyclic R. padi with different inducing time. This work will contribute to the comprehensive studies of the seasonal response of aphids, and to clarify the molecular mechanism of the aphid photoperiodism, which is important for elucidating the dynamics and micro-evolution of aphid population.

禾谷缢管蚜是重要的麦类作物害虫，其具有全周期型和不全周期型等生活史类型，全周期型蚜虫在短日照诱导时能从孤雌生殖转换为有性生殖，不全周期型只能进行孤雌生殖。申请者发现，短日照诱导在该蚜虫生殖转换中起决定作用，但具体机制不明。前人研究表明，隐花色素cry基因和miRNA在昆虫的光周期响应过程中有重要作用。申请者发现，cry基因在该虫生殖转换中发挥作用，干扰cry基因能够影响生殖转换过程中有性蚜的数量，靶向cry的5个miRNAs在该虫生殖转换过程中差异表达。在此基础上，本项目利用RNAi研究cry基因对性蚜首次产生时间的影响；利用双荧光素酶报告系统确证靶标为cry的miRNA；运用模拟物和抑制剂研究miRNA对靶向的cry基因mRNA和蛋白水平以及有性蚜产生的影响；分析诱导条件下cry基因和miRNA在该虫中的组织分布。研究结果对于深入阐释禾谷缢管蚜光周期现象和生殖转换的分子机理具有重要意义。

禾谷缢管蚜是重要的麦类作物害虫，其具有全周期型和不全周期型等生活史类型，全周期型蚜虫在短日照诱导时能从孤雌生殖转换为有性生殖，不全周期型只能进行孤雌生殖。本研究首次发现隐花色素cry基因在蚜虫光周期计时系统中具有非常重要的作用，分别干扰cry1和cry2基因能够显著影响孤雌蚜、产雌性母和雄蚜首次产生时间，同时也能够显著影响各蚜型数量；利用双荧光素酶报告系统确证miR-1000和novel-79靶向cry1基因且负调控cry1的表达，miR-3049和novel-7靶向结合 cry2的3′UTR且负调控cry2的表达。注射miR-1000和novel-79模拟物和抑制剂显著影响cry1基因的表达水平和蛋白含量；注射miR-3049和novel-7模拟物使cry2基因的表达水平显著降低和蛋白含量显著减少；注射miR-3049和novel-7抑制剂使cry2基因的表达水平显著升高。这四个miRNAs在不同诱导阶段的禾谷缢管蚜中的表达量存在显著差异，进一步确定这些miRNAs在禾谷缢管蚜生殖转换过程中具有重要的作用。与对照组相比，注射miRNA模拟物能够显著影响全周期型禾谷缢管蚜产生孤雌蚜、产雌性母和的雄蚜数量，同时还能影响产雌性母和雄蚜首次产生时间，孤雌蚜、产雌性母和雄蚜最终产生时间和总产生时间，这些结果表明miRNAs能够调控cry基因的表达从而影响禾谷缢管蚜生殖转换过程。研究结果对于深入阐释禾谷缢管蚜光周期现象和生殖转换的分子机理具有重要意义。