小菜蛾幼虫偏好表达的microRNA鉴定及功能研究  

Identification and functional characterization of larval-based microRNAsin the diamondback moth, Plutella xylostella

作　　者：白建林[1,2,3,4] 林桂芳 王月[1,2,3,4] 赵茜 彭露 谢苗 杨婕[1,2,3,4] 何玮毅[1,2,3,4] 尤民生[1,2,3,4] BAI Jianlin;LIN Guifang;WANG Yue;ZHAO Qian;PENG Lu;XIE Miao;YANG Jie;HE Weiyi;YOU Minsheng(State Key Laboratory for Ecological Pest Control of Fujian and Taiwan Crops,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China;Institute of Applied Ecology,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China;Joint International Research Laboratory of Ecological Pest Control,Ministry of Education,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China;Ministerial and Provincial Joint Innovation Centre for Safety Production of Cross-Strait Crops,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China;College of Life Sciences,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China)

机构地区：[1]福建农林大学闽台作物有害生物生态防控国家重点实验室,福建福州350002 [2]福建农林大学应用生态研究所,福建福州350002 [3]福建农林大学教育部害虫生态防控国际合作联合实验室,福建福州350002 [4]福建农林大学海峡两岸特色作物安全生产省部共建协同创新中心,福建福州350002 [5]福建农林大学生命科学学院,福建福州350002

出　　处：《生物安全学报》2022年第1期64-74,共11页Journal of biosafety

基　　金：国家自然科学基金项目(31701796、32172503);福建省自然科学基金项目(2018J01618);福建农林大学科技创新专项基金(KFA17319A)。

摘　　要：【目的】microRNA(miRNA)在昆虫生长发育中发挥重要功能,本研究拟通过鉴定小菜蛾不同发育阶段的miRNA,挖掘幼虫偏好表达的miRNA及其潜在功能。【方法】对小菜蛾卵、3龄幼虫、蛹和成虫的miRNA开展高通量测序,结合生物信息学分析方法,筛选在幼虫期偏好表达的miRNA;借助实时荧光定量PCR技术,验证候选miRNA在小菜蛾各发育阶段的表达量;利用软件RNAhybrid和miRanda预测候选miRNA的靶基因,筛选其中与生长发育相关的基因,并通过双荧光素酶报告系统验证靶向关系。【结果】从小菜蛾4个发育阶段中,共获得了2116个已知miRNA和189个新miRNA。经差异表达分析发现,幼虫阶段分别有265、228和132个miRNA的表达量显著高于卵、蛹和成虫。通过荧光定量PCR进一步筛选出了11个幼虫偏好表达的候选miRNA,与miRNA高通量测序结果一致;靶基因预测结果显示,pxy-miR-6094-3p、pxy-miR-750-3p、novelm502、miR-317-3p、miR-1175-3p、miR-750和miR-274这7个miRNA靶向保幼激素代谢通路,体外试验结果表明,pxy-miR-6094-3p、pxy-miR-750-3p、novel-m502和miR-274与保幼激素酯酶基因有靶向关系。【结论】小菜蛾幼虫偏好表达的miRNA可能参与调控了幼虫保幼激素代谢。【Aim】MicroRNAs(miRNAs)play important regulatory roles in insect growth and development.In this study,miRNAs preferentially expressed in Plutella xylostella larvae were identified,and their potential functions in growth and development were explored.【Method】The small RNA libraries of the eggs,3rd instar larvae,pupae,and adults of P.xylostella were sequenced.Based on bioinformatic analysis,miRNAs preferentially expressed in the larvae were selected and verified by quantitative reverse transcription-PCR.The target genes of the miRNAs were predicted using RNAhybrid and miRanda.miRNAs with potential target genes related to growth and development were selected,and an in vitro dual luciferase reporter assay was conducted to test miRNA-mRNA interactions.【Result】A small RNA library constructed from the four developmental stages of P.xylostella was sequenced on an Illumina sequencing platform,revealing 2116 known and 189 novel miRNAs.A total of 265,228,and 132 miRNAs showed significantly higher expression in larvae than in eggs,pupae,and adults,respectively.Further selection of 11 miRNAs that were highly expressed in the larvae was verified by quantitative reverse transcription-PCR,which exhibited a larval-based pattern and was consistent with the results of small RNA sequencing.Target gene prediction of these miRNAs showed that seven miRNAs may target the juvenile hormone metabolism pathway.In vitro analysis of the miRNA-mRNA interactions illustrated that pxy-miR-6094-3p,pxy-miR-750-3p,novel-m502,and miR-274 can target juvenile hormone esterase genes.【Conclusion】Larval-based miRNAs of P.xylostella may be involved in juvenile hormone metabolism regulation.

关 键 词：小菜蛾 生长发育 幼虫 MIRNA 保幼激素 

分 类 号：S433.4[农业科学—农业昆虫与害虫防治]