miR167在柑橘雄性不育中的功能及调控作用研究

Seedless breeding is very important for improving citrus quality. Male sterility is one of the main causes of seedless citrus fruits. In our previous study, two members of miR167 (miR167a and miR167c) were identified to be related to male sterility from two seedless citrus varieties, ‘Qiangyang’ seedless Ponkan (Citrus reticulate Blanco) and G1+HBP (Citrus grandis Osbeck), by high-throughput sRNA sequencing. In this project, some work will be done to validate the function of miR167a and miR167c in citrus male sterility. Firstly, the over-expression of miR167a and miR167c assays and silencing by Short Tandom Target Mimics (STTM) experiments in citrus will be conducted. Secondly, the over-expression and RNAi silencing of ARF6, which was target of miR167, will be also performed in citrus. Furthermore, the two-hybrid system will be put into effect to identify the downstream interaction protein of ARF6. The Digital Gene Expression (DGE) assays will be accomplished to explore the regulatory role of miR167 in citrus male sterility. This project will reveal the regulatory mechanism of miRNA in citrus male sterility and provide great theoretical and application value to citrus seedless varieties breeding.

无核育种是柑橘品质改良的重要内容，而雄性不育是导致柑橘无核的主要原因之一。申请人前期利用‘黔阳无核’椪柑和胞质杂种‘华柚2号’两个无核柑橘类型为试材，并分别以有核的‘鄂柑一号’椪柑和HB柚为对照，通过小RNA高通量测序及降解组测序筛选到miR167家族的两个成员（miR167a和miR167c）与柑橘雄性不育相关。本项目旨在通过拟南芥超量表达miR167a和miR167c及其靶基因ARF6，以及在短童期的山金柑中超量和抑制目的基因的表达，验证miR167-靶基因调控柑橘雄性育性的功能；并利用酵母双杂交筛选miR167靶基因互作蛋白；进一步利用miR167超量表达转基因材料，通过差异基因表达谱分析miR167对柑橘雄性不育的调控机制。本项目的实施有望揭示miR167在柑橘雄性不育中的作用，为柑橘无核机制研究提供良好的理论基础，对柑橘及其他果树无核品种选育也具有重要的理论和应用价值。

无核育种是柑橘品质改良的重要内容，雄性不育是导致柑橘无核的主要原因，研究柑橘雄性不育分子机理对柑橘无核育种具有重要意义。为探究phasiRNA调控网络及其在柑橘花发育和雄性不育中的作用，本项目利用雄性不育胞质杂种‘华柚2号’和HB柚的小RNA测序数据，鉴定到2235个phasiRNA，来自280个PHAS位点。利用降解组测序数据鉴定到可触发29个PHAS位点产生phasiRNA的9个miRNA和8个phasiRNA，以及67个phasiRNA的靶基因。由此构建10个miRNA/phasiRNA-PHAS(mRNA)-phasiRNA-mRNA调控网络。发现88个phasiRNA在‘华柚2号’和HB柚花发育过程中显著差异表达，其中miR167触发ARF6产生的3个phasiRNA在不育品种中下调表达，miR390触发一个非编码基因产生的12个phasiRNA在不育品种花发育后期上调表达，推测miR167c.1-ARF6-phasiRNA-target和miR390-ncRNA-phasiRNA-ARF调控路径可能参与柑橘雄性不育过程。项目负责人前期从无核椪柑‘黔阳无核’和雄性不育胞质杂种‘华柚2号’的花器官中鉴定到cga-miR167a.1、cga-miR167c.1和cga-miR156a，推测它们可能参与柑橘花发育和雄性不育。本项目将miR167a/c.1超量表达、STTM抑制表达载体及miR156a超量表达载体转化短童期山金柑。miR156a超量表达获得7棵转基因阳性苗，但未引起山金柑雄性不育表型。miR167a/c.1超量或抑制表达严重影响抗性再生芽的生长，均未能获得转基因阳性植株。转入诱导过量表达型pER8-miR167a.1载体，获得了11株转基因阳性植株，且发现喷施雌二醇诱导miR167a/c.1超量表达。本项目的实施筛选到可能与育性相关的phasiRNA及其调控网络，得到了pER8-miR167a.1诱导型超量表达转化山金柑阳性植株，为后续鉴定和解析miR167在柑橘花发育和雄性不育中的功能奠定了基础，积累了宝贵经验。已在Tree Genetics & Genomes和Plant Physiology上发表论文两篇，培养硕士生2人。