miR171调控柑橘愈伤组织体细胞胚发生的功能解析

项目名称： miR171调控柑橘愈伤组织体细胞胚发生的功能解析

项目编号： No.31501726

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 农业科学

项目作者： 伍小萌

作者单位： 华中农业大学

项目金额： 22万元

中文摘要： 愈伤组织是柑橘等果树离体保存与遗传改良的重要外植体，体细胞胚发生是愈伤组织再生的主要途径。愈伤组织经多次继代培养，其胚性减弱甚至丧失是柑橘等植物生物技术研究中的普遍问题。申请人前期从柑橘胚性愈伤组织中鉴定了csi-miR171c及其3个SCARECROW-LIKE（SCL）靶基因；发现csi-miR171c的表达水平与愈伤组织的胚性正相关。本申请项目拟分析CSMIR171c编码区和启动子区序列与柑橘愈伤组织体细胞发生能力的相关性，据此开发指示胚性的分子标记；通过超量与抑制表达，验证csi-miR171c是否促进柑橘愈伤组织产生体细胞胚；超量表达对miR171不敏感的SCL（rSCL）和抑制SCL表达，以鉴定在体细胞胚发生过程中起关键作用的“miR171c-SCL”组合。本项目的实施将明确csi-miR171c对柑橘体细胞胚发生的调控作用，为持续利用愈伤组织离体再生体系创新种质奠定基础。

中文关键词： 柑橘；体细胞胚发生；非编码RNA；分子标记；基因功能

英文摘要： Embryogenic callus (EC) is an important explant for germplasm conservation and improvement of fruit trees like citrus. Somatic embryogenesis (SE) is the main approach for EC to regenerate. However, it is a common problem in plant biotechnology that the SE capability of EC is always weakened or even lost during repetitive subculture. In our previous study, the conserved csi-miR171c and its three SCARECROW-LIKE (SCL) targets were identified from EC of Valencia sweet orange (Citrus sinensis), which maintains strong SE capability after over 30 years subculture. csi-miR171c was found to accumulate in high abundance in EC and during early SE stages. The expression level of csi-miR171c is positively correlated with the SE potential of citrus varieties. In this proposal, we plan to (i) compare the coding sequence of CSMIR171c and its promoter among citrus genotypes, to develop markers indicating SE capability, (ii) over-express csi-miR171c or block its function by Short Tandom Target Mimic (STTM) in citrus EC, to validate the function of csi-miR171c in SE, (iii) overexpress miR171-resistant form of SCL (rSCL) or silence it by RNA interference, to identify the key miR171c-SCL module regulating SE. The implementation of this proposal will provide new knowledge on regulatory function of csi-miR171c in SE. The long-term objective is to lay the foundation for continuous utilization of the callus regeneration system for germplasm enhancement.

英文关键词： Citrus;Somatic embryoenesis;Non-coding RNA;Molecular Marker;Gene function