miR171调控柑橘愈伤组织体细胞胚发生的功能解析

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.

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。

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。项目负责人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，推测其可能参与调控体细胞胚发生。本项目通过在柑橘胚性愈伤组织分别超量表达和采用STTM技术抑制csi-miR171c，获得了转化愈伤组织系，证实超量表达miR171c促进柑橘愈伤组织生胚，而抑制miR171c表达则减弱体细胞胚发生。在胚性弱的‘国庆1号’温州蜜柑（G1）愈伤组织同时干涉靶基因CsSCL2和CsSCL3（CsSCL2/3-RNAi）或单独干涉CsSCL1（CsSCL1-RNAi）均促进体细胞胚发生，其中CsSCL2/3-RNAi促进生胚的效率较高。为分析CSMIR171c基因序列与柑橘愈伤组织体细胞胚发生的相关性，并据此开发指示胚性的分子标记，从26个柑橘品种扩增MIR171c基因的编码区（550 bp）和约2 kb的启动子区，鉴定了多处序列差异，但均不与胚性直接相关。利用愈伤组织遗传转化体系验证了miR156与其2个靶基因CsSPL3和CsSPL14对体细胞胚发生的调控作用。通过两对柑橘单胚/多胚品种的胚珠转录组和小RNA组学测序和分析，鉴定了305个在单胚、多胚的胚珠间差异表达的基因以及150个miRNA，其中miRN23-5p及其靶基因可能参与珠心胚发生。本项目的实施明确了csi-miR171c与其3个靶基因CsSCLs对柑橘体细胞胚发生的调控作用，完成了项目目标，为持续利用愈伤组织离体再生体系创新柑橘种质奠定了基础。已在Journal of Experimental Botany、Plant Cell Tissue Organ Culture发表SCI论文3篇，培养博士生2人、硕士生2人。

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