麻疯树miR156及其靶基因的鉴定与生物学功能分析

Jatropha curcas L. is an important green energy species that is well developed for the production of biodiesel in China; however, the development and utilization of this plant has been limited to some extent due to low yield of seeds. Therefore it’s both necessary and important to carry out genetic improvement. The investigations thus far confined to traditional breeding and identification of the role of protein-coding genes related to lipid synthesis and metabolism etc. The role of small RNA is still unknown. Non-coding RNA-miR156 defines an age-dependent flowering pathway in plants that mainly control plant juvenile to adult phase transition and can be manipulated to control flowering time and enhance resistance to abiotic stress etc. In this study, we systematically identify miR156 and its targets in J.curcas by using high-throughput sequencing technology and bioinformatics analysis in combination with construction of transcriptome of high quality on the basis of genome sequence information of J.curcas. Then we analyze the expression patterns of miR156 and its targets in different tissues and developmental stages by quantitative PCR. Furthermore, we investigate the biological function of miR156 in the regulation of developmental timing and abiotic response of J.curcas at the level of phenotype, cell and molecules by strategy of genetic manipulation to control the level of expression of miR156 and its targets. These results are significantly meaningful in theory and practice for further research about genetic engineering and molecular assisted breeding.

麻疯树是我国重点开发的绿色能源树种，但因种子产量低而导致其推广应用受到一定程度限制，故亟待开展遗传改良。目前研究多集中于传统选育及油脂代谢相关编码蛋白的基因的分离和鉴定等分子辅助育种方面，而对小分子RNA的了解非常欠缺。非编码RNA-miR156主要控制植物年龄依赖的从营养期到生殖期的转变，并可用于调控花期和增强抗逆性等多个方面。基于麻疯树全基因组序列信息，本研究在构建高质量转录组的基础上，利用高通量测序技术，通过生物信息学分析，系统挖掘并鉴定麻疯树miR156及其靶基因；而后通过定量PCR检测技术，分析miR156及其靶基因在不同发育阶段及组织中的时空表达模式；最终利用遗传操作手段特异性调节miR156及其靶基因的表达量，从表型、细胞和分子水平分析并鉴定miR156在调控麻疯树生长发育和逆境响应中的生物学功能。研究结果对于进一步开展基因工程和加强分子辅助育种研究有着重要的理论和实践意义。

开发利用木本能源植物是解决粮食与能源争地的有效途经之一。麻疯树生长迅速、耐干旱贫瘠，果实出油率40%以上，是具有较高开发应用前景的工业油料树种。目前麻疯树面临生产性能低下，开花结实难及种子含油量低等问题，尚不能完全满足产业化发展的需要。小分子RNA-miR156及其靶基因SPL主要控制植物年龄依赖的从营养期到生殖期的转变，并可用于调控花期和增强抗逆性等多个方面。本项目基于麻疯树全基因组序列信息，利用生物信息学方法全面系统的挖掘了麻疯树基因组中miR156及其靶基因SPL基因家族，系统进化树分析表明麻疯树JcSPL与木本植物的SPL基因亲缘关系较近，而与拟南芥等草本植物距离较远。进一步通过细致分析15个JcSPLs的基因结构、染色体位置、保守结构域和重复序列特征等揭示了JcSPL基因功能的多样性和特殊性。通过降解组测序、RLM-RACE和小分子RNA定量实验验证了miR156对JcSPL基因的切割和转录调控作用。亚细胞定位实验证实JcSPL3主要分布于细胞核中。定量表达分析发现JcSPL3在7年生和10年生植株的根、茎和叶中表达最高，且其表达量随植物年龄增加而显著提高，表明JcSPL3在调控麻疯树年龄发育中的功能。过表达JcSPL3植株表现出花期提前、叶片数目减少、果荚长度降低等表型，说明JcSPL3可能作为正调控因子参与调控麻疯树开花时间及果实发育。该成果丰富了对林木花期转变和果实发育调控机制的认识，为通过基因工程手段改良麻疯树提供科学依据。