短营养期凤梨突变体的早花机理研究

Pineapple is both an important tropical flower and herb fruit tree in the world, but its vegetative growth period is much longer than most other ones. It takes as long as 18 to 48 months under natural flowering conditions, and there is also a great inconsistency between individuals of the same variety. Flower-inducing with ethylene can shorten the period of vegetative growth to about 12 months, the storage performance and the decline of fresh food quality have always been important factors affecting the development of the pineapple industry. Therefore, the research on the early flowering mechanism of short vegetative growth period pineapple mutant is of great significance for the creation of new varieties of short vegetative growth and the improvement of economic benefits of pineapple production. In this application, we will use the short vegetative mutant ‘14-1’ as the material to get the hybrid population of F1 and BC1 to analyze the inheritance of early flower traits. Screening of 9 early-flower-related differentially expressed genes were verified by qRT-PCR, and the key genes of early flowering were screened by using the over-expression and CRISPR/Cas9 technologies in the original varieties and mutants, respectively, and then correlated with the F1 generation early flower phenotype. Studying on the mechanism of early flower trait formation will lay a foundation for further development of genetic map construction, gene mapping of early flower traits and in-depth discussion of the molecular regulation mechanism of pineapple conversion from vegetative growth to reproductive growth.

凤梨是世界重要的热带花卉和草本果树，但其营养期长达18～48个月，较一般草本果树和花卉更长，且同品种个体间相差较大。虽然人工乙烯催花也能使营养生长期缩短至12个月左右，但催花果实的贮藏性能和鲜食品质下降等问题一直是影响凤梨产业发展的重要因素。因此，开展短营养期凤梨突变体创制及其早花机理研究对短营养期新品种培育和提高凤梨生产效益有着重要意义。本申请项目将以筛选得到的短营养期突变体‘14-1’为材料，将其与野生型杂交构建F1代和BC1代群体来分析早花性状遗传规律；通过对前期筛选的9个早花相关差异表达基因进行qRT-PCR验证，利用转化使其在原始品种中超量表达和利用CRISPR/Cas9技术靶向编辑进行功能鉴定来筛选获得早花关键基因，再与F1代早花表型相关联，以此探讨早花性状形成的机理，为进一步开展遗传图谱构建、早花性状的基因定位以及深入探讨凤梨从营养生长向生殖生长转换的分子调控机制打下基础。

凤梨是世界重要的热带草本果树，但其营养期却长达1年半以上，缩短营养期一直是凤梨生产中瓶颈问题。虽然人工乙烯催花能缩短营养期，但因催花不当造成严重经济损失的现象常有发生。因此，利用短营养期凤梨突变体研究早花性状的分子机制，对进一步创制早花早果新品种具有重要意义。本项目从短营养期突变体‘玉玲珑’中筛选出了能正向调控转基因凤梨提早成花的转录因子AcERF24b，转基因结果表明该基因具有促进凤梨早花的功能；与野生型中的同源基因AcERF24a序列相比，AcERF24b中存在3个碱基的突变，均位于其5’上游调控序列，推测其在突变体与野生型中的表达差异可能与序列变异相关；亚细胞定位表明AcERF24b为核定位基因，在细胞核中接受信号发挥功能；通过qRT-PCR分析F1代植株营养期（长池和短池）花芽分化不同阶段AcERF2b4基因的表达差异分析发现，AcERF24b与F1代早花性状显著相关；通过杂交群体构建和单株早花性状调查统计，发现正、反交对F1早花性状没有明显影响，明确杂种F1营养生长期早花性状为显性遗传，初步厘清突变体早花性状遗传规律，以上研究结果揭示了短营养期凤梨突变体的早花机理，为通过基因工程手段定向改良凤梨成花提供分子依据。