miR319a/PmTCP15在果梅雌蕊花柱败育中的协同调控研究

The normal development of fruit pistil is an important factor in fruit formation and yield. The phenomenon of pistil abortion is very common in Prunus mume, but the mechanism of abortion is not clear now. Our previous studies showed that miR319a and its target PmTCP15 expressed differentially between perfect and imperfect flowers, and the content of auxin also showed significantly differential. We hypothesize that miR319a could regulate the auxin distribution and polar auxin transport by repressing the expression of its target gene, PmTCP15 in Prunus mume. Extreme distribution of auxin could cause the degradation of cell wall, which probably induces pistil abortion in Prunus mume. Through further studying the mechanism of pistil abortion in Prunus mume by RLM-RACE, LCM, ChIP, ChIP-seq and yeast one-hybrid, to identify the differential expression of miR319a and its target PmTCP15, and the relationship between PmTCP15 and its downstream genes in the process of pistil abortion, besides, the relationship between auxin distribution and pistil abortion. Consequently，make sure the molecular mechanism of miR319a/PmTCP15 coordinately regulates polar auxin transport of style abortion in Prunus mume.

果树雌蕊发育是否正常是果实形成和产量影响的重要因素。果梅雌蕊败育现象十分普遍，但败育的机理还不清楚。前期研究发现miR319a及其靶基因PmTCP15在完全花和败育花中表达量及生长素含量均呈显著性差异。因此推测miR319a可能通过抑制其靶标PmTCP15家族调控了果梅内源生长素分布及极性运输，生长素的极端分布有可能引起细胞壁的降解，从而参与调控了果梅雌蕊败育的发生。因此，为验证这一假设，拟利用RLM-RACE、LCM、ChIP-seq和酵母单杂等技术，深入研究果梅雌蕊败育的发生机理，以明确miR319a及其靶基因PmTCP15在果梅雌蕊发育中的差异表达；确定果梅雌蕊发育过程中PmTCP15与下游相关基因的互作关系；获得果梅雌蕊内源生长素的分布和含量变化及其与雌蕊花柱败育的关系，最终揭示miR319a与其靶基因PmTCP15参与调控生长素极性运输在果梅雌蕊发育障碍中的分子机制。

果树雌蕊发育是否正常是果实形成和产量影响的重要因素。果梅雌蕊败育现象十分普遍，但败育的机理还不清楚。前期研究发现miR319a及其靶基因PmTCP15在完全花和败育花中表达量及生长素含量均呈显著性差异。因此推测miR319a可能通过抑制其靶标PmTCP15家族调控了果梅内源生长素分布及极性运输，生长素的极端分布有可能引起细胞壁的降解，从而参与调控了果梅雌蕊败育的发生。本项目首先鉴定了完全花与不完全花中内源激素含量及分布，其中正常雌蕊的生长素、细胞分裂素和乙烯含量分别为50.03、424.43和25.87 ng/g，显著高于败育雌蕊。败育雌蕊中赤霉素和脱落酸的含量分别显著高于败育雌蕊的赤霉素和脱落酸含量，分别为234.05和130.28 ng/g。在败育雌蕊中发现生长素、细胞分裂素和乙烯含量较低，分别为28.29、332.78和19.73 ng/g，而在正常雌蕊中发现赤霉素和脱落酸的含量较低，分别记录为172.87和69.81 ng/g。利用5’RACE试验方法检测Pm-miR319a切割的靶基因，结果表明PmTCP15受Pm-miR319a的调控。Pm-miR319a可以在两个切割位点10-11nt和12-13nt处切割靶基因PmTCP15的表达。Pm-miR319a在正常雌蕊与褐色雌蕊中表达量相近，无明显差别。在两个品种雌蕊的柱头、花柱和子房中，PmTCP15在‘大嵌蒂’中的表达量高于‘龙眼’中的表达量，并且后期的表达量要高于前期的。PmTCP15在两个品种雌蕊的柱头、花柱和子房中的表达量在十二月份明显上调，而十二月也是梅雌蕊发生败育的关键时期，说明在梅雌蕊的生长发育过程中，Pm-TCP15的高表达可能促使梅雌蕊败育。ChIP-Seq数据分别在败育雌蕊和正常雌蕊中鉴定出720个和251个潜在的峰相关基因，这些基因受PmTCP15转录因子调控，确定了果梅雌蕊发育过程中PmTCP15与下游相关基因的互作关系。最终揭示了miR319a与其靶基因PmTCP15参与调控内源生长素合成在果梅雌蕊发育障碍中的分子机制。