生长素调控水通道蛋白AQP诱导番茄花柄脱落的作用机制研究

In this study, tomato flower pedicel was used as experimental material. Plant organs abscission could divided in two phases, the first phase of which is starting to abscising stage which might cause by cell expansion manipulate through auxin-dependent induced aquaporin AQP, but the mechanism is unknown. First, identified the change of auxin vertically and horizontally in pedicel explants during the abscission, and established the routine of auxin concentration and aquaporin(AQP) distribution for regulating the abscission according to AQP spatial-temporal expression, protein content and positioning change pattern, therefore screen out the candidate AQP members which possible mediate abscission; second, identified the key AQP genes involved in abscission via activity identification and the effects of RNAi silencing and over-expressing candidate genes on cell expansion and abscission; third, obtain the interacting proteins of AQP proteins and screen out the AQP protein which is oligomerized and its corresponding phosphorylated kinase and phosphorlated phosphatase by the yeast two-hydridsystem analysis of selected key AQP genes and pedicel abscission-specific cDNA library, and confirm their function through RNAi technology; forth, yeast one-hybrid system was performed in search of auxin transcription factor(ARF) located upstream of abscission key AQP promoter, and verify its roles in regulating AQP by RNAisilencing and over-expression; finally, with the combination of above conclusion and the use of auxin treatment and ARF which delayed abscission to figure out the way how auxin influences the state of cell expansion through AQP protein content, phosphorylation and oligomerization, and establish the regulatory mode of auxin-ARF-AQP.

植物器官脱落分两个阶段，其中第一阶段为脱落启始阶段，有报道称这一阶段可能由生长素调控水通道蛋白AQP导致离区细胞膨胀引起的，但其机理不详。本研究以番茄花柄为试材，在明确其脱落过程中外植体生长素浓度空间变化、AQP蛋白时空表达及定位变化以及生长素-AQP蛋白分布规律的基础上，筛选出调控花柄脱落的AQP候选基因；并采用RNAi沉默和超表达，明确AQP候选基因对细胞膨大和脱落的作用，从而筛选出调控脱落的关键AQP基因；进一步通过关键AQP蛋白与花柄脱落特异cDNA文库酵母双杂交，筛选出与其发生寡聚化的AQP蛋白和发生磷酸化及脱磷酸化的激酶/磷酸酶；利用关键AQP启动子以酵母单杂交获得生长素转录因子ARF，采用RNAi沉默和超表达验证ARF对AQP蛋白的调控作用；最后利用生长素和生长素响应因子ARF，弄清生长素调控AQP蛋白含量、磷酸化和寡聚化导致细胞膨大，从而确定生长素-ARF-AQP调控模式。

番茄花柄正常条件下细胞维持在小而圆的状态，而在干旱，盐胁迫等不适合条件下细胞膨大发生脱落，而调控离区细胞水分状态，可以影响细胞膨大调控脱落进程，这一作用依赖于乙烯，但调控机制不清楚。我们发现，正常条件下生长素诱导SlARF3表达，其可以与SlERF52互作抑制其激活SlTIP1;1, SlPIP1;3和SlPIP1;5的表达。而胁迫条件下离区生长素信号下降后，乙烯通过SlERF52促进SlTIP1;1, SlPIP1;3和SlPIP1;5的表达。原位杂交和免疫杂交证实SlTIP1;1, SlPIP1;3和SlPIP1;5在离区特异表达，与脱落发生密切相关。进一步研究发现在离区细胞中SlPIP1;3和SlPIP1;5互作形成异源四聚体和SlTIP1;1形成同源四聚体。SlTIP1;1, SlPIP1;3和SlPIP1;5一方面通过运输水分给离区细胞提供膨压为细胞分离提供动力，更重要的是通过提高细胞质中过氧化氢含量抑制生长素信号促进乙烯的生成量，细胞壁水解和细胞程序性死亡导致脱落发生。