【作者】 王平；

【导师】 陈昆松；

【作者基本信息】 浙江大学 ， 果树学， 2011， 博士

【摘要】 以枇杷(Eriobotrya japonica Lindl.)冷敏果实‘洛阳青’(LYQ)、‘大红袍’(DHP)和非冷敏果实‘白沙’(BS)品种为试材,结合乙烯、1-MCP、程序降温(LowTemperature Conditioning, LTC)和热激(Heat shock, HS)等处理,研究了乙烯信号转导元件调控枇杷果实成熟衰老和冷害木质化的分子机制。主要结果如下：1.利用简并引物和抑制性差减杂交(Suppression Subtractive Hybridization, SSH) cDNA文库,从枇杷果实中克隆得到了9个乙烯信号转导元件基因,包括3个EjETRs基因,1个EjCTRl基因,1个EjEIL1基因和4个EjERFs基因。这9个基因在枇杷幼根、幼茎、幼叶、盛花期花瓣和不同发育阶段果实等组织中均广泛表达。EjERS1a、EjCTR1、EjERF2和EjERF3基因在枇杷果实发育的8 WAA到12 WAA过程中维持着较高的表达丰度,表明这些基因可能与该阶段的果实快速生长有关。2.采后枇杷果实成熟衰老过程中,9个乙烯信号转导元件基因均呈稳定或下降的表达模式,尤其是在LYQ品种中,可能与枇杷果实采后低浓度的内源乙烯有关。外源乙烯(100μll-1)可强烈诱导EjERS1a、EjERS1b、EjCTR1、EjERF2和EjERF3基因的表达,表明外源乙烯可以通过乙烯信号转导调控非跃变型果实采后成熟衰老进程。3.采后枇杷果实低温贮藏过程中,EjETR1、EjCTR1、EjEIL1、EjERF1和EjERF2基因的转录本丰度呈增加的趋势,其中EjETR1、EjEIL1和EERF1基因的上调模式在冷害木质化的LYQ果实中尤为明显。LTC和1-MCP(5μll-1)处理均能减轻LYQ枇杷果实冷害木质化程度,但不同的处理可能通过不同的乙烯信号转导元件起作用,即LTC可能通过调控EjEIL1的基因表达来减轻冷害,而1-MCP可能通过调控EjETR1的转录丰度来减轻冷害,进一步表明EjEIL1可能直接响应低温逆境,而EjETR1可能直接响应乙烯。LTC处理减轻冷害木质化的效果优于1-MCP处理,表明EjEIL1可能是更重要的一个调控位点。4.高温处理导致LYQ和DHP枇杷果实乙烯释放量呈峰型变化。EjERS1a、EjERS1b和EjERF2在高温及其处理后的常温恢复中的表达模式与高温逆境导致的乙烯变化呈现一定的正相关性,表明这些基因在高温逆境响应中的重要作用。此外,EjHSP70和EjERF2基因在HS效果明显的DHP果实中维持较高的表达水平,这与枇杷果实冷害程度呈现一定的负相关性。综上所述,乙烯信号转导元件不仅参与了非跃变型枇杷果实的成熟衰老,还通过不同调控位点参与了果实冷害木质化进程,研究结果丰富了乙烯信号转导元件调控非跃变型果实成熟衰老与冷害发生的相关生物学理论。更多还原

【Abstract】 Loquat (Eriobotrya japonica Lindl.) is a non-climacteric fruit, with some cultivars such as ’Luoyangqing’(LYQ) and ’Dahongpao’(DHP) susceptible to chilling injury (CI), while others such as’Baisha’(BS) are resistant. Different levels of ethylene signal transduction elements were isolated and their expression patterns were investigated in loquat fruit at different developmental stages and under different postharvest conditions. These genes were differentially responsive to ethylene,1-methycyclopropene (1-MCP), low temperature conditioning (LTC) and heat shock (HS) treatments. Main results are as follows:1. By using degenerate primers and suppression subtractive hybridization (SSH) cDNA library, three ethylene receptor genes, one EjCTRl gene, one EjEIL1 gene, and four EjERFs were isolated and characterized in ripening loquat fruit. All of these genes were expressed in all tissues tested, including young root, young stem, young leaf, petal in full flowering stage, and fruit at different developmental stages. EjERS1a, EjCTR1, EjERF2 and EjERF3 maintained higher expression levels from 8 to 12 WAA, suggesting an important role that ethylene signalling played during the rapid fruit growth stages.2. During loquat fruit ripening, all nine genes showed constant or decreased expression patterns in reponse to low level of endogenous ethylene, especially in LYQ cultivar. However, transcripts of EjERS1a, EjERS1b, EjCTR1, EjERF2 and EjERF3 were significantly induced by exogenous ethylene, indicating that external ethylene can stimulate non-climacteric fruit ripening through ethylene signalling.3. During low temperature storage, EjETR1, EjCTR1, EjEIL1, EjERF1 and EjERF2 genes showed increased expression pattern and this was particularly notable for EjETR1, EjEIL1 and EjERF1 during CI development in LYQ fruit. Though both LTC and 1-MCP treatments can alleviate chilling-induced lignification, regulation of CI development by ethylene signalling may differ in these two treatments:LTC may alleviate CI through the regulation of EjEILl while 1-MCP may alleviate CI through the regulation of EjETRl at the transcriptional level. These results further indicate that EjEIL1 may directly respond to low temperature, while EjETR1 may directly responed to ethylene. Furthermore, the effect of CI alleviation was more pronounced with LTC treatment than that with 1-MCP treatment, suggesting EjEIL1 might be a more important regulation site for CI prevention in LYQ fruit.4. In response to high temperature, ethylene production increased and showed a peak in both LYQ and DHP loquat fruit. Expression patterns of EjERS1a, EjERS1b and EjERF2 during or after high temperature treatment were paralleled with the changes in such ethylene production, suggesting an important role of these genes played in high temperature stress response. Higher expression levels of EjHSP70 and EjERF2 genes were observed in HS treatment, especially in DHP cultivar, which showed better alleviation effect of CI symptom by HS.In conclusion, ethylene signalling regulates not only the development and postharevest ripening of non-climacteric loquat fruit, and it regulates chilling-induced lignification by different elements as well. Our study may fullfil the biological theory of ethylene transduction in the regulation of ripening and CI of non-climacteric fruit.更多还原