【作者】 殷学仁；

【导师】 陈昆松； Ian B.Ferguson；

【作者基本信息】 浙江大学 ， 果树学， 2010， 博士

【摘要】 本文以猕猴桃(Actinidia)果实为试材,研究乙烯信号转导途径相关编码基因在猕猴桃果实生长发育和后熟软化进程中的表达模式,以及对乙烯(100μl／L)、1-MCP(0.5μl/L).低温(0℃)、高温(35℃)、高CO2(5%)和快速失水(～10%RH空气)等采后处理的应答模式,鉴别参与猕猴桃后熟软化及采后逆境胁迫应答的相关成员；开展转录因子级别元件与果实后熟软化相关基因的互作模式研究,探索基因的作用方向；进行部分基因的功能验证,明确其与猕猴桃果实后熟软化及逆境胁迫应答相关的功能。主要结果如下：1、基于猕猴桃EST数据库,分离得到猕猴桃5个AdETRs基因、2个AdCTRs基因、4个AdEILs基因和14个AdERFs基因。组织和阶段特异性分析结果表明,除AdERF2不在果实组织中表达外,其他成员在根、茎、叶、花和果实等不同组织中均有分布,这与乙烯的广泛生理功能相一致；另外,信号转导元件编码基因的大部分成员在果实发育初期表达较强,它们可能介导了乙烯参与果实快速生长过程。2、AdETRs家族成员的AdERS1a、AdETR2和AdETR3在果实后熟软化中表达增强,且受外源乙烯上调,可能是直接“绑定”乙烯的结合子(binder);而AdETR1表达伴随果实后熟软化进程下降,并受外源乙烯下调,可能是果实乙烯敏感性感受器(sensor);首次发现的呈乙烯下调模式的AdETR1基因,可部分解释猕猴桃果实乙烯超敏感性的内在分子机制。AdERS1b对外源乙烯无应答,它在果实快速软化前表达较强,而在果实完成快速软化之后表达下降,并维持在较低水平,表明AdERS1b可能与果实软化相关。3、AdEILs基因家族成员在果实后熟进程中表达水平较稳定,Transient Assay结果显示AdEIL2/3可增强AdACO1和AdXET5启动子活性,表明AdEIL2/3行使转录激活子功能。转基因结果显示,过量表达AdEIL2和AdEIL3均可增加拟南芥乙烯合成。AdXET5启动子删截实验表明,AdEIL2识别AdXET5启动子的第1和第3个结构域,而AdEIL3识别第1和第2结构域,认为同一启动子区不同结构域在基因互作中具有不同功能,且同一转录因子家族不同成员的识别位点也可存在差异。4、除AdERF2外(不在果实中表达),其他13个AdERFs家族成员在猕猴桃果实后熟软化进程中呈4种表达模式,其中AdERF10/14随果实后熟软化表达增强,AdERF4/6转录本在乙烯跃变高峰后的果实中大量积累,而AdERF5/9呈组成型表达,其余7个AdERFs成员在果实后熟软化进程中表达下降。分析乙烯、1-MCP处理及ACO敲除的果实,7个表达下调的AdERFs中,AdERF1/7/8/11/12与乙烯无显著联系,表现为果实后熟软化进程特异性模式。AdERFs与后熟软化相关基因互作结果表明AdERFs对含有GCC盒的AdEXP1启动子无显著调控效应,但AdERF9可抑制AdXET5启动子活性,表明其行使转录抑制子功能。进一步的启动子删截研究表明,AdERF9始终抑制AdXET5启动子活性,认为未知的AdERF9结合位点可能位于AdXET5启动子的-265至-1区间。5、低温可显著延缓猕猴桃果实后熟软化,且可诱导大部分乙烯信号转导基因的表达。这些乙烯信号转导元件对于低温的应答模式,与果实后熟软化无直接联系,更可能是相关基因的逆境应答。但AdEILs对低温处理的独特应答模式,表明AdEILs可能是调控果实低温应答的关键级别之一。转基因结果显示,AdEIL3、AdERF1/6/9/13可增强拟南芥植株的低温抗性。依据相关基因对低温的应答模式,进一步分析了AdETRs和AdERFs基因家族对采后逆境(高温、高CO2和快速失水等)的应答模式,发现AdETR3、AdERF1/3/4/11/12/14对不同的采后非生物逆境的应答模式一致,它们可能是猕猴桃AdETRs和AdERFs级别参与采后逆境应答的重要成员。上述结果表明,猕猴桃乙烯信号转导途径不同成员不仅参与调控果实后熟软化进程,还参与果实对采后非生物逆境胁迫的应答机制。更多还原

【Abstract】 Kiwifruit(Actinidia) was used to study ethylene signaling related genes expression during kiwifruit development, ripening and softening. Treatment of fruit with ethylene (100μl/L),1-MCP (0.5μl/L), low-temperature (0℃), high-temperature (35℃), high CO2 (5%) and dry air (～10%RH) was used to follow responses of these genes in relation to fruit ripening and postharvest stress. Transcription factor and promoter interaction and functional transgenic works were also included in the experiment. The main results are as follows:1. Five ethylene receptor genes, two CTR1 like genes, four AdEILs and fourteen AdERFs were isolated based on kiwifruit EST database. Among the 25 genes, only AdERF2 was not detected in fruit tissue, while the others were widely expressed in various kiwifruit plant tissues. Temporal expression patterns of the genes indicated that most of them were strongly expressed at fruit early stages of development. This is consistent with our knowledge of ethylene-dependent activity in early stages of organ development.2. Expression studies of the five ethylene receptor genes showed that AdERS1a, AdETR2 and AdETR3 were increased during fruit ripening, oppositely, AdETR1 was down-regulated by ethylene. According to ethylene receptor negative regulation model, AdERS1a, AdETR2 and AdETR3 might act as ethylene binder, while AdETR1 might act as sensor with implications for responsiveness of the ethylene signalling pathway. The expression patterns of AdETR1 also could partially explain the mechanism of kiwifruit high ethylene sensitivity. The relative insensitivity to ethylene of AdERS1b and its pattern associated with softening invites the speculation that it might have a separate involvement in promoting some aspect of later fruit softening.3. Contrast to AdETRs, AdEILs were constitutively expressed during fruit ripening. Gene interaction experiments indicated that AdEIL2/3 act as transcription activators by inducing AdACO1 and AdXET5 promoter activity. Further study on AdEIL2/3 function found that ethylene production was triggered in AdEIL2/3 over-expressed Arabidopsis plants. In order to study the function of different motifs in gene interaction, four deletions were designed in promoter of AdXET5. AdEIL2 might recognize the first and third motif in AdXET5 promoter, while the first and second motifs were important for AdEIL3. These results indicated AdEIL2/3 could transcriptionally regulate ripening related genes, but the target motifs might be different.4. Within AdERFs gene family, AdERF2 was excluded in fruit regime experiment. The other thirteen AdERFs genes were differentially expressed during fruit ripening, which could be divided into four patterns:(Ⅰ) AdERF10 and 14 were increased during fruit ripening, (Ⅱ) AdERF4 and 6 expression was relatively high at the post-climacteric stage, (Ⅲ) Expression of AdERF5 and 9 was constant and not affected by exogenous ethylene, (Ⅳ) the other seven AdERFs genes followed a decreasing pattern during ripening. Combined results from ethylene treatment,1-MCP treatment and ACO knock-out fruit, decreases of AdERF1/7/8/11/12 transcripts were likely to be ripening specific, and be independent of ethylene. Promoter of AdEXP1 contains a GCC box, however, no significant interaction was found between AdERFs and AdEXPl promoter. Interestingly, AdERF9 was confirmed as a transcriptional repressor by repressing AdXET5 promoter, although no GCC box was found in this promoter. Based on AdXET5 promoter deletion results, the unknown target for AdERF9 located at-265 to-1 region of AdXET5 promoter.5. Low-temperature (0℃) significantly delay kiwifruit ripening and softening, while low temperature generally resulted in increased gene expression, it is not yet clear how much it is associated with ripening changes including softening, whilst that response is more likely to be stress response. It is worth emphasizing the novel response of EIN3-like genes to low temperature, which provides some insight into the relationship between ethylene signalling and low temperature. AdEIL3 and AdERF1/6/9/13 were over-expressed in Arabidopsis respectively, and all of the transgenic lines manifest higher low-temperature tolerance. More postharvest abiotic stresses were applied to kiwifruit, and AdETRs and AdERFs were choosed to study the genes stress response. AdETR3 and AdERF1/3/4/11/12/14 had similar response to different abiotic stress.In conclusion, different ethylene signalling components of kiwifruit are not only involved in regulation on kiwifruit ripening, but also involved in response to fruit postharvest abiotic stress.更多还原