【作者】 李清清；

【导师】 李德全； 李大鹏；

【作者基本信息】 山东农业大学 ， 植物学， 2010， 硕士

【摘要】 醇酰基转移酶(Alcohol acvltransferase，AAT)是生物体内酯类挥发性成分合成的关键酶。前期研究发现，水杨酸(salicvlic acid，SA)、乙烯利(Ethephon，ETH)和茉莉酸甲酯(methyI iasInonate，MeJA)可以不同程度的诱导节果果实中醇酰基转移酶基因(MdAAT2)的表达，这表明该基因可能参与了对胁迫信号的响应。目前，对醇酰基转移酶基因的研究主要集中在果实和花香气合成调控方面，其转录调控的机制及上游转录因子尚不清楚。本试验通过生物信息学技术，筛选并克隆了六个胁迫相关的转录因子(MdMYB1，MdMYB2,MdMYB6，MdERFl，MdERF2和WRKY)，通过半定量RT-PCR技术分析了这些转录因子在金帅节果不同组织、发育阶段以及不同信号物质诱导后的表达特性，分析了其与MdAAT2基因表达的相关性。主要结果如下：1、利用PLANTCARE等相关数据库，对MdAAT2启动子的顺式元件进行了分析。研究发现，MdAAT2启动子区域存在两个水杨酸响应元件W-box和一个茉莉酸甲酯响应元件TGACG-motif，分别位于转录起始位点上游-66lbp、-1130 bp和-1129bP)处。结合同源性分析，在金帅苹果中克隆了六个候选的转录因子：MdMYBl (GU013684)、MdMYB2(GU013681)、MdMYB6(GU013682)、MdERF1(AB288347)、MdERF2(AB288348)和WRKY(GUO13683)。2、通过RT-PCR技术分析了候选转录因子和MdAAT2在节果果实和花器官中的表达特性。研究发现六个候选转录因子在柱头中均有显著表达，其中MdERF1和MdERF2在果实和花器官中均有不同程度的表达。序列分析发现MdAAT2启动子中存在两个与花粉特异表达有关的元件，POLLENILELAT52，分别位于-82bp和-882bp处。这表明所分析的转录因子可能通过调控功能基因的表达或与MdAAT2相互作用，参与果实和花器官中挥发性酯类成分的合成。3、通过RT-PCR技术分析了MdAAT2和候选转录因子在果实发育阶段的表达特性。相关性分析发现WRKY、MdMYB6与MdAAT2的表达模式相关性不显著，而MdMYB1、MdMYB2、MdERFl、MdERF2与MdAAT2表达特性具有显著的相关性。这表明WRKY和MdMYB6可能不参与对MdAAT2的转录调控，而MdMYBl、MdMYB2、MdERFl三个转录因子可能通过调控MdAAT2的表达，参与了挥发性酯类物质的代谢。4、水杨酸(2.0mM)处理发现,MdERFl与MdAAT2的表达模式具有极显著的相关性，MdMYB6与MdAAT2也具有显著的相关性。这表明MdERFl和MdMYB6可能在水杨酸信号转导途径中，调控了MdAAT2的表达。5、乙烯利(2.0mM)处理发现,MdMYB1、MdMYB6和MdAAT2的表达模式具有显著的相关性。这表明MdMYBl、MdMYB6很可能参与乙烯信号转导途径，作为MdAAT2表达的激活因子。6、茉莉酸甲酯(2.0mM)处理发现，MdMYB2、MdERF2与MdAAT2的表达呈显著的相关性，这表明MdMYB2、MdERF2可能在茉莉酸甲酯信号转导途径中，调控了MdAAT2的表达。7、通过序列分析发现，MdAAT2启动子含有CAAT盒子、CCAAT盒子和许多MYB转录因子结合元件，而MdMYBl和MdMYB6转录因子具有CAAT盒子和CCAAT盒子的结合位点。结合相关性分析的结果，我们推测MdMYBl和MdMYB6可能是调控MdAAT2表达的上游转录因子，参与了对胁迫信号的响应。更多还原

【Abstract】 Alcohol acyltransferase (AAT), as the key enzyme, plays important roles in ester biogenesis. In our previous reports, the expression of apple AAT gene (MdAAT2) was shown to be induced on different levels by salicylic acid (SA), Ethephon (ETH), and methyl jasmonate (MeJA). This indicated that MdAAT2 gene may be involved in defense signaling pathways. At present, the research of AAT genes are mostly foucs on fruit and flower aroma synthesis. But the mechanism of transcription factors on regulation of MdAAT2 promoter has not been well established. Using bioinformatics technology, we isolated and investigated six candidate stress-induced transcription factors, i.e., MdMYB1, MdMYB2, MdMYB6, MdERF1, MdERF2 and WRKY. In order to investagate the transcript correlation between these TFs and MdAAT2 gene during fruit development series and different hormone treatments, reverse transcription-polymerase chain reaction (RT-PCR) and statistical analysis were performed. The main results are as follows:1.To identify potential cis-acting regulatory elements in MdAAT2 gene romoter, Plant CARE and other available databases were performed. Sequence analysis showed that the transcription factor binding sites of two salicylic responsive element W-box and a methyl jasmonate (MeJA) responsive element TGACG-motif were found in MdAAT2 promoter region, located at -661bp, -1130bp and -1129bp,respectively. Based on the results presented here and homologous analyze, we isolated six candidate transcription factors MdMYB1 (GU013684), MdMYB2 (GU013681), MdMYB6 (GU013682), MdERF1 (AB288347), MdERF2 (AB288348) and WRKY (GU013683) from “Golden delicious”apple.2.The transcript pattern of these TFs and MdAAT2 gene was analyzed by RT-PCR in apple fruit and flower tissues. The results indicated that the transcript expression of six TF genes was all detected in stigma.The transcript expression of MdERF1 and MdERF2 were also detected in fruit and flower tissues. Sequence analysis showed the MdAAT2 promoter contained two pollen-relative elements POLLEN1LELAT52 which located at the -82 and -882. Accordingly, in apple fruit and flower tissues, these TF genes may be involved in the regulation of functional genes or interacted with the MdAAT2 gene during volatile ester biosynthetic pathway.3.The transcript pattern of these TFs and MdAAT2 gene during fruit ontogeny was analyzed by RT-PCR. There were comparatively low correlation of the transcript levels between two transcription factors WRKY, MdMYB6 and MdAAT2 gene by correlation analysis. But as compared with the MdAAT2 gene expression, the transcript levels of MdMYB1, MdMYB2, MdERF1 and MdERF2 showed a significant correlation. Therefore, MdMYB6 (GU013682) and WRKY (GU013683) may not affect in fruit development. On the contrary, MdMYB1, MdMYB2 and MdERF1 may be responsible for the regulating the expression of MdAAT2 gene which results in the volatile esters metabolizes.4.Transcript pattern of MdERF1 was almost consistent with MdAAT2 and the correlation analysis also showed a high degree after application of 2.0 mM SA treatment. And there was a well correlation between the gene of MdMYB6 expression and MdAAT2 gene expression. These results indicate that MdERF1 and MdMYB6 probably play an important role in regulation of MdAAT2 gene expression during the SA defense signaling pathway.5.Transcript pattern of two TF genes MdMYB1 and MdMYB6 showed a significant correlation with MdAAT2 gene expression after 2.0 mM ETH treatment. Therefore, MdMYB1 and MdMYB6 might serve as a candidate activator of MdAAT2 gene expression in the ethylene signaling pathway. 6.Under 2.0mM MeJA treatment, the transcript levels of MdMYB2 and MdERF2 were observed to be a significant correlative with MdAAT2 expression. Thus, MdMYB2 and MdERF2 probably regulated the expression of MdAAT2 gene after MeJA inducement.7.Sequence analysis indicated CAAT、CCAAT elements and several MYB transcription factor binding sites exist in MdAAT2 promoter region. And the MdMYB1 and MdMYB6 sequences in“Golden Delicious”revealed similarities to CAAT or CCAAT consensus sequence of two protein complexes. Thus, the transcription factors MdMYB1 and MdMYB6 might serve as activators of MdAAT2 gene involved in stress signaling pathways.更多还原