【作者】 阮颖；

【导师】 周朴华；

【作者基本信息】 湖南农业大学 ， 植物学， 2006， 博士

【摘要】 十字花科植物中存在一个特殊的底物—酶系统，即硫苷—黑芥子酶系统(Glucosinolate-Myrosinase system)。在植物体内，黑芥子酶往往与黑芥子酶结合蛋白和黑芥子酶协助蛋白一起以复合体的形式存在。iMyAP是一种存在于营养器官中的诱导表达的蛋白。创伤、MeJA，JA和ABA处理能使iMyAP启动子启动iMyAP基因转录。为了进一步了解iMyAP基因启动子的功能结构特征，确定iMyAP基因启动子的核心启动子(core promoter)大小，我们将油菜(Brassica napus)iMyAP基因转录起始位点上游-1868bp区段的全部和部分(分为5个不同长度)插到GUS报告基因的前面，并将融合子转入模式植物拟南芥(Arabidopsis thalina)基因组中。在拟南芥的遗传背景下来剖析iMyAP基因启动子的功能结构。研究结果显示，甲基茉莉酸和创伤处理能使iMyAP启动子启动GUS基因在植株体内表达。地上部分与地下部分相比较，地上部分染色较深，而地下部分染色较浅；微管组织染色较深，薄壁组织染色较浅；茎叶与根的接合区染色最深。通过缺失分析发现只需要iMyAP启动子全长序列中从-1到-370区段的370bp的长度就可以实现全长启动子的诱导功能。带有的370bp长片段的M4-5植株受创伤和茉莉酸处理后，都能启动GUS基因的表达，并且GUS基因在组织中的表达部位和强度基本一致；进一步缺失到只有153bp长时，创伤和茉莉酸处理不能诱导GUS基因的表达，说明这段DNA序列不具备在诱导条件下启动基因表达的功能。通过在PLACE网站上对这段370bp的序列进行基序分析，发现在占总长约为19％的这370bp长的序列上，包含有iMyAP全部8类诱导基序，并拥有全长iMyAP启动子唯一的1个与茉莉酸诱导相关的基序；唯一的1个与生长素诱导有关的基序；与创伤诱导有关的基序两个中的1个，唯一的1个与生长素诱导有关的基序；唯一的1个与糖诱导有关的基序；与创伤诱导有关的基序两个中的1个；与无氧诱导有关的基序ANAERO1CONSENSUS四个中的3个；与干旱脱水诱导有关的6种基序，其中MYCATERD1和MYCATRD22是全长启动子中唯一的基序。这些数据与GUS组织化学染色的结果一起充分说明了370bp长启动子片断代表了诱导型iMyAP启动子的基本特征，是iMyAP启动子的核心结构，即iMyAP启动子的核心启动子。这是首次确定iMyAP启动子的核心启动子大小。370bp长的核心启动子不仅能受甲基茉莉酸和创伤诱导启动基因的表达，而且还具有细胞组织特异型启动子的特征。该核心启动子在正常生长条件下，能启动GUS基因在气孔器保卫细胞中特异表达，成为了一个典型的细胞组织特异型启动子。但是，我们目前已知的器官组织细胞特异性表达的基序通常都不在370bp的范围之内。这种表面看似矛盾的现象暗示：是否可能存在一种未知的保卫细胞特异表达的基序，这有待我们进一步的实验来证实。这种因为缺失一定长度后获得的“启动子”在保持原有启动子功能的同时，又能产生出新的启动功能，这种现象尚未见有报道。对M4-1到M4-6进行的GUS定量分析结果显示：在—1868～—1506bp之间的363bp长的核苷酸序列上存在增强子，因为这段序列的缺失导致GUS活性有一个较大幅度的下降。这是首次在iMyAP启动子上发现有增强子的存在。核心启动子能在高温胁迫和正常条件下都能启动GUS基因在气孔保卫细胞里表达。但是从被染色气孔保卫细胞的数量来讲，高温下气孔染色的数量明显低于正常条件下气孔染色的数量，说明高温有抑制370bp片段启动GUS基因在保卫细胞中表达的作用。核心启动子能在低温胁迫和正常条件下都能启动GUS基因在气孔保卫细胞里表达。但是气孔保卫细胞的染色数量也明显低于正常条件下气孔染色的数量。通过PLACE对启动子全长序列分析，在370bp的序列上没有找到目前已知的与低温反应有关的基序，所以通过核心启动子的进一步实验解剖分析，将会找到新的与低温反应有关的基序。受低温处理的M4-1植株其根部只在维管束部分和第三层柱细胞(third story columella cell，S3)中部的2个细胞有深的GUS染色，而维管束外围的薄壁组织和根尖伸长区不染色。蓝色在根尖弯曲的相反方向一侧，伸长区与成熟区的交界部位，其中柱部分大约一半有蓝色，另一半没有蓝色；就柱细胞而言，S3的中间两个有蓝色，其中与弯曲方向相反的一个柱细胞染色明显比另一个强，这两个观测事实似乎暗示iMyAP可能参与根向重力性(gravitropism)的某个过程。这个推测有待进一步的实验证实。更多还原

【Abstract】 In the Cruciferae plant there is a special substrate - enzyme system, namely glucosinolate -myrosinase system. In vivo, Myrosinases usually occur in complexes with myrosinase-bingingprotein and myrosinase-associated protein, iMyAP is one of myrosinase-associated proteinspresent in vegetative organ, which can be promoted by iMyAP promoter induced by wounding,MeJA, JA and the ABA treatment.In order to further understand the function- structure characteristic of iMyAP genepromoter, to identify the size of core promoter of iMyAP promoter, we divided the whole-1868bp promoter into 5 fragments, inserted the 5 fragments and whole promoter just beforeGUS gene and transferred the fusions into Arabidopsis genome. Then the functional structure ofiMyAP gene promoter was analyzed on the arabidopsis heredity background.The results showed that the methyl jasmonnic acid(MeJA) and wounding processing canmake iMyAP promoter propel the GUS gene expression in the whole plant. The up-ground wasstained deeper than the underground and the vascular tissue deeper than the parenchyma. Thejoint area of leafstalk and root was stained the deepest.370bp fragment is enough for finishing the induced function of whole promoter by deletionanalysis. M4 - 5 with 370bp fragment. can start up the GUS gene expression after being treatedby wounding and methyl jasmonic acid and the blue position and blue intensity is consistent withone of M4-1. When the whole promoter was deleted to 153bp, the wounding and the jasmonicacid treatment cannot induce the GUS gene expression in M4-6, which means that this 153bpDNA sequence does not have the induction function after treated by wounding and MeJA. Byuse of the the PLACE, we analyzed the motif in the 370 fragment and found that the fragmentaccounting for 19% of whole promoter has all kinds of 8 motifs relative to induction of wholeiMyAP promoter and includes a sole JA-responsive motif in the whole promoter; a soleauxin-responsive motif, a sole sugar-responsive motif, one of two wounding-responsive motifs,three of four anaerobically-responsing motif, all 6 type motifs relative to dehydration-responsive(among the 6 type motifs MYCATERD1 and MYCATRD22 are sole). These data and the resultsof GUS histochemical stain together fully showed that the 370bp fragment has represented thebasic characteristic of induction function of iMyAP promoter, namely the cor promoter of theiMyAP promoter. This is the first time to identified the size of the iMyAP core promoter.Not only the 370bp core promoter can start the gene expression after wounding and MeJAtreatment, but also the core promoter has the characteristic of cell-specific promoter. The corepromoter can start the GUS gene expression specifically in the guard-cell under normal growthcondition, ands act as a typical tissue-specific promoter. But all known tissue-specific motifs arenot found in the 370bp core promoter. Under the ostensible contradictory phenomenon, it is hintsthat there is an unknown guard-cell-specific motif in the core promoter. Further experimentalneed to be done for this deduction. This phenomenon has not been reported until now that the new promoter from the primary promoter deletion not only maintains the function of primaryiMyAp, but brings novel promoter function.The GUS quantitative analysis results from M4-1 to M4-6 demonstrated that at less there isan enhance element between - 1868～- 1506bp(363bp), because the 363bp loss caused GUSactivity decrease in larger scale. It is first time to find enhance element in iMyAP promoter.The core promoter can propel the GUS gene expression in guard cell under highertemperature stress and normal growth condition. But based on the stained guard cell numbers,the blue guard cell numbers are fewer under higher temperature than normal temperature. It issuggested that higher temperature inhibits the GUS gene expression promoted by 370bp in guardcell.The core promoter can also propel the GUS gene expression in guard cell under lowertemperature stress and normal growth condition. But the stained guard-cell numbers are fewerunder lower temperature stress than normal growth condition. No known motif related to lowertemperature response had been found in the 370bp by PLACE. So further deletion analysis of thecore promoter will be helpful to find new motif responsive to lower temperature stress.At the root tip of M4-1 treated by lower temperature treatment, deeper GUS dye werealong the vascular bundle and in two central cell of the third story columella cell(S3), and theparenchyma tissue around the vascular bundle and the elongation zone were stained nothing.Opposite the curving direction, in the elongation zone, half central cylinder was stained blue,another half wasn’t stained. Speaking of the S3 columella, two have the blue color, but the cellopposite curving direction was obviously stained more intensive and the size was biggercompared to another one. According to the two observation facts, it is inferred that iMyAPpossibly participates in the response of root gravitropism. This extrapolation waits for the furtherexperimental confirmation.更多还原