大蒜中植物络合素合酶基因的克隆及功能分析

【作者】 姜瑛楠；

【导师】 麻密；

【作者基本信息】 中国科学院研究生院（植物研究所） ， 细胞生物学， 2003， 硕士

【摘要】 随着现代工业的发展，重金属污染已经成为一个非常严重的问题。 传统的重金属治理方法花费较高并且过程非常繁琐。植物修复技术是一种经济并且有效的利用植物进行环境污染治理的新方法。植物络合素是一类植物体内络合重金属离子的多肽，它已经发现于多种植物与微生物中。植物络合素合酶是催化GSH合成植物络合素的关键酶。因此揭示植物络合素合酶的分子机理对于了解植物对重金属抗性的机制又很重要的意义。迄今为止，关于植物络合素合酶基因的研究主要集中在两种非重金属的植物中：拟南芥与小麦。许多关于该基因结构与功能的问题依然很不清楚。大蒜是一种能够抗很高浓度重金属的植物。在本研究中，我们利用大蒜这种重金属抗性植物对以下问题做了较为系统的研究：1． 测量了大蒜在重金属胁迫下的生理表现，并得出大蒜是一种具有重金属抗性的植物；2． 我们从大蒜中克隆出一个新的植物络合素合酶基因。该基因全长1868bp，包含一个506个氨基酸的开放读码框并编码一个55． 8KD的蛋白。该基因转译的氨基酸序列与其他十二种物种的植物络合素合酶氨基酸序列具有很高的同源性；3． 酵母功能互补试验证明表达AsPCS的酵母可以比对照耐受更高浓度的镉与砷。这表明AsPCS的转译产物在酵母与植物的重金属的耐受过程中起很重要的作用；4． RT-PCR的结果表明，经过重金属Cd2+的胁迫，AsPCS在根中与茎中的表达量都有提高，这说明AsPCS的调控是发生在转录水平上的。另外通过比较该基因在相同处理条件下根中与茎中的表达量，我们发现AsPCS在根中的表达量远高于茎中；5． 原位杂交显示AsPCS主要表达于根的表皮、顶端分生组织、韧皮部，并且当重金属压力提高后，表皮的表达量明显提高。更多还原

【Abstract】 Heavy metals have become one of the most serious environmental pollution problems worldwide. Traditional methods to remove the heavy metals from environments are very expensive and inconvenient. Phytoremediation is a new cheap and effective way that depends plants to clean the environmental pollution. Phytochelatins (PCs) are a type of polypeptide in plant to bind heavy metals. They have been identified in a wide variety of plant species and in some microorganisms. PCs are synthesized from GSH by the catalysis of phytochelatin synthase. So revealing the molecular base of the phytochelatins synthase is very important to understand the mechanism of heavy metal resistance in plants. Until now, the research about phytochelatins synthase genes only focuses on two heavy metal sensitive plant: Arabidopsis thaliana and Triticum aestivum. Many problems about the genes’ structures and functions of transcript products are still unclear. Allium Sativum L. (garlic) is a kind of plant which can tolerate much heavy metal stress. In this study, we used this plant and make some research below:1. The physiological performance of garlic under heavy metal stress was determined and we get a conclusion that garlic is one of heavy metal tolerance plants.2. A new phytochelatin synthase cDNA from Allium Sativum L. (AsPCS) was cloned. This cDNA sequence with a full length of 1868 bp contains an open reading frame (ORF) of 506 amino acids and encodes a protein with 55.8 kDa molecular weight. The predicted polypeptide shows high homology with PC synthase from twelve other species;3. The AsPCS-expression yeast cells can tolerate more cadmium and arsenite stress. It suggests that as a key enzyme, the transcript product of AsPCS plays an important role in different heavy metals tolerance.4. The results of RT-PCR showed that the expression of AsPCS was enhanced by Cd2+ in both roots and shoots in garlic. It means that AsPCS was regulated at thetranscriptional level. Moreover, comparing the expression of AsPCS in the roots and shoots, we found that the roots display a much higher expression level than the shoots;5. The results of in situ hybridization demonstrated that AsPCS was found to be expressed in the apical meristem, epidermis, and vascular cylinder of roots. In addition, the expression quality of this gene in epidermis was increased under Cd2+ stress.更多还原