Studies on gene structure,enzymatic activity and regulatory mechanism of acetohydroxy acid isomeroreductase from G2 pea

TheAAIR genomic DNA of G2 pea (Pisum sativum L.) was amplified by PCR method. Sequence analysis showed that it was composed of 8 introns and 9 exons with three of the introns containing specific A/T-rich endogenous promoter regions. Molecular hybridization experiments revealed that the expression of AAIR remained at a high level before and after flowering if grown in short day growth chambers. However, when grown under long day conditions, the level of AAIR expression declined very rapidly after flowering. This variation of AAIR expression is consistent with the change of enzymatic activity of acetohydroxy acid isomeroreductase. Functional complementation experiments carried out using an acetohydroxy acid isomeroreductase deficientE. coli strain showed that these cells could not grow on M9 medium without addition of branched-chain amino acids unless they were transformed with theAAIR expression vector. Further study revealed that overexpression of the peaAAIR cDNA in acetohydroxy acid isomeroreductase deficientE. coli strain enhanced significantly its branched-chain amino acid biosynthetic capacity. Results from gel shift experiments showed that fractions of pea nuclear protein extracts could bind specifically to some A/T rich regions present in introns of theAAIR gene. The A/T-rich-region-binding proteins remained at a steady level in the non-senescing apical buds of short-day grown G2 pea. In the rapid-senescing apical buds of long-day grown G2 pea, the levels of these proteins declined rapidly after flower initiation. Therefore, the nuclear protein binding capacities to endogenous promoter regions may constitute an important mechanism to regulateAAIR gene expression.     

L-SOD发挥活性作用的可能机制

氧化剂、还原剂处理前后,Ｌ－ＳＯＤ的活性及紫外光谱发生变化,Ｈ２Ｏ２使Ｆｅ（Ⅲ）吸收增强,同时钝化Ｌ－ＳＯＤ的活性;加入保险粉后,Ｌ－ＳＯＤ重新活化,Ｆｅ（Ⅲ）吸收减弱．ＮＥＭ封闭Ｃｙｓ后,Ｌ－ＳＯＤ紫外吸收谱发生变化,且活性减弱．说明Ｆｅ辅基及Ｃｙｓ是活性发挥的必需基团．     

光敏色素与光调控

生物体的新陈代谢和生长发育主要受遗传信息及环境信息的调控,遗传信息规定了个体发育的潜在模式,但它的实现在很大程度上受控于环境信息。光作为主要的环境因子,不仅提供光合作用所需的能量,而且触发植物形态变化、质体分化、新陈代谢等重要反应(统称为光形态建成)。光形态建成至少与四个不同类型光受体相关:光敏色素、蓝光受体、UV-A受体、UV-B受体,其中研究最深入的当属光敏色素。自1983年Vierstra和Quail分离到完整的光敏色素蛋白质以来,科学家相继对光敏色素的分子种类、生物合成与调控及生理机制展开了广泛深入的研究,并且取得了令人瞩目的进展。时至今日,随着新方法、新技术的应用,从光敏色素感受光刺激到基因在细胞核中表达,再到光形态建成的整个信号传递途径已逐步为人们所认识,许多与光敏色素调节有关的顺式因子及相应的DNA结合蛋白也已被确定。功能研究发现,没有哪一个反式作用因子在光调节的表达中单独起作用,可见光敏色素调控的基因表达是相当复杂的。本文拟就光敏色素分子、光敏色素基因家族、光敏色素所激活的信号传递途径及光敏色素与基因表达的关系等方面做一综述。     

用两步PCR法克隆全长cDNA

采用两步 PCR法成功地克隆了一个全长的 c DNA.首先 ,用差式分析法克隆得到差别表达的 c DNA片段 ,再分别用这些片段内部的特异序列及 c DNA两端不同接头的序列为引物进行第一步 PCR扩增 ,得到差别 c DNA片段的上游和下游序列 .然后 ,根据第一步 PCR扩增得到的上游和下游序列设计基因特异的引物进行第二步 PCR,从而得到全长的 c DNA.     

豌豆乙酰羟酸还原异构酶基因结构、蛋白活性分析及其调控机制研究

用PCR方法获得了G2豌豆AAIR基因的核DNA序列, 分析表明该基因由8个内含子和9个外显子组成, 其中3个内含子带有特征性的富A/T内源启动子区. 分子杂交试验表明, 短日照条件下G2豌豆中AAIR的表达不受生殖生长的影响, 开花前后都维持较高水平的表达. 长日照条件下, 植株开花后AAIR的表达水平迅速下降. AAIR基因表达强度的变化与豌豆乙酰羟酸还原异构酶的活性变化规律相一致. 用乙酰羟酸还原异构酶缺陷型大肠杆菌做功能互补实验发现, 带有豌豆AAIR cDNA的细菌能够在缺少分枝氨基酸的M9培养基上生长, 表明该基因产物确实具有乙酰羟酸还原异构酶活性. 进一步的研究表明, 豌豆AAIR基因产物能显著提高野生型大肠杆菌合成分枝氨基酸的能力. 凝胶阻滞实验表明, AAIR基因的内含子中的富A/T区能与豌豆核蛋白提取物发生特异性结合. 该蛋白在不衰老的短日照G2豌豆顶芽中一直保持稳定表达, 但在迅速衰老的长日照豌豆顶芽中, 开花后该蛋白就迅速降低直至完全消失. 因此, AAIR基因内源启动子区与核蛋白的特异性结合可能是调控该基因表达的重要机制.     

一种类SOD物质的基本性质与结构研究

玫瑰果（Ｒｏｓａｒｕｇｏｓａｖａｒ．ｃｈａｍｉｓｏｎｉａｎａ）果实中有一种具有高ＳＯＤ活性的成分,系统研究表明,这是一个小肽,定名为类ＳＯＤ物质。用有机溶剂分级、聚几内酰胺、ＤＥＡＥ１１、Ｚｅｒｏｌｉｔ２２５柱分离纯化,提纯物经紫外光谱、圆二色谱分析、氨基酸分析、邻菲咯啉定铁、ＤＮＳＣｌ法检测末端氨基酸等实验,证实类ＳＯＤ物质的等电点为５．２,每分子肽含一个Ｆｅ原子,有７０个氨基酸左右,分子量约为７０００,可能是一种结合多肽,末端氨基酸被封闭,富含ｃｙｓ,呈无规卷曲状态,这与其很高的温度稳定性及ｐＨ稳定性是一致的。     

Studies on gene structure, enzymatic activity and regulatory mechanism of acetohydroxy acid isomeroreductase from G2 pea

Using cDNA representational difference analysis (cDNA RDA), a cDNA whose expression was induced by gibberellins was cloned in our lab from G2 pea[1]. Sequence analysis showed that it shares high homology with acetohydroxy acid isomeroreductase (also known as ketol-acid reductoisomerase, EC1.1.1.86) in branched-chain amino acids biosynthetic pathway. Previous experiments confirmed that when expressed in E. coli cells, it was able to catalyze the reduction of AHB (2-aceto-2-hydroxybutyrat…