定位于类囊体膜的PPF1在转基因拟南芥中的表达影响叶绿体的发育

PPF1是一个与植物营养生长相关的基因.它编码的产物可能是一个膜蛋白并与拟南芥叶绿体中的类囊体蛋白ALB3有很高的同源性.免疫电镜分析表明PPF1蛋白同样主要定位于类囊体膜,而且在短日照G2豌豆开花两周后仍发育良好的叶绿体中有很高的表达,在长日照豌豆同时期非正常叶绿体中丰度非常低.对转基因拟南芥和野生型植株的叶片衰老进程比较发现, PPF1在拟南芥中的过量表达可以延缓叶片的衰老,而用PPF1反义mRNA抑制拟南芥中的同源基因ALB3则明显加快叶片衰老速度.对转基因拟南芥的超微结构分析显示,PPF1在拟南芥中过量表达时,转基因植株的叶绿体比野生型植株的叶绿体大并含有更多的基粒和基质类囊体膜;相反,反义PPF1表达抑制其在拟南芥中的同源物时,转基因植株的叶绿体比野生型植株的叶绿体小并含有较少的基粒和发育较差的类囊体膜系统.这些数据表明叶绿体的发育状况与PPF1或拟南芥同源物ALB3的表达水平呈正相关.我们的结果提示PPF1基因可能通过控制叶绿体的发育状况来调节植物的发育.     

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.     

定位于类囊体膜的PPF1在转基因拟南芥中的表达影响叶绿体的发育

PPF1是一个与植物营养生长相关的基因。它编码的产物可能是一个膜蛋白并与拟南芥叶绿体中的类囊体蛋白ALB3有很高的同源性。免疫电镜分析表明PPF1蛋白同样主要定位于类囊体膜 ,而且在短日照G2豌豆开花两周后仍发育良好的叶绿体中有很高的表达 ,在长日照豌豆同时期非正常叶绿体中丰度非常低。对转基因拟南芥和野生型植株的叶片衰老进程比较发现 ,PPF1在拟南芥中的过量表达可以延缓叶片的衰老 ,而用PPF1反义mRNA抑制拟南芥中的同源基因ALB3则明显加快叶片衰老速度。对转基因拟南芥的超微结构分析显示 ,PPF1在拟南芥中过量表达时 ,转基因植株的叶绿体比野生型植株的叶绿体大并含有更多的基粒和基质类囊体膜 ;相反 ,反义PPF1表达抑制其在拟南芥中的同源物时 ,转基因植株的叶绿体比野生型植株的叶绿体小并含有较少的基粒和发育较差的类囊体膜系统。这些数据表明叶绿体的发育状况与PPF1或拟南芥同源物ALB3的表达水平呈正相关。我们的结果提示PPF1基因可能通过控制叶绿体的发育状况来调节植物的发育。     

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.     

PPF1基因C端片段在大肠杆菌中的融合表达和纯化以及抗体制备

PPF1是与G2豌豆短日不衰老现象紧密相关的基因之一 .以pSK PPF1为模板 ,用PCR方法得到了编码该蛋白C端的基因片段 ,称为PPFC .进一步将该基因片段克隆到中间载体pGEM T easy ,再酶切得到PPFC片段插入到pGEX 4T 1载体中 ,构建成表达质粒pGEX 4T PPFC .在BL2 1细胞中经IPTG诱导表达 ,得到GST PPFC融合蛋白 .用GST亲和柱纯化得到PPFC蛋白 ,将其作为抗原得到兔源的多克隆抗体 .Western杂交分析表明所得到的抗体具有较强的特异性 ,ELISA分析证实其效价为 10 6.该抗体的获得为用免疫沉淀等免疫分析技术研究PPF1与其它蛋白质的相互作用 ,从而阐明PPF1在延缓G2豌豆衰老中的分子机制提供了可能     

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.     

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…