拟南芥未知功能基因At4g22890编码蛋白的叶绿体定位

蛋白质的亚细胞定位信息对于深入了解该蛋白质的功能具有重要意义。本文对一个预测的拟南芥叶绿体未知功能基因At4g22890编码蛋白进行了叶绿体定位研究。我们克隆了该基因5′端长208bp的DNA片段,与绿色荧光蛋白(GFP)基因构建重组表达载体pMON530-cTP-GFP,经农杆菌介导转化拟南芥。转基因植株经激光共聚焦显微镜观察,GFP荧光仅在叶绿体中观察到,表明所克隆的DNA序列编码的多肽能够将At4g22890编码蛋白质引导进入叶绿体,由此推测该蛋白质为叶绿体蛋白质。     

拟南芥未知功能基因At3g61870编码蛋白的叶绿体定位研究

利用反向遗传学研究方法对1个预测的拟南芥叶绿体未知功能基因At3g61870编码蛋白进行了亚细胞定位研究.通过克隆At3g61870基因5′端长229 bp的DNA片段,与绿色荧光蛋白(GFP)基因构建重组表达载体pMON530-CP-TP-GFP,经农杆菌介导转化拟南芥.转基因植株的叶肉细胞经激光共聚焦显微镜观察,叶绿素自发荧光与GFP荧光共定位于叶绿体中.结果表明,未知功能基因At3g61870编码的蛋白质为叶绿体蛋白质.     

两个拟南芥未知功能蛋白的亚细胞定位研究

蛋白质的亚细胞定位对于深入了解该蛋白质所行使的生理功能具有重要意义。经生物信息学预测,两个拟南芥未知功能基因At4g16410与Atl gI8060编码蛋白含有叶绿体定位信息。我们分别克隆了这两个基因5’端长199bp与220bp的DNA片段,与绿色荧光蛋白（GFP）基因构建重组表达载体pMON530-cTP1-GFP与pMON530-cTP2-GFP,经农杆菌介导转化拟南芥。两种转基因植株经激光共聚焦显微镜观察,GFP荧光仅在叶绿体中观察到,表明所克隆的两段DNA序列编码的多肽能够将At4gl6410与Atlgl8060编码蛋白质引导进入叶绿体,确定这两个蛋白质均为叶绿体蛋白质。     

拟南芥中一个未知功能蛋白的叶绿体亚细胞定位研究

生物信息学分析表明, 模式植物拟南芥叶绿体中含有大约4 000多种蛋白质, 目前只分离得到1 000多种, 其他预测的叶绿体蛋白的实验验证对叶绿体功能研究有重要意义。本文对一个预测的叶绿体未知功能蛋白AT5G48790进行了亚细胞定位研究。我们克隆了该基因5'端长178 bp的DNA片段, 与绿色荧光蛋白(GFP)基因构建重组载体pMON530-cTP-GFP。转基因植株通过激光共聚焦显微镜观察, GFP只在叶绿体中特异表达。实验结果表明, AT5G48790的确为叶绿体蛋白。本实验方法也可用于其他预测的蛋白质的实验验证。     

拟南芥中一个未知功能蛋白的叶绿体亚细胞定位研究

生物信息学分析表明,模式植物拟南芥叶绿体中含有大约4000多种蛋白质,目前只分离得到1000多种,其他预测的叶绿体蛋白的实验验证对叶绿体功能研究有重要意义。本文对一个预测的叶绿体未知功能蛋白AT5G48790进行了亚细胞定位研究。我们克隆了该基因5端长178bp的DNA片段,与绿色荧光蛋白（GFP）基因构建重组载体pMON530-cTP-GFP。转基因植株通过激光共聚焦显微镜观察,GFP只在叶绿体中特异表达。实验结果表明,AT5G48790的确为叶绿体蛋白。本实验方法也可用于其他预测的蛋白质的实验验证。     

拟南芥砷诱导基因At4g13180的表达模式研究

拟南芥(Arabidopsis thaliana)砷诱导基因At4g13180编码蛋白是短链脱氢酶(Short-Chain Dehydrogenase/Reductase Superfamily,SDR)家族的成员之一,其过表达可以增强植物对过氧化氢的耐受性。该实验通过半定量RT-PCR,构建ProAt4g13180:GUS、At4g13180-EGFP和At4g13180-OE表达载体,获得At4g13180基因过表达转基因株系,并研究了At4g13180基因的表达模式及其编码蛋白的亚细胞定位。结果显示,At4g13180基因在根尖、叶脉、萼片和花丝等组织都强烈表达,该基因编码蛋白主要定位于胞质和核中。该研究结果为深入探究拟南芥砷诱导基因At4g13180的功能奠定了一定的基础。     

拟南芥醛还原酶编码基因At3g04000表达模式分析及过量表达转基因植株获得

植物体内的α,β-不饱和活性醛类化合物对植物细胞具有毒害作用,清除这些α,β-不饱和活性醛类化合物对于植物细胞维持正常的生命活动至关重要。前人研究报道通过体外酶活测定和异源瞬时表达鉴定拟南芥 At3g04000基因编码的蛋白为 NADPH 依赖的叶绿体醛还原酶(Arabidopsis NADPH-dependent chloroplastic aldehyde reductases, AtChlADRs),推测其在清除叶绿体中长链(≥5)α,β-不饱和醛类物质中具有重要的功能。该研究主要构建了拟南芥 At3g04000基因的表达模式分析载体 ProAt3g04000：GUS、亚细胞定位分析载体At3g04000-EGFP 和过量表达载体 At3g04000-OE,并获得了转基因拟南芥,并通过实时定量 PCR 分析了At3g04000基因在拟南芥不同组织中的转录水平。结果表明：拟南芥 At3g04000基因在幼苗中的转录水平最高,在莲座叶、茎生叶、花序和角果中均有较高的转录水平;而在根部和茎秆中的转录水平较低。通过对ProAt3g04000：GUS 转基因植株的 GUS 染色分析可知,At3g04000基因在子叶、莲座叶和萼片的维管组织和保卫细胞中均有较强的表达,在根的维管组织中有较弱的表达。通过共聚焦显微镜对 At3g04000-EGFP 转基因植株的观察和分析发现,At3g04000不是定位于叶绿体中,而是定位在细胞质和细胞核中。该研究结果为深入研究拟南芥醛还原酶编码基因 At3g04000的功能奠定了基础。     

鸭梨PPO与GFP融合基因的烟草转化及亚细胞定位观察

将鸭梨PPO基因与绿色萤光蛋白GFP基因相融合共同进行遗传转化的方式,对鸭梨多酚氧化酶开展细胞定位研究。通过克隆该酶基因除终止密码子TAA外长度为1 779bp的CDS序列,与绿色荧光蛋白基因重组构建了荧光表达载体pBI121-PPO-GFP,借助农杆菌转化烟草,转基因烟草叶片细胞经激光扫描共聚焦显微镜观察,绿色荧光蛋白荧光与叶绿体自发荧光相重合。结果表明鸭梨多酚氧化酶为叶绿体蛋白质。     

拟南芥膜联蛋白2的亚细胞定位研究

膜联蛋白(annexin)是一类依赖钙离子的多功能磷脂结合蛋白家族,在进化上高度保守,但不同的膜联蛋白基因的表达模式和蛋白质的亚细胞定位具有特异性。拟南芥中已经鉴定出8个编码膜联蛋白的基因,在生长发育和对逆境胁迫响应过程中起作用。已知拟南芥膜联蛋白2参与根的分泌活动和生长素介导的根的向地性反应,但作用机制不清楚。蛋白质的亚细胞定位能为研究其功能和作用机制提供重要参考信息。将编码膜联蛋白2的序列克隆到植物双元表达载体p CAMBIA1300-m Cherry上,在拟南芥中表达Ann At2-m Cherry。利用荧光蛋白技术、m Cherry与绿色荧光蛋白标记的细胞器标记物共定位技术以及细胞器特异性荧光染料染色技术,作者研究了膜联蛋白2的亚细胞定位。结果显示,膜联蛋白2定位于细胞质、细胞核、高尔基体和内质网中,表明该蛋白质可能具有非常重要的功能和复杂的蛋白质翻译与转运调控机制。更多结果发现,转基因拟南芥中膜联蛋白2与绿色荧光蛋白标记的微丝骨架存在共定位现象,推测该蛋白可能通过微丝骨架调节及微丝骨架介导的囊泡运输参与细胞分泌活动。该文为进一步研究膜联蛋白2蛋白质的翻译与转运调控以及作用机制提供了实验依据。     

小立碗藓叶绿体分裂相关基因PpMinE的克隆及其功能与进化分析

用RT-PCR技术从小立碗藓中(Physcomitrella patens)克隆了核编码的MinE基因,命名为PpMinE,并克隆了该基因的基因组DNA。序列比对显示该基因编码的蛋白质与真细菌和绿藻叶绿体编码的MinE蛋白具有较高的相似性。pMinE-EGFP融合蛋白在烟草中的瞬时表达证明该蛋白定位于叶绿体内。在大肠杆菌中过量表达PpMinE导致细胞不正常分裂,产生无染色体的小细胞,这表明MinE的功能在进化上是保守的。在系统发育树中,PpMinE和高等陆生植物有较近的亲缘关系。在已知的陆生植物的叶绿体基因组中没有找到MinE的同源蛋白,这暗示在进化过程中MinE从叶绿体到细胞核的水平转移可能发生在陆生植物发生以前。     

一个推测的拟南芥漆酶基因在巴斯德毕赤酵母中的表达

At5g01040基因是一个推测的拟南芥漆酶基因。根据其编码序列设计引物,利用RT—PCR方法扩增出1755bp的片段。测序结果表明,该片段存在3个突变位点,其中一个突变位点改变了所编码的氨基酸。将该片段克隆到表达载体pPICZaB上,电击转化毕赤酵母。经筛选获得80个候选重组菌株,其中18个菌株的培养液中存在漆酶活性,说明所克隆的基因在毕赤酵母中实现了分泌表达,证实了At5g01040编码的蛋白具有漆酶活性。     

Characterization of Geranium (Pelargonium graveolens) Chloroplast EF-Tu cDNA

A geranium (Pelargonium graveolens) chloroplast translational elongation factor EF-Tu (tufA) cDNA was isolated. The geranium tufA cDNA is 1,584 bp long with 20 bp of 5 untranslated region (UTR) and 139 bp of 3 UTR. It encodes 474 amino acids including a putative chloroplast transit peptide of 65 amino acids. The deduced polypeptides of the geranium tufA cDNA contains four GTP binding sequences in its N-terminal region and two chloroplast EF-Tu signature regions in the C-terminal region. The predicted molecular weight of the mature geranium chloroplast EF-Tu protein was about 45,000 and its amino acid sequence identity with the chloroplast EF-Tu proteins of tobacco, pea, Arabidopsis, rice, and soybean ranges from 85% to 91%. The geranium tufA appears to exist as a single copy gene like Arabidopsis and rice, whereas other known dicot plants have more than one copy in their nuclear genomes.     

A plant-specific protein essential for blue-light-induced chloroplast movements

In Arabidopsis (Arabidopsis thaliana), light-dependent chloroplast movements are induced by blue light. When exposed to low fluence rates of light, chloroplasts accumulate in periclinal layers perpendicular to the direction of light, presumably to optimize light absorption by exposing more chloroplast area to the light. Under high light conditions, chloroplasts become positioned parallel to the incoming light in a response that can reduce exposure to light intensities that may damage the photosynthetic machinery. To identify components of the pathway downstream of the photoreceptors that mediate chloroplast movements (i.e. phototropins), we conducted a mutant screen that has led to the isolation of several Arabidopsis mutants displaying altered chloroplast movements. The plastid movement impaired1 (pmi1) mutant exhibits severely attenuated chloroplast movements under all tested fluence rates of light, suggesting that it is a necessary component for both the low- and high-light-dependant chloroplast movement responses. Analysis of pmi1 leaf cross sections revealed that regardless of the light condition, chloroplasts are more evenly distributed in leaf mesophyll cells than in the wild type. The pmi1-1 mutant was found to contain a single nonsense mutation within the open reading frame of At1g42550. This gene encodes a plant-specific protein of unknown function that appears to be conserved among angiosperms. Sequence analysis of the protein suggests that it may be involved in calcium-mediated signal transduction, possibly through protein-protein interactions.     

Identification, expression, and functional analyses of a thylakoid ATP/ADP carrier from Arabidopsis

In plants the chloroplast thylakoid membrane is the site of light-dependent photosynthetic reactions coupled to ATP synthesis. The ability of the plant cell to build and alter this membrane system is essential for efficient photosynthesis. A nucleotide translocator homologous to the bovine mitochondrial ADP/ATP carrier (AAC) was previously found in spinach thylakoids. Here we have identified and characterized a thylakoid ATP/ADP carrier (TAAC) from Arabidopsis.(i) Sequence homology with the bovine AAC and the prediction of chloroplast transit peptides indicated a putative carrier encoded by the At5g01500 gene, as a TAAC. (ii) Transiently expressed TAAC-green fluorescent protein fusion construct was targeted to the chloroplast. Western blotting using a peptide-specific antibody together with immunogold electron microscopy revealed a major location of TAAC in the thylakoid membrane. Previous proteomic analyses identified this protein in chloroplast envelope preparations. (iii) Recombinant TAAC protein specifically imports ATP in exchange for ADP across the cytoplasmic membrane of Escherichia coli. Studies on isolated thylakoids from Arabidopsis confirmed these observations. (iv) The lack of TAAC in an Arabidopsis T-DNA insertion mutant caused a 30-40% reduction in the thylakoid ATP transport and metabolism. (v) TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants.