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.     

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208 bp 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。     

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。     

叶绿体分裂相关蛋白CrMinD的保守功能

细胞或质体中部正确分裂位点的选择是MinD蛋白与其他Min蛋白（MinC／E）相互作用的结果,MinD蛋白在原核细胞以及植物叶绿体的分裂过程中发挥着重要的作用。细胞中MinD蛋白浓度的明显升高可影响正常细胞的分裂过程而产生丝状体细胞。为了研究叶绿体分裂蛋白CrMinD的保守功能,构建了衣藻CrMinD-gfp的原核表达重组质粒进行了原核功能验证。试验结果表明,衣藻CrMinD蛋白的过量表达严重影响了大肠杆菌的分裂,其在原核细胞中运动和定位与用GFP标记的原核细胞MinD蛋白具有相似性。更进一步证明了叶绿体分裂同源物CrMinD蛋白与原核细胞MinD蛋白有着相似的功能,是一个进化上功能保守的蛋白。同时,这一结果也为研究植物细胞中质体的分裂机制奠定了一定的基础。     

衣藻叶绿体分裂相关基因CrFtsZ3的克隆及其原核表达

FtsZ蛋白在原核细胞以及植物细胞叶绿体的分裂过程中发挥着重要作用。为了研究叶绿体分裂装置的进化 ,运用RT PCR方法从莱茵衣藻中克隆了叶绿体分裂相关基因CrFtsZ3。由于已经从衣藻细胞中克隆了一个ftsZ基因 ,所以CrFtsZ3的克隆表明衣藻中已经存在两类不同的 ftsZ基因 ,这说明 ftsZ基因的复制与分歧发生于绿藻的分化之前。序列分析结果显示 ,CrFtsZ3所编码的蛋白质具有FtsZ蛋白的典型模体。进一步的原核表达与定位分析表明CrFtsZ3 GFP融合蛋白沿着宿主菌体的纵轴方向有规律地聚集成荧光点或荧光带 ,并且CrFtsZ3蛋白过量表达明显干挠了宿主菌正常的细胞分裂过程 ,说明衣藻CrFtsZ3蛋白能够识别宿主细胞内的分裂位点并影响细胞分裂过程 ,从而初步验证了它的生物学功能     

拟南芥3个蛋白磷酸酶2C基因编码蛋白的亚细胞定位及组织表达分析

利用gateway技术从拟南芥中克隆了3个蛋白磷酸酶2C基因At5G66080、At1G68410和At5G06750,3个基因的ORF全长分别为1 158 bp、1 311 bp和1 182 bp,分别编码一条385、376和393个氨基酸残基的多肽.构建了3个基因的植物表达载体35S:GFP:At5G66080、35S:GFP:At1G68410和35S:GFP:At5G06750,采用基因枪法进行的洋葱表皮细胞GFP瞬时表达实验表明,荧光信号主要分布在细胞核上,显示这3个基因的产物可能在细胞核上发挥作用.利用实时荧光定量PCR研究At5G66080、At1G68410和At5G06750基因在不同组织中的表达特性,结果表明:3个基因在各个器官均有表达,但表达量不同;At5G66080、At1G68410和At5G06750基因在花中表达量最大;At5G66080和At5G06750基因在根、叶和叶柄中的表达量次之,在茎中的表达量最低;At1G68410基因在根中的表达量次之,在茎、叶和叶柄中的表达量较低.     

基于四环素调控系统的叶绿体启动子在大肠杆菌中活性检测

旨在研究四环素调控系统在叶绿体基因工程中调控启动子活性。以四环素基因及四环素特异性识别序列的核心操控区为基础,首先合成带有四环素核心操控区的prrn O1O2启动子,通过酶切连接的方法连接到表达载体Bio3-GFP,并在大肠杆菌中验证该启动子的活性。结果表明,在该启动子的驱动下GFP基因表达使菌落呈明亮绿色;构建四环素诱导下GFP基因的表达载体Bio3-Tet R-prrn O1O2-GFP,筛选出适应大肠杆菌生长的最高四环素使用浓度为5μg/m L;然后构建四环素调控系统下的GFP表达载体,在未加入四环素时,prrn O1O2启动子的功能被抑制,加入四环素后,GFP基因表达出绿色荧光蛋白。说明利用四环素调控系统可以在大肠杆菌中控制叶绿体启动子prrn O1O2的活性,从而避免了核基因组对质体基因组的调控干扰,为进一步利用质体基因工程育种提供有效的方法和途径。     

利用IE基因表达外源抗原的HSVⅠ重组病毒的构建

目的 研究ICP22蛋白缺失是否影响病毒的感染复制以及ICP22蛋白在病毒水平上功能.方法 利用同源重组方法用绿色荧光蛋白（green fluorescent protein,GFP）的编码基因取代ICP22蛋白的编码基因,以流式细胞仪分选结合蚀斑筛选的方法得到ICP22蛋白缺失的单克隆重组病毒.结果 对ICP22蛋白缺失重组病毒进行绿色荧光的表达情况的镜下观察、GFP基因序列的测定、重组病毒中GFP基因的定量PCR检测、GFP蛋白表达的Western 印迹 检测等方法进行了验证,成功构建了ICP22蛋白缺失的重组病毒,并在研究过程中发现ICP22蛋白缺失的重组病毒出现感染复制停滞的现象.结论 ICP22蛋白缺失的重组病毒出现感染复制停滞的现象提示其在细胞中具有重要的功能.     

A protein oxidase catalysing disulfide bond formation is localized to the chloroplast thylakoids

In chloroplasts, thiol/disulfide-redox-regulated proteins have been linked to numerous metabolic pathways. However, the biochemical system for disulfide bond formation in chloroplasts remains undetermined. In the present study, we characterized an oxidoreductase, AtVKOR-DsbA, encoded by the gene At4g35760 as a potential disulfide bond oxidant in Arabidopsis. The gene product contains two distinct domains: an integral membrane domain homologous to the catalytic subunit of mammalian vitamin K epoxide reductase (VKOR) and a soluble DsbA-like domain. Transient expression of green fluorescent protein fusion in Arabidopsis protoplasts indicated that AtVKOR-DsbA is located in the chloroplast. The first 45 amino acids from the N-terminus were found to act as a transit peptide targeting the protein to the chloroplast. An immunoblot assay of chloroplast fractions revealed that AtVKOR-DsbA was localized in the thylakoid. A motility complementation assay showed that the full-length of AtVKOR-DsbA, if lacking its transit peptide, could catalyze the formation of disulfide bonds. Among the 10 cysteine residues present in the mature protein, eight cysteines (four in the AtVKOR domain and four in the AtDsbA domain) were found to be essential for promoting disulfide bond formation. The topological arrangement of AtVKOR-DsbA was assayed using alkaline phosphatase sandwich fusions. From these results, we developed a possible topology model of AtVKOR-DsbA in chloroplasts. We propose that the integral membrane domain of AtVKOR-DsbA contains four transmembrane helices, and that both termini and the cysteines involved in catalyzing the formation of disulfide bonds face the oxidative thylakoid lumen. These studies may help to resolve some of the issues surrounding the structure and function of AtVKOR-DsbA in Arabidopsis chloroplasts.     

AKR2A-mediated import of chloroplast outer membrane proteins is essential for chloroplast biogenesis

In plant cells, chloroplasts have essential roles in many biochemical reactions and physiological responses. Chloroplasts require numerous protein components, but only a fraction of these proteins are encoded by the chloroplast genome. Instead, most are encoded by the nuclear genome and imported into chloroplasts from the cytoplasm post-translationally. Membrane proteins located in the chloroplast outer envelope membrane (OEM) have a critical function in the import of proteins into the chloroplast. However, the biogenesis of chloroplast OEM proteins remains poorly understood. Here, we report that an Arabidopsis ankyrin repeat protein, AKR2A, plays an essential role in the biogenesis of the chloroplast OEM proteins. AKR2A binds to chloroplast OEM protein targeting signals, as well as to chloroplasts. It also displays chaperone activity towards chloroplast OEM proteins, and facilitates the targeting of OEP7 to chloroplasts in vitro. AKR2A RNAi in plants with an akr2b knockout background showed greatly reduced levels of chloroplast proteins, including OEM proteins, and chloroplast biogenesis was also defective. Thus, AKR2A functions as a cytosolic mediator for sorting and targeting of nascent chloroplast OEM proteins to the chloroplast.     

Tic21 is an essential translocon component for protein translocation across the chloroplast inner envelope membrane

An Arabidopsis thaliana mutant defective in chloroplast protein import was isolated and the mutant locus, cia5, identified by map-based cloning. CIA5 is a 21-kD integral membrane protein in the chloroplast inner envelope membrane with four predicted transmembrane domains, similar to another potential chloroplast inner membrane protein-conducting channel, At Tic20, and the mitochondrial inner membrane counterparts Tim17, Tim22, and Tim23. cia5 null mutants were albino and accumulated unprocessed precursor proteins. cia5 mutant chloroplasts were normal in targeting and binding of precursors to the chloroplast surface but were defective in protein translocation across the inner envelope membrane. Expression levels of CIA5 were comparable to those of major translocon components, such as At Tic110 and At Toc75, except during germination, at which stage At Tic20 was expressed at its highest level. A double mutant of cia5 At tic20-I had the same phenotype as the At tic20-I single mutant, suggesting that CIA5 and At Tic20 function similarly in chloroplast biogenesis, with At Tic20 functioning earlier in development. We renamed CIA5 as Arabidopsis Tic21 (At Tic21) and propose that it functions as part of the inner membrane protein-conducting channel and may be more important for later stages of leaf development.     

Is There a COPII‐Mediated Membrane Traffic in Chloroplasts?

COPII proteins facilitate membrane transport from the endoplasmic reticulum (ER) to the Golgi. They are highly conserved, although there are variations in their subcellular localization across plant, animal and yeast cells. Such variations may be needed to suit the unique organization of the ER and Golgi in the different cell systems. Earlier bioinformatics analyses have indicated that the Arabidopsis nuclear genome may encode chloroplast isoforms of the cytosolic trafficking protein machineries, including COPI and COPII, for vesicular transport within chloroplasts. These analyses suggest the intriguing possibility that plants may have evolved or adapted COP-like proteins to suit membrane trafficking events within specialized organelles. Here, we discuss recent data on the distribution and activity of the product of the At5g18570 locus, which encodes a putative chloroplast isoform of Sar1, the GTPase that regulates COPII assembly on the surface of the ER. Evidence is accumulating that the protein is targeted to the chloroplasts, that it has GTPase activity and that it may have a role in thylakoid membrane development, supporting the possibility that COPII-like trafficking machinery may be active in chloroplasts.     

RASPBERRY3 gene encodes a novel protein important for embryo development

We identified a new gene that is interrupted by T-DNA in an Arabidopsis embryo mutant called raspberry3. raspberry3 has "raspberry-like" cellular protuberances with an enlarged suspensor characteristic of other raspberry embryo mutants, and is arrested morphologically at the globular stage of embryo development. The predicted RASPBERRY3 protein has domains found in proteins present in prokaryotes and algae chloroplasts. Computer prediction analysis suggests that the RASPBERRY3protein may be localized in the chloroplast. Complementation analysis supports the possibility that the RASPBERRY3 protein may be involved in chloroplast development. Our experiments demonstrate the important role of the chloroplast, directly or indirectly, in embryo morphogenesis and development.