GFP基因转化香樟胚性愈伤组织的研究

以香樟胚性愈伤组织作为受体,利用根癌农杆菌介导法进行了绿色荧光蛋白基因(GFP)的遗传转化研究。经农杆菌侵染后的胚性愈伤组织通过共培养、选择培养后获得抗性愈伤组织和体胚,对抗性愈伤组织及体胚的诱导过程进行了GFP荧光检测。结果表明,GFP基因能在抗性愈伤组织和体胚中强烈表达,证明GFP基因能够在香樟遗传转化中得到应用。对抗性愈伤组织的PCR检测初步证实外源GFP基因已整合到香樟胚性愈伤组织的基因组中。     

GFP基因转化藿香的研究

以藿香无菌苗幼嫩茎段为受体,利用根癌农杆菌介导法进行了绿色荧光蛋白基因(GFP)的遗传转化研究。经农杆菌侵染,通过共培养、选择培养后获得其抗性愈伤组织,对抗性愈伤组织的诱导过程进行了GFP荧光检测。结果表明,GFP基因能在抗性愈伤组织中强烈表达,证明GFP基因能够在藿香遗传转化中得到应用。对抗性愈伤组织的PCR检测初步证实外源GFP基因已整合到藿香愈伤组织的基因组中。     

水稻愈伤组织形态发生中的MADS盒基因的差异表达

采用MADS(MCM1-Agamous-Deficiens-SRF)盒基因家族功能区保守序列PCR引物,将水稻(Oryzasativa L.ssp indica)“珍汕97B”悬浮细胞、愈伤组织、分化愈伤组织和再生试管苗等不同形态发生的组织的mRNA反转录后选择性放大,经测序胶分离鉴定出一组差异表达的cDNA。对命名为RM1 cDNA的5＇端序列测定表明,RM1与典型的MADS基因——拟南芥agamous蛋白保守区一级结构同源性达63％,模拟二级结构相似性显著,初步确认RM1属于MADS基因家族成员。分子杂交证实,RM1在悬浮培养细胞中不表达,而在愈伤组织、分化愈伤组织和再生试管苗中活跃表达。     

水稻OsRab7耐盐功能的初步鉴定及其表达载体的构建

Rab7是一个GTP结合蛋白,隶属于Rab家族,该家族在调控膜泡的运输、结合以及细胞内膜结构的组织中行使重要作用.本实验通过RT-PCR技术从盐敏感水稻品种'日本晴'('Nipponbare')获得了OsRab7基因全长序列,成功构建了原核表达载体pET-32a-OsRab7.在IPTG诱导下,该蛋白高效表达,并明显缓解了高盐环境(4.5%～8.5% NaCl)对大肠杆菌的生长胁迫.为进一步研究该基因的功能,将OsRab7在大肠杆菌中的表达蛋白纯化, 并利用p1301B(pCAMBIA1301改造载体)构建了植物表达载体,成功转化来源于水稻株系'中花11'(盐敏感)的愈伤组织,为探讨其在水稻中的表达模式和功能分析及应用于水稻耐盐品种的改良奠定了一定的基础.     

绿色荧光蛋白与人肾脏NaDC3融合基因的构建及其在肾小管上皮细胞中的定位

本采用RT-PCR方法从人的正常肾组织中扩增出hNaDC3基因全长cDNA序列,采用基因重组技术将hNaDC3基因和绿色荧光蛋白基因融合,通过真核表达质粒—脂质体介导,导入LLC-PKl细胞系,激光共聚焦显微镜动态观察GFP—hNaDC3的定位情况。结果显示PKGFP-C3空白质粒转染的LLC—PKl细胞表达了GFP,GFP在胞内分布以核浆为主,呈均匀致密的细颗粒荧光,胞核染色强,核仁荧光稀少,界线清楚。而pKGFP-C3-hNaDC3转染细胞株的绿色荧光蛋白,转染后第1天混合分布于细胞浆和细胞膜,以粗颗粒荧光为主,两界限不清楚,胞浆中可见未染色的圆形空洞,未见胞核染色;第3天主要聚集于细胞膜,核周的胞浆中可见粗颗粒荧光物质,胞浆和胞膜界线清楚,胞核亦未见绿色荧光蛋白;第5天清晰聚集于细胞膜,细胞膜连接至网状。因此hNaDC3蛋白在细胞质中生成后,定位于细胞膜并稳定表达。     

绿色荧光蛋白基因导入胡萝卜愈伤组织并获得表达

目的:将绿色荧光蛋白基因(green fluorescent protein,GFP)重组到胡萝卜愈伤组织细胞中,使其获得表达,为今后利用GFP基因作为植物报告基因提供条件。方法:通过冻融法将含有GFP基因的重组表达载体PBI1121转入到根癌农杆菌EHA105中,再利用根癌农杆菌介导的方法将GFP基因导入到胡萝卜愈伤组织细胞中,经过除菌和抗性筛选后观测转化结果。结果:荧光显微镜观测到被转化的愈伤组织在受蓝光激发后发出绿色荧光,利用PCR法扩增出约740bp的目的基因片断。结论:GFP基因在胡萝卜愈伤组织细胞中获得了表达。     

OsPIN1a基因在水稻植株不同组织表达与定位研究

目的:为了探讨水稻生长素极性运输输出载体蛋白OsPIN1a的在水稻不同组织的作用和分布。方法:以带有GFP和GUS标记的转OsPIN1a基因水稻和野生型中花11水稻为研究材料。通过PCR方法检测抗性标记基因潮霉素基因判断植株是否阳性;通过半定量RT-PCR方法分析OsPIN1a基因在转基因植株幼穗、叶片、根系的表达;通过GUS组织染色检测OsPIN1a在幼穗、叶片、根系中的活性;通过激光共聚焦显微镜观察OsPIN1a-GFP蛋白在根尖亚细胞定位情况。结果:实验所用的水稻株系均为转基因株系;RT-PCR检测发现,在根尖、叶片和幼穗中hpt和OsPIN1a基因均有表达;GUS组织染色结果表明,幼穗、叶片、根系均有GUS活性,其中在幼嫩的组织中表达量最大,如刚萌发种子胚芽鞘、花药和柱头;GUS活性受外源生长素诱导而受生长素极性运输抑制剂(TIBA)的抑制;激光共聚焦显微镜观察发现,OsPIN1a-GFP蛋白主要分布在根尖细胞膜上。结论:实验表明OsPIN1a基因参与了水稻各个器官和组织的发育,这些组织发育可能都受到生长素极性运输的调控。     

用一种新型基因枪将外源基因转入水稻细胞(简报)(英文)

本文介绍了一种设计上改进的基因枪,用这种新型基因枪将外源基因转移到水稻细胞获得了表达。用包有pBI 121质粒DNA的钨微弹轰击水稻悬浮细胞以及成熟种子胚后,在悬浮细胞中检测到了外源基因(GUS)的表达。胚诱导的愈伤组织在无抗生素选择的条件下扩增并分化、再生,共获得30株绿色小植株。经DNA斑点杂交测得其中两株的植物总DNA中存在GUS基因。Southern杂交分析证实GUS基因整合到了水稻基因组.     

金鱼草愈伤组织过氧化物酶活性及其同工酶变化

在不同激素比例的MS培养基上 ,金鱼草 (Antirrhinummajus)茎愈伤组织主要有三种分化状态 :a愈伤组织不分化 ;b以器官发生途径分化出不定芽和不定根 ;c通过胚胎发生途径形成胚状体。过氧化物酶活性与愈伤组织分化程度呈正相关。未分化愈伤组织与分化芽、分胚状体及分化芽和根的愈伤组织酶活性呈线性递增关系 ,相对酶活力为 1:3:5 :6。在其同工酶谱中 ,三种状态的愈伤组织都具有酶带I和II,表明这两种同工酶与细胞的增殖生长有联系。分化的愈伤组织比未分化愈伤组织多出现酶带Ⅲ、Ⅳ和Ⅴ ,说明这三条酶带是影响分化的特异蛋白质。在分化芽的愈伤组织中 ,还存在酶带Ⅳ ,说明它是催化器官发生途径的特有同工酶。     

龙眼体胚发生过程生长素响应因子DlARF5a的克隆及表达分析

以龙眼松散型胚性愈伤组织为材料,采用RT-PCR和RACE技术克隆获得生长素响应因子ARF5acDNA全长序列,利用实时荧光定量法检测其在龙眼体胚不同发育时期的转录水平,并将其与绿色荧光蛋白(GFP)构建成亚细胞定位载体侵染洋葱表皮细胞。结果表明:龙眼ARF5a(命名为DlARF5a,GenBank登录号为KF739401)的cDNA序列全长为3 322bp,其中开放阅读框为2 829bp,212bp 5′非编码区,258bp 3′非编码区,编码942个氨基酸。生物信息学预测显示,DlARF5a编码蛋白具有B3、Auxin-resp和AUX-IAA保守区与结构功能域,中间区域富含谷甘氨酸、丝氨酸和亮氨酸,是一个定位于细胞质的具有促进转录活性功能的水溶性蛋白。系统进化树分析表明,该基因编码的氨基酸序列与大豆的亲缘关系最近。qRT-PCR检测显示,DlARF5a在龙眼球形胚和鱼雷形胚时期表达显著增强,而在6个发育阶段中子叶形胚的表达量最低,推测DlARF5a参与龙眼体胚中鱼雷胚时期的形态建成。共聚焦显微镜观察结果显示,未添加外源生长素IAA的DlARF5a蛋白定位于细胞质中,而经外源生长素处理的DlARF5a蛋白在细胞膜、细胞质和细胞核中均有表达,推测龙眼生长素响应蛋白DlARF5a能够响应外源生长素IAA而改变其在细胞中的空间位置。     

Molecular and biochemical analyses of OsRab7, a rice Rab7 homolog

Rab7 is a small GTP-binding protein important in early to late endosome/lysosome vesicular transport in mammalian cells. We have isolated a Rab7 cDNA clone, OsRab7, from a cold-treated rice cDNA library by the subtraction screening method. The cDNA encodes a polypeptide of 206 amino acids with a calculated molecular mass of about 23 kDa. Its predicted amino acid sequence shows significantly high identity with the sequences of other Rab7 proteins. His-tagged OsRab7 bound to radiolabeled GTPgammaS in a specific and stoichiometric manner. Biochemical and structural properties of the Rab7 wild type (WT) protein were compared to those of Q67L and T22N mutants. The detergent 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate (CHAPS) increased the guanine nucleotide binding and hydrolysis activities of Rab7WT. The OsRab7Q67L mutant showed much lower GTPase activity compared to the WT protein untreated with CHAPS, and the T22N mutant showed no GTP binding activity at all. The OsRab7Q67L mutant was constitutively active for guanine nucleotide binding while the T22N mutant (dominant negative) showed no guanine nucleotide binding activity. When bound to GTP, the Rab7WT and the Q67L mutants were protected from tryptic proteolysis. The cleavage pattern of the Rab7T22N mutant, however, was not affected by GTP addition. Northern and Western blot analyses suggested that OsRab7 is distributed in various tissues of rice. Furthermore, expression of a rice Rab7 gene was differentially regulated by various environmental stimuli such as cold, NaCl, dehydration, and ABA. In addition, subcellular localization of OsRab7 was investigated in the Arabidopsis protoplasts by a double-labeling experiment using GFP-fused OsRab7 and FM4-64. GFP-OsRab7 is localized to the vacuolar membrane, suggesting that OsRab7 is implicated in a vesicular transport to the vacuole in plant cells.     

OsGAP1 functions as a positive regulator of OsRab11-mediated TGN to PM or vacuole trafficking

The Ypt/Rab family of small G-proteins is important in regulating vesicular transport. Rabs hydrolyze GTP very slowly on their own and require GTPase-activating proteins (GAPs). Here we report the identification and characterization of OsGAP1, a Rab-specific rice GAP. OsGAP1 strongly stimulated OsRab8a and OsRab11, which are homologs of the mammalian Rab8 and Rab11 proteins that are essential for Golgi to plasma membrane (PM) and trans-Golgi network (TGN) to PM trafficking, respectively. Substitution of two invariant arginines within the catalytic domain of Oryza sativa GTPase-activating protein 1 (OsGAP1) with alanines significantly inhibited its GAP activity. In vivo targeting experiments revealed that OsGAP1 localizes to the TGN or pre-vacuolar compartment (PVC). A yeast expression system demonstrated that wild-type OsGAP1 facilitates O. sativa dissociation inhibitor 3 (OsGDI3)-catalyzed OsRab11 recycling at an early stage, but the OsGAP1(R385A) and (R450A) mutants do not. Thus, GTP hydrolysis is essential for Rab recycling. Moreover, expression of the OsGAP1 mutants in Arabidopsis protoplasts inhibited the trafficking of some cargo proteins, including the PM-localizing H+-ATPase-green fluorescent protein (GFP) and Ca2+-ATPase8-GFP and the central vacuole-localizing Arabidopsis aleurain-like protein (AALP)-GFP. The OsGAP1 mutants caused these proteins to accumulate at the Golgi apparatus. Surprisingly, OsRab11 overproduction relieved the inhibitory effect of the OsGAP1 mutants on vesicular trafficking. OsRab8a had no such effect. Thus, the OsGAP1 mutants may inhibit TGN to PM or central vacuole trafficking because they induce the sequestration of endogenous Rab11. We propose that OsGAP1 facilitates vesicular trafficking from the TGN to the PM or central vacuole by both stimulating the GTPase activity of OsRab11 and increasing the recycling of inactive OsRab11.     

Disorder of trafficking system of plasma membrane and vacuole antiporter proteins causes hypersensitive response to salinity stress in <Emphasis Type="Italic">Arabidopsis Thaliana</Emphasis>

Rab GTPases play an important role in regulating intracellular vesicular trafficking in eukaryotic cells. Previously, we found that Oryza sativa rice Rab11 (OsRab11) is required for the regulation of vesicular trafficking from the trans- Golgi network (TGN) to the plasma membrane (PM) and/or vacuoles. To further elucidate the relationship between vesicular trafficking and abiotic and biotic stresses, we determined OsRab11 expression levels under several environmental stress conditions. OsRab11 expression was induced by pathogens, jasmonic acid (JA), and high salt treatment. Under high salt conditions, dominant negative OsRab11(S28N) mutant plants exhibited a hypersensitive phenotype similar to that of sos1-1, whereas overexpressed-OsRab11 plants showed resistance to high salt stress. When the expression of vacuolar and PM Na⁺/H⁺ antiporter genes such as AtNHX1, AtNHX2, and AtSOS1 was examined, there was no significant difference between the wild-type and OsRab11(S28N) mutant plants. However, PM trafficking of AtSOS1-green fluorescent protein (GFP) in 35S::AtSOS1-GFP sos1-1 plants was severely impaired by T7-OsRab11(S28N) expression. Similarly, vacuolar trafficking of AtNHX2-GFP was inhibited by T7-OsRab11 (S28N) expression. These results indicate that trafficking of PM and vacuolar antiporter proteins by OsRab11 is important for high salt stress resistance.     

UNC-108/Rab2 regulates postendocytic trafficking in Caenorhabditis elegans

After endocytosis, membrane proteins are often sorted between two alternative pathways: a recycling pathway and a degradation pathway. Relatively little is known about how trafficking through these alternative pathways is differentially regulated. Here, we identify UNC-108/Rab2 as a regulator of postendocytic trafficking in both neurons and coelomocytes. Mutations in the Caenorhabditis elegans Rab2 gene unc-108, caused the green fluorescent protein (GFP)-tagged glutamate receptor GLR-1 (GLR-1::GFP) to accumulate in the ventral cord and in neuronal cell bodies. In neuronal cell bodies of unc-108/Rab2 mutants, GLR-1::GFP was found in tubulovesicular structures that colocalized with markers for early and recycling endosomes, including Syntaxin-13 and Rab8. GFP-tagged Syntaxin-13 also accumulated in the ventral cord of unc-108/Rab2 mutants. UNC-108/Rab2 was not required for ubiquitin-mediated sorting of GLR-1::GFP into the multivesicular body (MVB) degradation pathway. Mutations disrupting the MVB pathway and unc-108/Rab2 mutations had additive effects on GLR-1::GFP levels in the ventral cord. In coelomocytes, postendocytic trafficking of the marker Texas Red-bovine serum albumin was delayed. These results demonstrate that UNC-108/Rab2 regulates postendocytic trafficking, most likely at the level of early or recycling endosomes, and that UNC-108/Rab2 and the MVB pathway define alternative postendocytic trafficking mechanisms that operate in parallel. These results define a new function for Rab2 in protein trafficking.     

Overexpression of Constitutively Active OsRab11 in Plants Enhances Tolerance to High Salinity Levels

Rab proteins are key regulators of intracellular trafficking between specific compartments in a cell. Among them, Rab11, a widely conserved sub-group, mainly regulates plasma membrane (PM) trafficking. Previously, we reported that Oryza sativa Rab11 (OsRab11) plays an important role in the intracellular trafficking from the trans-Golginetwork (TGN) to the plasma membrane (PM) and prevacuolar compartments (PVCs), and in the plant’s response to high salt stress. In this study, when the constitutively active mutant of OsRab11, (CA OsRab11(Q73L)) was co-transformed with Arabidopsis Ca²⁺-ATPase8-GFP (ACA8-GFP) or sporamin-GFP (Spo-GFP) into Arabidopsis protoplasts, the PM or vacuolar trafficking proportion of the reporter proteins was highly increased. Transgenic Arabidopsis plants overexpressing (OE) CA OsRab11(Q73L) exhibited enhanced tolerance to high salt stress and exogenous abscisic acid (ABA) compared to Col plants. Moreover, certain stress-responsive genes were expressed under high salt stress and ABA treatment in OEOsRab11(Q73L) plants. Thus, these results suggest that the active conformation of OsRab11 may be required to modulate plant responses to salt and ABA via the regulation of the expression of stress-responsive genes.     

OsPRA1 plays a significant role in targeting of OsRab7 into the tonoplast via the prevacuolar compartment during vacuolar trafficking in plant cells

In yeast and mammals, the Yip/PRA1 family of proteins has been reported to facilitate the delivery of Rab GTPases to the membrane by dissociating the Rab–GDI complex during vesicle trafficking. Recently, we identified OsPRA1, a plant Yip/PRA1 homolog, as an OsRab7-interacting protein that localizes to the prevacuolar compartment, which suggests that it plays a role in vacuolar trafficking of plant cells. Here, we show that OsPRA1 is essential for vacuolar trafficking and that it has molecular properties that are typical of the Yip/PRA1 family of proteins. A trafficking assay using Arabidopsis protoplasts showed that the point mutant OsPRA1_(Y94A) strongly inhibits the vacuolar trafficking of cargo proteins, but has no inhibitory effect on the plasma membrane trafficking of H⁺-ATPase-GFP, suggesting its specific involvement in vacuolar trafficking. Moreover, OsPRA1 was shown to be an integral membrane protein, suggesting that its two hydrophobic domains may mediate membrane integration, and its cytoplasmic N- and C-terminal regions were found to be important for binding to OsRab7. OsPRA1 also interacted with OsVamp3, implying its involvement in vesicle fusion. Finally, we used a yeast expression system to show that OsPRA1 opposes OsGDI2 activity and facilitates the delivery of OsRab7 to the target membrane. Taken together, our results support strongly that OsPRA1 targets OsRab7 to the tonoplast during vacuolar trafficking.     

Plant PRA plays an important role in intracellular vesicular trafficking between compartments as GDF

Rab GTPases like Ras-related monomeric GTPases are well known to regulate intracellular vesicle trafficking by cycling between membrane-bound and cytosolic states. The functions of these proteins are controlled by upstream regulators and downstream effectors. Ypt/Rabs transmit signals to downstream effectors in a GTP-dependent manner. GDP-bound Rab proteins are extracted from their target membrane by cytosolic proteins known as GDP dissociation inhibitors (GDIs), and the Rab GTPase is recruited to the membrane compartment following dissociation from the GDI by GDI displacement factor (GDF). Now, we''re going to discuss the role of plant PRA concerted with Rab and GDI proteins by recycling Rab between membrane and cytosol for intracellular trafficking of cargo proteins.Key words: GDF, GDI, PRA1, Rab, vacuolar trafficking, vesicle traffickingAlthough Rabs appear to undergo multiple cycles of GDI-mediated delivery to, and extraction from membranes,¹ the mechanisms underlying Rab membrane delivery and association by GDI and other factors remain unclear. GDP-GTP exchange occurs at the target membrane, catalyzed by a guanine nucleotide exchange factor (GEF),^2,3 and the GTP-bound Rab transmits signals to downstream effectors and associates with the membrane to ensure proper docking and fusion of transport vesicles.⁴ After vesicle fusion on its target membrane, subsequently hydrolysis of GTP by the Rab is facilitated by GTPase-activating proteins (GAPs).⁴ The resulting GDP-bound Rab is subsequently retrieved from the membrane by GDI, which then maintains Rab in the cytosol to complete the cycle.Many research groups isolated PRAs, a homolog of human YIP3, in several two-hybrid screenings as interacting with multiple Rabs in their GTP- or GDP-bound form.^5,6 PRA contains two extensive hydrophobic domains which may form a membrane-spanning domain or the inner hydrophobic core of the protein.⁷ PRA1 is localized to the Golgi and late endosomes,⁸ and the related PRA2 is present in the endoplasmic reticulum.⁹Recently it has been shown that the human YIP3 stimulates the rate of nucleotide binding to Rab9 when added to prenyl Rab9-GDI complex and catalyzes the dissociation of the endosomal Rab-GDI complex, indicating that YIP3 is a GDI displacement factor that recruits Rab to membranes.¹⁰ According to the Gougeon et al. report (2002),¹¹ PRA1 inhibits the extraction of membrane-bound Rab3A by GDI1, suggesting that recycling of Rab depends on the opposing actions of PRA and GDI, with PRA favoring membrane retention but GDI favoring solubilization.Moreover, mammalian PRA1 is required for vesicle formation from the Golgi complex and might influence the recruitment of Rab effectors during cargo sequestration as well as proteins required for subsequent vesicle docking and fusion.¹¹ This is consistent with its transport function based on interaction of yeast homologue Yip3p with proteins in the secretory pathway.⁷ Yip1-Yif1p complex binds to the ER and to the Golgi SNAREs, Bos1p and Sec22p, and is required for membrane fusion machinery.⁵ In addition, a role of Yip1p had also been proposed in COPII vesicle biogenesis.¹²To our current knowledge there is no report on the physiological role of a GDF in plants. Aims to enrich the understanding of the mechanism of Rab recycling and trafficking pathways in plant, we identified and characterized OsPRA1, a rice homolog of PRA. OsPRA1, isolated by yeast two-hybrid screening using OsRab7 as bait, localized to the prevacuolar compartment as a membrane protein.¹³ Additionally, through western blot and protoplast transient assays it was confirmed that OsPRA1 has GDF activity, which dissociates the Rab7-GDI2 complex and recruits dissociated Rab from the Rab7-GDI2 complex to the donor membrane (unpublished data). When yeast two-hybrid interaction assay between OsPRA1 and OsGDI2 was performed, OsPRA1 interacted with OsGDI2 weakly (unpublished data), supporting our proposition that OsPRA1 dissociates the OsRab7-OsGDI2 complex.Furthermore, by using yeast two-hybrid and co-immunoprecipitation assays it was demonstrated that OsPRA1 interacted with dominant negative OsRab7 (T22N) which has no GTP binding activity, but not the constitutively active OsRab7 (Q67L),¹³ indicating that OsPRA1 may interact with GDP-bound OsRab7 at the donor membrane, PVC. These results support that OsPRA1 is a GDF for OsRab7.Subsequently, in order to find its interacting proteins implicated in vesicular trafficking, such as t- or v-SNAREs, yeast two-hybrid screening using OsPRA1 as bait was performed. Interestingly, a t-SNARE, OsVam3p, homolgous to AtVam3p involved in vacuolar trafficking and localizing to both PVC and vacuole membranes in Arabidopsis,¹⁴ was isolated (unpublished data). This suggests that OsPRA1 may be a component of the vesicle fusion machinery. To further strengthen our hypothesis, we examined whether or not mutant OsPRA1 (Y94A) and OsRab7 interact. Mutant OsPRA1 (Y94A) showed weak and no interaction with OsRab7 and OsVam3p, respectively, indicating that mutant OsPRA1 (Y94A) may lose its activity for recruiting Rab GTPase and Rab effector proteins and fusing vesicles to the vacuolar membrane. Actually, when OsPRA1 was mutated, its GDF activity was reduced to less than 50%, and its localization was changed from the PVC to the cytosol. These results are consistent with the assigned transport function of OsPRA1. Besides, our data from transient expression assay using vacuole markers suggested a direct involvement of OsPRA1 in the trafficking of vacuolar proteins.In summary, OsPRA1, a Yip homologous protein, may function in regulating vacuolar trafficking as a GDF dissociating OsRab7-OsGDI2 complex in plant cells.     

Rab5b localization to early endosomes in the protozoan human pathogen Leishmania donovani

Leishmania donovani is a primitive trypanosomatid pathogen of humans. This protozoan is apically polarized such that the flagellar reservoir, the exclusive site of endocytosis and exocytosis, is situated at the anterior end. Recent evidence for the existence of an endocytic pathway in Leishmania has prompted us to investigate candidate temporal markers for endocytosis. In this study we identify the L. donovani Rab5b gene, and demonstrate the localization of a Rab5b chimera to early endosomes. A full-length Rab5b protein was fused to green fluorescent protein (GFP) to generate a chimeric protein GFP::Rab5b. Transfected L. donovani promastigotes carrying this chimeric construct displayed GFP::Rab5b localization. Additionally, incubation of transfected promastigotes with the fluid-phase marker Texas Red dextran demonstrated anterior co-localization of GFP::Rab5b and dye. This suggests Rab5b may act as a marker for early endosomes in L. donovani. Note. Nucleotide sequence data reported in this paper are available in the GenBank^TM, EMBL and DDBJ databases under the accession numbers AY357217, AL359774, AF007547, BC032740.     

水稻rab5B基因在大肠杆菌中的表达、纯化和GTP结合分析

Rab5B类蛋白因为其编码产物的N端具有特殊结构而被认为是一类特殊的蛋白质.水稻rab5B基因Osrab5B是这类蛋白质基因在单子叶植物中的首例发现.将Osrab5B基因的编码序列按正确读码框重组到具有谷胱甘肽硫转移酶（glutathione S-transferase, GST）融合标签的pGEX-4T1表达载体中,转化大肠杆菌,获得了稳定表达目标融合蛋白的菌株,经GSTrap^TM柱纯化,获得了纯化的目标融合蛋白.GTP结合试验表明,在原核细胞中表达出的GST-OsRab5B融合蛋白具有体外结合GTP的能力.     

The Anaplasma phagocytophilum‐occupied vacuole selectively recruits Rab‐GTPases that are predominantly associated with recycling endosomes

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to reside within a host cell‐derived vacuole. The A. phagocytophilum‐occupied vacuole (ApV) fails to mature along the endocytic pathway and is non‐fusogenic with lysosomes. Rab GTPases regulate membrane traffic. To better understand how the bacterium modulates the ApV's selective fusogencity, we examined the intracellular localization of 20 green fluorescent protein (GFP) or red fluorescent protein (RFP)‐tagged Rab GTPases in A. phagocytophilum‐infected HL‐60 cells. GFP‐Rab4A, GFP‐Rab10, GFP‐Rab11A, GFP‐Rab14, RFP‐Rab22A and GFP‐Rab35, which regulate endocytic recycling, and GFP‐Rab1, which mediates endoplasmic reticulum to Golgi apparatus trafficking, localize to the ApV. Fluorescently tagged Rabs are recruited to the ApV upon its formation and remain associated throughout infection. Endogenous Rab14 localizes to the ApV. Tetracycline treatment concomitantly promotes loss of recycling endosome‐associated GFP‐Rabs and acquisition of GFP‐Rab5, GFP‐Rab7, and the lysosomal marker, LAMP‐1. Wild‐type and GTPase‐ deficient versions, but not GDP‐restricted versions of GFP‐Rab1, GFP‐Rab4A and GFP‐Rab11A, localize to the ApV. Strikingly, GFP‐Rab10 recruitment to the ApV is guanine nucleotide‐independent. These data establish that A. phagocytophilum selectively recruits Rab GTPases that are primarily associated with recycling endosomes to facilitate its intracellular survival and implicate bacterial proteins in regulating Rab10 membrane cycling on the ApV.