植物小G蛋白功能的最新研究进展

小G蛋白超家族包括Ras、Rab、Rho、Arf和Ran亚家族,不同成员间在结构和功能方面又呈现明显的多样性。小G蛋白作为重要的分子开关,其结构域主要包括4个鸟苷酸（GTP／GDP）结合域、1个效应区,其鸟苷酸结合域起着关键作用且保守性最高,在植物、动物和酵母中均有较高的保守性。小G蛋白成员凭借其多样性和不同鸟苷酸结合态的功能调控（结合GTP时为活性状态,而结合GDP时为非活性状态）,参与多种细胞生命活动,履行不同的生物学功能,例如基因表达调控,细胞骨架重组,囊泡运输的调节,出芽过程,核一质运输及微管形成。本文主要阐述植物中研究小G蛋白各个家族功能的最新进展。     

土壤颗粒态有机质中铜和铅的结合特征

为了解土壤颗粒态有机质(POM)中重金属的富集机理,采用化学试剂选择提取方法,把POM中铜和铅分为可交换态、络合态、有机质紧结合态和矿质态等4种形态.结果表明,与细土(＜0.05 mm)相比,POM中铜和铅的有机质紧结合态、可交换态和络合态比例较高;可交换态重金属比例随POM粒径减小而下降,络合态重金属比例却随POM粒径减小而增加.污染土壤中分离获得的POM有较高比例有机质紧结合态铜和铅,而人为添加的铜和铅在POM中主要向络合态和交换态转化.研究认为,污染土壤中有机质紧结合态重金属大多可能是经植物吸收进入土壤后分解残留所致;而POM中络合态和交换态重金属主要通过有机质与土壤重金属的交换和络合作用而富集.     

添加化学改良剂对海南岛砖红壤中铅的化学形态与转化的影响

采用土培试验和连续提取-原子吸收分光光度法,研究了海南岛花岗岩砖红壤中重金属Pb的6种形态(水溶态Pb、交换态Pb、碳酸盐结合态Pb、铁锰氧化物结合态Pb、有机质结合态Pb和残余态Pb)、外源Pb污染对土壤Pb形态的影响以及化学修复剂磷、钙、硫对土壤Pb形态的影响.结果表明,在供试原土壤中,重金属Pb的化学形态以结合态和残余态为主,土壤有效态Pb含量较低,其中:残余态Pb有机质结合态Pb铁锰氧化物结合态Pb碳酸盐结合态Pb交换态Pb水溶态Pb,说明土壤Pb的环境风险较低.当外源Pb以离子态污染土壤时,土壤中6种形态Pb的含量均有明显上升,且随时间推移,外源Pb污染土壤中Pb的化学形态发生了连续变化,有:水溶态Pb和交换态Pb的比例持续下降,碳酸盐结合态Pb和铁锰氧化物结合态Pb的比例持续上升,有机质结合态Pb和残余态Pb的比例则有先升后略有下降再趋于稳定的趋势;土壤中Pb主要以铁锰氧化结合态累积,并有:铁锰结合态Pb残余态Pb有机态Pb碳酸盐结合态Pb交换态Pb水溶态Pb的趋势.向污染土壤施加化学改良剂过磷酸钙、硫化钠和石灰,能显著降低水溶态Pb和交换态Pb的含量,可使有机结合态Pb、碳酸盐结合态Pb和铁锰氧化物结合态Pb含量下降,而残余态Pb有增加的趋势.化学改良剂的加入可使土壤Pb的有效态含量降低、活性下降,从而可抑制重金属Pb由土壤向植物体系的迁移,有利于植物安全生产.     

ROP蛋白研究进展

ROP(Rho-related GT Pases from plants)蛋白作为高等植物体内广泛存在的一类GTP结合蛋白,是近些年植物信号转导方面研究的热点。它调控了肌动蛋白细胞骨架的形成、物质的膜泡运输、细胞极性的形成、胞内氧化态环境的形成、脱落酸的信号转导等诸多途径。为此,着重从分类、结构、转导途径及功能方面对其进行了阐述。     

辣椒小G蛋白CaRab11基因全长cDNA的分离及序列分析

通过分析克隆获得的辣椒Rab11全长cDNA及其编码的氨基酸序列结构特征,为进一步研究辣椒Rab11功能奠定基础.通过对辣椒均一化cDNA文库的筛选分离获得了一个与葡萄Rab11小G蛋白VvRabAlf高度同源的全长cDNA,命名为CaRab11.序列分析结果表明:该cDNA包含有1164 bp的完整开放阅读框,编码217个氨基酸.该蛋白含有Rab GTP酶超家族保守的RabSF模体;Rab亚家族蛋白所特有的五个氨基酸序列RabF模体(RabF1-RabF5);参与GTP/Mg++结合及GTP水解的G结构域(G1-G5:GDSGVGKS,T,DTAGQE,GNKADL及ETSAL);两个构象变构域(Switch Ⅰ和SwitchⅡ)及与异戊二烯化相关的CCX序列.氨基酸同源性及进化分析同样表明CaRab11为辣椒小G蛋白Rab11家族新成员.     

土壤中镉的赋存形态及生物有效性研究

为研究镉在土壤-植物系统中的赋存形态和生物有效性,以棕壤为供试土壤,添加1、3、8、15、30mg·kg-1等5个不同浓度的外源镉,测定不同浓度和不同处理时间下土壤中镉的存在形态及质量分数。结果表明随外源镉处理浓度的升高,土壤中碳酸盐结合态和有机结合态的镉向交换态、残渣态方向转化,铁锰氧化物结合态变化不大;随处理时间的延长,土壤中交换态和碳酸盐结合态的镉向残渣态、铁锰氧化物结合态和有机结合态方向转化,镉的生物有效性逐渐降低。     

改良剂对Pb污染土壤中Pb形态及植物有效性的影响

通过盆栽试验和土壤连续浸提-原子吸收法探讨了过磷酸钙、石灰、硫化钠对Pb污染土壤中Pb存在的6种化学形态(水溶态、可交换态、碳酸盐结合态、铁锰氧化物结合态、有机物结合态和残渣态)及植物有效性的影响.结果表明,改良剂对水溶态Pb修复的效果:硫化钠＞石灰＞过磷酸钙;对交换态Pb的修复效果:石灰＞过磷酸钙＞硫化钠;对碳酸盐结合态Pb修复效果:石灰＞硫化钠＞过磷酸钙;对铁锰氧化态Pb的修复效果:过磷酸钙＞硫化钠＞石灰;对有机结合态与残渣态Pb的修复效果:硫化钠＞石灰＞过磷酸钙.改良剂的施加可使土壤Pb的有效态含量降低、活性下降,抑制了Pb在土壤-植物体系的迁移,从而有利于植物安全生产.     

施用污泥堆肥对土壤中Cu Zn形态分布的影响研究

采用小区试验区连续提取方法,研究了施用污泥堆肥后土壤中Cu,Zn的形态分布状况,影响因素,各形态之间的关系,以及在土壤中的累积.结果表明①施用污泥堆肥后土壤中的Cu,Zn有效态含量和其他各形态含量均有所增加,且其增加程度随污泥堆肥施人最的增加而增加;②各处理土壤中Cu的各种形态的关系为残渣态＞铁锰氧化物结合态＞有机结合态＞可交换态＞碳酸盐结合态,其中可交换态Cu对生物有效态Cu的贡献是主要的;土壤中的Cu大部分以不能被植物利用的残渣态形式存在;在Zn的各形态中残渣态占绝大部分,其次为铁锰氧化物结合态,碳酸盐结合态和有机结合态次之;③不同蔬菜地土壤中Cu,Zn的累积有很大差异.生菜地土壤中Cu的累积高于油菜地土壤,油菜地土壤中Zn的累积高于生菜地土壤.     

蔬菜根际环境钒的形态变化及植物有效性

通过盆栽试验和Tessier连续提取法,研究了蔬菜根际和非根际土壤中钒的形态分布与植物有效性.结果表明,钒在根际和非根际土壤中主要以残渣态和铁锰氧化物结合态存在,钒的含量随其形态不同而变化:当土壤中钒添加量从25 mg·kg-1增加到200mg·kg-1时.根际土壤交换态钒的百分率增加了9.89%,碳酸盐结合态钒增加了9.38%,铁锰氧化物结合态钒增加了9.99%,残渣态钒的百分率下降了33.6%.土壤添加的钒最增加时,钒的迁移能力增强,使蔬菜的生物最显著降低,蔬菜对钒的吸收作用增强:钒添加量从0增至200mg·kg-1时,蔬菜鲜质量降低了41.65%,而蔬菜中85%以上的钒积累在根部.根际土壤中碳酸盐结合态钒含量对植物的根、茎叶中钒含量有显著影响,碳酸盐结合态钒与蔬菜鲜质量呈显著负相关.     

酵母双杂交系统在植物病毒学中的应用

酵母双杂交系统应用有效的酵母遗传学方法分析蛋白质问相互作用,已成为鉴定蛋白质相互作用强有力的方法之一。文章从植物病毒编码的蛋白质间、植物病毒蛋白与植物蛋白间、植物病毒蛋白与介体蛋白间等3个方面阐述了酵母双杂交系统在植物病毒学中的应用现状,并对其应用前景进行了展望。     

一个假定的稻瘟病菌RhoGEF蛋白参与营养生长和产孢过程的调控

对稻瘟病菌致病机制的认识将有助于更好地防治水稻稻瘟病.在明确多个Rho族小G蛋白在该菌营养生长、分化孢子形成以及侵染致病过程中起到重要作用的基础上,进一步研究Rho族小G蛋白调控因子-鸟苷酸交换因子(GEF)的功能.以生物信息学和分子遗传学方法,研究了稻瘟病菌基因位点Mgg_11178.6所编码蛋白的功能.结果表明Mgg_11178.6编码一个假定的Rho族小G蛋白鸟苷酸交换因子(MoRGF1),与多种丝状子囊菌RhoGEF亲缘关系密切;基因敲除突变体表现为生长速度减慢,产孢量明显减少,但是致病力没有变化.说明该假定的Rho族小G蛋白鸟苷酸交换因子(MoRGF1)可能参与稻瘟病菌营养生长与产孢过程的调控.     

A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants

The role of guanine nucleotides in ras p21 function was determined by using the ability of p21 protein to induce maturation of Xenopus oocytes as a quantitative assay for biological activity. Two oncogenic mutant human N-ras p21 proteins, Asp12 and Val12, actively induced maturation, whereas normal Gly12 p21 was relatively inactive in this assay. Both mutant proteins were found to be associated with guanosine triphosphate (GTP) in vivo. In contrast, Gly12 p21 was predominantly guanosine diphosphate (GDP)-bound because of a dramatic stimulation of Gly12 p21-associated guanosine triphosphatase (GTPase) activity. A cytoplasmic protein was shown to be responsible for this increase in activity. This protein stimulated GTP hydrolysis by purified Gly12 p21 more than 200-fold in vitro, but had no effect on Asp12 or Val12 mutants. A similar factor could be detected in extracts from mammalian cells. It thus appears that, in Xenopus oocytes, this protein maintains normal p21 in a biologically inactive, GDP-bound state through its effect on GTPase activity. Furthermore, it appears that the major effect of position 12 mutations is to prevent this protein from stimulating p21 GTPase activity, thereby allowing these mutants to remain in the active GTP-bound state.     

A cytosolic protein catalyzes the release of GDP from p21ras

The rate of release of guanine nucleotides from the ras proteins (Ras) is extremely slow in the presence of Mg2+. It seemed likely, therefore that a factor might exist to accelerate the release of guanosine diphosphate (GDP), and hence the exchange of GDP for guanosine triphosphate (GTP). Such a factor has now been discovered in rat brain cytosol. Brain cytosol was found to catalyze, by orders of magnitude, the release of guanine nucleotides from recombinant v-H-Ras protein bound with [alpha-32P]GDP. This effect occurred even in the presence of a large excess of Mg2+, but was destroyed by heat or by incubation of the cytosol for an hour at 37 degrees C in the absence of phosphatase inhibitors. The effect was observed with either v-H-Ras or c-H-Ras, but not with p25rab3A, a small G protein with about 30% similarity to Ras. The effect could not be mimicked by addition of recombinant Ras-GAP or purified GEF, a guanine nucleotide exchange factor involved in the regulation of eukaryotic protein synthesis. By gel filtration chromatography, the factor appears to possess a molecular size between 100,000 and 160,000 daltons. This protein (Ras-guanine nucleotide-releasing factor, or Ras-GRF) may be involved in the activation of p21ras.     

Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts

The nuclear envelope (NE) forms a controlled boundary between the cytoplasm and the nucleus of eukaryotic cells. To facilitate investigation of mechanisms controlling NE assembly, we developed a cell-free system made from Xenopus laevis eggs to study the process in the absence of chromatin. NEs incorporating nuclear pores were assembled around beads coated with the guanosine triphosphatase Ran, forming pseudo-nuclei that actively imported nuclear proteins. NE assembly required the cycling of guanine nucleotides on Ran and was promoted by RCC1, a nucleotide exchange factor recruited to beads by Ran-guanosine diphosphate (Ran-GDP). Thus, concentration of Ran-GDP followed by generation of Ran-GTP is sufficient to induce NE assembly.     

Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus egg extracts

The small guanosine triphosphatase Ran is loaded with guanosine triphosphate (GTP) by the chromatin-bound guanine nucleotide exchange factor RCC1 and releases import cargoes in the nucleus during interphase. In mitosis, Ran-GTP promotes spindle assembly around chromosomes by locally discharging cargoes that regulate microtubule dynamics and organization. We used fluorescence resonance energy transfer-based biosensors to visualize gradients of Ran-GTP and liberated cargoes around chromosomes in mitotic Xenopus egg extracts. Both gradients were required to assemble and maintain spindle structure. During interphase, Ran-GTP was highly enriched in the nucleoplasm, and a steep concentration difference between nuclear and cytoplasmic Ran-GTP was established, providing evidence for a Ran-GTP gradient surrounding chromosomes throughout the cell cycle.     

Structure of Galphaq-p63RhoGEF-RhoA complex reveals a pathway for the activation of RhoA by GPCRs

The guanine nucleotide exchange factor p63RhoGEF is an effector of the heterotrimeric guanine nucleotide-binding protein (G protein) Galphaq and thereby links Galphaq-coupled receptors (GPCRs) to the activation of the small-molecular-weight G protein RhoA. We determined the crystal structure of the Galphaq-p63RhoGEF-RhoA complex, detailing the interactions of Galphaq with the Dbl and pleckstrin homology (DH and PH) domains of p63RhoGEF. These interactions involve the effector-binding site and the C-terminal region of Galphaq and appear to relieve autoinhibition of the catalytic DH domain by the PH domain. Trio, Duet, and p63RhoGEF are shown to constitute a family of Galphaq effectors that appear to activate RhoA both in vitro and in intact cells. We propose that this structure represents the crux of an ancient signal transduction pathway that is expected to be important in an array of physiological processes.     

Asef, a link between the tumor suppressor APC and G-protein signaling

The adenomatous polyposis coli gene (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors. Here the APC gene product is shown to bind through its armadillo repeat domain to a Rac-specific guanine nucleotide exchange factor (GEF), termed Asef. Endogenous APC colocalized with Asef in mouse colon epithelial cells and neuronal cells. Furthermore, APC enhanced the GEF activity of Asef and stimulated Asef-mediated cell flattening, membrane ruffling, and lamellipodia formation in MDCK cells. These results suggest that the APC-Asef complex may regulate the actin cytoskeletal network, cell morphology and migration, and neuronal function.     

Polo-like kinase Cdc5 controls the local activation of Rho1 to promote cytokinesis

The links between the cell cycle machinery and the cytoskeletal proteins controlling cytokinesis are poorly understood. The small guanine nucleotide triphosphate (GTP)-binding protein RhoA stimulates type II myosin contractility and formin-dependent assembly of the cytokinetic actin contractile ring. We found that budding yeast Polo-like kinase Cdc5 controls the targeting and activation of Rho1 (RhoA) at the division site via Rho1 guanine nucleotide exchange factors. This role of Cdc5 (Polo-like kinase) in regulating Rho1 is likely to be relevant to cytokinesis and asymmetric cell division in other organisms.     

鸡胸腺重和脾脏重性状的全基因组关联

【目的】全基因组关联分析（genome-wide association study,GWAS）是复杂性状和疾病相关基因定位的新策略。【方法】试验利用鸡60K SNP芯片对来自50个公鸡家系的728只北京油鸡进行SNP分型检测,采用全基因组关联分析方法对影响100日龄胸腺重和脾脏重的基因进行定位研究。【结果】结果发现24个达5%全基因组水平显著的位点,与100日龄胸腺重和脾脏重显著相关,并在这些位点附近发现Janus kinase 1（JAK1）、 zinc finger DHHC-type containing 8（ZDHHC8）、vav 3 guanine nucleotide exchange factor（VAV3）、SATB homeobox 1（SATB1）等候选基因;84个与这两个性状潜在关联同时达到5%染色体水平显著的位点。【结论】利用GWAS分析策略筛选和鉴定的重要突变位点及候选基因,将为揭示鸡免疫器官发育的分子调控机制和抗病育种分子标记辅助选择提供必要的分子基础。     

辣椒NBS类抗病基因同源序列的克隆与分析

辣椒RGA的分离将为进一步从辣椒中分离功能性抗病基因和抗病机理分析打下基础,为辣椒相关抗病基因的克隆提供依据。根据已知抗病基因保守氨基酸结构域设计简并引物,以高抗辣椒品种88为试材,通过PCR扩增抗病基因同源序列。结果表明:从辣椒基因组DNA中分离出1条辣椒抗病基因同源序列WD1。对其编码的氨基酸序列进行分析表明:该序列同时含有P-环(GGVGKTT)、kinase-2(VLDD)、kinase-3(GSRII)及HD(即GLPLAL)保守域结构,分离的辣椒抗病基因同源序列(RGA)与已报道的辣椒抗病基因同源序列A5和A13都有99%的同源性。