橡胶树白粉菌分支酸变位酶基因克隆及蛋白表达纯化

由橡胶树白粉菌引起的橡胶树白粉病是橡胶树的重要的叶部病害之一,严重影响橡胶的产量。然而目前对橡胶树白粉菌的致病机理研究匮乏。分支酸变位酶是莽草酸途径的关键酶,能够将分支酸转化为预苯酸,为植物提供氨基酸及大量代谢产物,在植物抗病中起到非常重要的作用。而植物病原物在致病过程当中能够分泌分支酸变位酶影响植物莽草酸途径,从而抑制植物的防卫反应。因此研究橡胶树白粉菌分支酸变位酶在其致病过程中的功能具有一定的意义。试验利用同源比对分析在橡胶树白粉菌基因组中获得一个分支酸变位酶同源蛋白,并用PCR克隆获得橡胶树白粉菌分支酸变位酶基因,命名为OHCmu。后续构建GST-OHCmu融合原核表达载体,筛选最优诱导条件,并利用GST亲和层析柱对蛋白进行纯化。结果表明橡胶树白粉菌OHCmu基因大小843 bp,具有1个内含子,编码263个氨基酸;具有d5csma_结构域,属于Chorismate mutaseⅡ蛋白家族;GST-OHCmu融合蛋白外源诱导表达在供试条件下(IPTG:0.8 mmol/L, 16℃)可以有较好的表达,获得融合蛋白大小约为56 kD。经过GST亲和层析柱纯化、切割GST标签后,顺利获得浓度较高、较纯的橡胶树白粉菌OHCmu蛋白。研究结果为后续OHCmu蛋白的特性及致病机理研究提供参考。     

启动子探针载体的构建及橡胶树白粉菌启动子筛选鉴定

旨在构建适于快速检测橡胶树白粉病菌(Oidium heveae)HO-73启动子的探针载体。以卡那霉素抗性基因Kan作为报告基因,在pUC19骨架载体,构建获得启动子探针载体pUC19-K,连入CaMV35S启动子做启动子探针载体功能验证;利用启动子探针载体pUC19-K对预测的启动子片段LY1、LY2、LY3、LY4进行筛选鉴定。将CaMV35S启动子连入到启动子探针载体中,得到可检测启动子活性的启动子探针载体pUC19-K;应用生物信息学软件对部分橡胶树白粉菌HO-73全基因组数据预测,得到4个理论上具有活性的启动子序列LY1、LY2、LY3、LY4,利用构建的启动子活性探针载体进行活性比较,卡那耐受性实验检测发现含LY2和LY3的菌株随卡那霉素浓度的升高耐受性更强,最终得到2个活性较强的启动子LY2和LY3。以上结果表明,构建的启动子活性探针载体可以有效、灵敏地用于HO-73强启动子的筛选和启动子活性检测。     

非经典蛋白分泌研究进展

蛋白分泌是细胞间信息交流的重要途径。通常所指的蛋白分泌是经典分泌,即有信号肽的分泌蛋白会进入内质网,通过内质网-高尔基体运输释放。没有信号肽的胞质蛋白可以通过非经典的途径分泌,统称为非经典蛋白分泌。这些分泌蛋白在多个生物学过程中发挥作用,包括发育、代谢和炎症反应等,并且与人类重大疾病相关,如神经退行性疾病和癌症等。虽然对经典蛋白分泌途径已经有较深的了解,且主要内容已列入教科书,但是对非经典蛋白分泌的认识还处于初级阶段。本文就非经典蛋白分泌的研究进展作简要总结。     

小鼠新分泌蛋白基因mBolA1的克隆与鉴定

采用生物信息学工具预测与实验相结合的方法得到了一个新的小鼠分泌蛋白基因mBolA1。该基因定位于染色体3F2,cDNA全长为730bp,编码137个氨基酸的蛋白,该蛋白含有一个保守的BolA结构域,等电点为9.05。用RT-PCR方法从鼠的混合cDNA库中克隆到mBolA1。Western blot实验表明mBolA1能从瞬转的COS 7细胞中分泌到细胞培养液中。亚细胞定位显示mBolA1定位于细胞浆,且与高尔基体不共定位,提示它是个非经典分泌途径的分泌蛋白。RT PCR显示mBolA1在组织中广泛表达。它的具体功能有待进一步研究。     

真核细胞非经典蛋白分泌途径

在生物体中, 细胞间的信息传递是细胞生长、分化、发育、增殖、凋亡等生命活动的基本保证, 而蛋白分泌是细胞间信息传递的重要方式。大多数分泌蛋白都是通过内质网-高尔基体(ER-Golgi)途径分泌的。然而越来越多的研究表明, 存在着一类无信号肽的分泌蛋白, 这类蛋白不依赖ER-Golgi途径就能分泌到细胞外发挥功能, 被称为非经典分泌蛋白。非经典蛋白的分泌有其特有的机制, 它对ER-Golgi分泌途径是一种必要和有益的补充。非经典分泌与细胞增殖、免疫反应、肿瘤形成、传染病病理学等密切相关。文章旨在对非经典分泌蛋白的特点、分泌机制及生物学意义进行概述。     

登革2型病毒E蛋白在酶母菌中的分泌表达

以pPICZαB为载体,应用RT-PCR从感染D2V的C6/36病变细胞中克隆全长E基因,电转化法将重组质粒整合入巴斯德毕赤氏酵母菌,经抗生素筛选、表型鉴定和PCR分析得到Mut^ 型的多拷贝整合菌,经甲醇诱导培养可产生69KD的融合蛋白,与含组氨酸尾的D2V包膜糖蛋白分子量理论值相符;免疫印迹证实该表达产物可与D2V E特异性单抗和D2V多抗进行反应;表达产物经金属螯合亲和层析可获得纯化的含组氨酸尾的E融合蛋白并保留其免疫反应性。研究显示克隆的全长D2V E基因可在毕赤氏酵母菌中高效分泌表达,E融合蛋白最大表达量0.1g/L。     

马铃薯晚疫病菌全基因组分泌蛋白的初步分析

利用马铃薯晚疫病菌全基因组测序结果,结合计算机技术和生物信息学的方法,对马铃薯晚疫病菌的蛋白进行分析,为明确该病原菌与寄主互作的分子机制奠定基础。文章应用信号肽预测软件SignalP v3.0和PSORT,跨膜螺旋结构预测软件TMHMM-2.0和THUMBUP,GPI锚定位点预测软件big-PI Predictor,亚细胞器中蛋白定位分布预测软件TargetP v1.01,对已经公布的马铃薯晚疫病菌全基因组22 658个蛋白质氨基酸序列进行分析。结果发现,晚疫病菌全基因组编码蛋白中有671个为潜在的分泌型蛋白,占编码蛋白总数的3.0%。其中有45个分泌蛋白有功能方面的描述,其功能涉及细胞代谢、信号转导等方面;此外,还有一些与激发子类似的分泌蛋白,它们可能与晚疫病菌的毒性有关。     

橡胶树白粉菌(HO-73)启动子WY172不同长度片段的克隆及表达活性分析

旨在克隆橡胶树白粉菌启动子WY172及其上游2K序列上4个不同长度缺失片段,以分析启动子各片段的表达活性。基于实验室前期研究基础,以WY172上游2K序列作为研究对象进行渐变缺失突变,得到4个不同长度的可能具有启动子活性的片段,结合WY172,选用pBI121载体作为骨架,分别替换GUS基因前的CaMV35S启动子,并分别构建重组表达载体,通过ATMT法转化农杆菌;利用GUS染色法和酶活性检测,分析WY172启动子及不同长度片段的酶活性。分别构建了pBI121-WY172、pBI121-WY172Q、pBI121-WY172Q1、pBI121-WY172Q2、pBI121-WY172Q3共5个重组的植物表达载体,所有植物表达载体烟草瞬时表达GUS染色均有蓝色出现,且蓝色程度均强于阳性对照CaMV35S启动子,其中pBI121-WY172Q3的GUS染色相对最深;GUS酶活性测定结果显示所有缺失突变片段都具有调控基因表达的启动子活性,且启动活性均强于CaMV35S启动子,WY172Q3调控GUS基因表达的活性最高。因此我们判断WY172及其上游2K序列上4个不同长度缺失片段均具有启动子活性,其中以WY172Q3启动子片段的表达活性最强。     

香薷白粉病菌及其重寄生菌的分离鉴定

对采自中国长春香薷白粉病菌进行系统观察和测量,鉴定其为本间白粉菌(Erysiphe hommae Braun),同时发现了重寄生菌——白粉寄生孢（Ampelomyces quisqualis Ces.）,并对重寄生茵的叶部寄生特征及培养特征进行了描述.室内检测结果表明,重寄生菌的重寄生强度较高,可抑制白粉病的发生.     

肝细胞生成素——一种非经典分泌蛋白

肝细胞生成素(hepatopoietin ,HPO)是一种分泌蛋白.为了研究肝细胞生成素的分泌途径,利用SignalP软件分析了HPO的氨基酸序列,但HPO序列中没有经典分泌蛋白的信号肽.Western印迹实验证明,HPO能以双体形式从细胞中分泌出来.特异性体外阻断实验表明,布雷菲尔德菌素A(brefeldinA)和莫能菌素(monensin)都不能阻断HPO的分泌,说明HPO并不通过经典的内质网 高尔基体(ER -Golgi)途径分泌;优降糖(glyburide)对HPO的分泌没有抑制作用,说明HPO的分泌并不是由ABC1(ATP bindingcassette)转运子介导的;DNP和NH4Cl也不能刺激HPO的分泌,说明内体 溶酶体系统不参与HPO的分泌.上述结果表明,HPO是一种非经典分泌蛋白(non classicalsecretoryprotein) ,能以双体形式从细胞中分泌出来.但和已知的非经典分泌蛋白IL -1β不同,HPO的分泌并不是通过ABC1转运子介导的,内体 溶酶体系统也不参与其分泌.     

Physiological Regulation of Membrane Protein Sorting Late in the Secretory Pathway of Saccharomyces cerevisiae

In mammalian cells, extracellular signals can regulate the delivery of particular proteins to the plasma membrane. We have discovered a novel example of regulated protein sorting in the late secretory pathway of Saccharomyces cerevisiae. In yeast cells grown on either ammonia or urea medium, the general amino acid permease (Gap1p) is transported from the Golgi complex to the plasma membrane, whereas, in cells grown on glutamate medium, Gap1p is transported from the Golgi to the vacuole. We have also found that sorting of Gap1p in the Golgi is controlled by SEC13, a gene previously shown to encode a component of the COPII vesicle coat. In sec13 mutants grown on ammonia, Gap1p is transported from the Golgi to the vacuole, instead of to the plasma membrane. Deletion of PEP12, a gene required for vesicular transport from the Golgi to the prevacuolar compartment, counteracts the effect of the sec13 mutation and partially restores Gap1p transport to the plasma membrane. Together, these studies demonstrate that both a nitrogen-sensing mechanism and Sec13p control Gap1p transport from the Golgi to the plasma membrane.     

A Recombinant Saccharomyces cerevisiae Strain Overproducing Mannoproteins Stabilizes Wine against Protein Haze

Stabilization against protein haze was one of the first positive properties attributed to yeast mannoproteins in winemaking. In previous work we demonstrated that deletion of KNR4 leads to increased mannoprotein release in laboratory Saccharomyces cerevisiae strains. We have now constructed strains with KNR4 deleted in two different industrial wine yeast backgrounds. This required replacement of two and three alleles of KNR4 for the EC1118 and T73-4 backgrounds, respectively, and the use of three different selection markers for yeast genetic transformation. The actual effect of the genetic modification was dependent on both the genetic background and the culture conditions. The fermentation performance of T73-4 derivatives was clearly impaired, and these derivatives did not contribute to the protein stability of the wine, even though they showed increased mannoprotein release in vitro. In contrast, the EC1118 derivative with both alleles of KNR4 deleted released increased amounts of mannoproteins both in vitro and during wine fermentation assays, and the resulting wines were consistently less susceptible to protein haze. The fermentation performance of this strain was slightly impaired, but only with must with a very high sugar content. These results pave the way for the development of new commercial strains with the potential to improve several mannoprotein-related quality and technological parameters of wine.     

Protein phosphatase type 1 regulates ion homeostasis in Saccharomyces cerevisiae

Protein phosphatase type 1 (PP1) is encoded by the essential gene GLC7 in Saccharomyces cerevisiae. glc7-109 (K259A, R260A) has a dominant, hyperglycogen defect and a recessive, ion and drug sensitivity. Surprisingly, the hyperglycogen phenotype is partially retained in null mutants of GAC1, GIP2, and PIG1, which encode potential glycogen-targeting subunits of Glc7. The R260A substitution in GLC7 is responsible for the dominant and recessive traits of glc7-109. Another mutation at this residue, glc7-R260P, confers only salt sensitivity, indicating that the glycogen and salt traits of glc7-109 are due to defects in distinct physiological pathways. The glc7-109 mutant is sensitive to cations, aminoglycosides, and alkaline pH and exhibits increased rates of l-leucine and 3,3'-dihexyloxacarbocyanine iodide uptake, but it is resistant to molar concentrations of sorbitol or KCl, indicating that it has normal osmoregulation. KCl suppresses the ion and drug sensitivities of the glc7-109 mutant. The CsCl sensitivity of this mutant is suppressed by recessive mutations in PMA1, which encodes the essential plasma membrane H(+)ATPase. Together, these results indicate that Glc7 regulates ion homeostasis by controlling ion transport and/or plasma membrane potential, a new role for Glc7 in budding yeast.     

Evidence for a Nonendosomal Function of the Saccharomyces cerevisiae ESCRT-III-Like Protein Chm7

Endosomal sorting complex required for transport (ESCRT) proteins are involved in a number of cellular processes, such as endosomal protein sorting, HIV budding, cytokinesis, plasma membrane repair, and resealing of the nuclear envelope during mitosis. Here we explored the function of a noncanonical member of the ESCRT-III protein family, the Saccharomyces cerevisiae ortholog of human CHMP7. Very little is known about this protein. In silico analysis predicted that Chm7 (yeast ORF YJL049w) is a fusion of an ESCRT-II and ESCRT-III-like domain, which would suggest a role in endosomal protein sorting. However, our data argue against a role of Chm7 in endosomal protein sorting. The turnover of the endocytic cargo protein Ste6 and the vacuolar protein sorting of carboxypeptidase S (CPS) were not affected by CHM7 deletion, and Chm7 also responded very differently to a loss in Vps4 function compared to a canonical ESCRT-III protein. Our data indicate that the Chm7 function could be connected to the endoplasmic reticulum (ER). In line with a function at the ER, we observed a strong negative genetic interaction between the deletion of a gene function (APQ12) implicated in nuclear pore complex assembly and messenger RNA (mRNA) export and the CHM7 deletion. The patterns of genetic interactions between the APQ12 deletion and deletions of ESCRT-III genes, two-hybrid interactions, and the specific localization of mCherry fusion proteins are consistent with the notion that Chm7 performs a novel function at the ER as part of an alternative ESCRT-III complex.     

Stm1 Modulates mRNA Decay and Dhh1 Function in Saccharomyces cerevisiae

The control of mRNA degradation and translation are important for the regulation of gene expression. mRNA degradation is often initiated by deadenylation, which leads to decapping and 5′–3′ decay. In the budding yeast Saccharomyces cerevisae, decapping is promoted by the Dhh1 and Pat1 proteins, which appear to both inhibit translation initiation and promote decapping. To understand the function of these factors, we identified the ribosome binding protein Stm1 as a multicopy suppressor of the temperature sensitivity of the pat1Δ strain. Stm1 loss-of-function alleles and overexpression strains show several genetic interactions with Pat1 and Dhh1 alleles in a manner consistent with Stm1 working upstream of Dhh1 to promote Dhh1 function. Consistent with Stm1 affecting Dhh1 function, stm1Δ strains are defective in the degradation of the EDC1 and COX17 mRNAs, whose decay is strongly affected by the loss of Dhh1. These results identify Stm1 as an additional component of the mRNA degradation machinery and suggest a possible connection of mRNA decapping to ribosome function.     

Protein composition of Saccharomyces cerevisiae mitochondrial ribosomes

Protein composition of mitochondrial ribosomes of the yeast Saccharomyces cerevisiae was analysed by two-dimensional electrophoresis. The small (37S) mitoribosomal subunit contains 36 different polypeptides with molecular weights ranging from 10,000 to 60,000. The large (50S) subunit is composed of 41 proteins with molecular weights from 10,000 to 43,000. The molecular weights of mitoribosomal small and large subunits are 1.85 MDa and 2.35 MDa, respectively. Proteins represent 60-62% and 42-45% of the total mass of 37S and 50S subunits respectively. On the basis of the protein content and molecular weights of individual proteins we conclude that all mitoribosomal proteins are present in the mitoribosome in equimolar proportions.     

拟康氏木霉eg1基因的克隆及在酿酒酵母中的分泌表达

从拟康氏木霉3.3002基因组中克隆了内切葡聚糖酶EGI基因,该基因全长1566 bp,由3个外显子2个内含子组成,编码461个氨基酸.编码蛋白EGI的N端为22aa组成的信号肽,其后依次为催化结构域、连接肽和结合结构域.采用重叠PCR法获得无内含子的内切葡聚糖酶基因eg1,并将其成熟肽编码序列插入酿酒酵母分泌型表达载体pYEα中,构建成pYEα-Peg1重组质粒,转化酿酒酵母.重组转化子经β-半乳糖诱导,检测表达产物的分子大小以及酶活,结果表明,转化子在刚果红平板上可产生明显的水解圈;酶活检测显示该基因能在酿酒酵母中表达有生物活性的EG I并分泌到胞外;SDS-PAGE电泳显示EGI蛋白分子量比预期目的蛋白稍偏大.