Rubisco副合基因的构建及其在E.coli中的表达

将编码番茄核酮糖－１,５－二磷酸羧化酶／加氧酶小亚基转运肽的一段ＤＮＡ序列与菠菜Ｒｕｂｉｓｃｏ大亚基的编码区连接,构建了一个Ｒｕｂｉｓｃｏ融合基因。限制性内切酶图谱和ＤＮＡ序列分析证明副合部位的核苷酸序列符合构建前的三联密码子框架。将Ｒｕｂｉｓｃｏ融合基因转入Ｅ．ｃｏｌｉ,用ＩＰＴＧ进行诱导表达。利用蛋白质印迹技术检测到诱导产物的存在。     

不同生态型芦苇Rubisco免疫学特性差异

以河西走廊荒漠地区不同生态型芦苇为研究材料,提取并纯化得Rubisco蛋白,经SDS-PAGE凝胶电泳将Rubisco大、小亚基分离,用Rubisco全酶蛋白及其大、小亚基分别注射昆明系雄性小白鼠制备抗体,经Western-blotting鉴定结果表明:(1)水芦Rubisco全酶抗体可与水芦、沙芦及菠菜Rubisco大亚基发生反应,而与小亚基均未见显色反应,且水芦显色最深,沙芦略浅,菠菜最浅;(2)水芦、沙芦Rubisco大亚基抗体可与水芦、沙芦、菠菜大亚基发生抗原交叉反应,且均不与小亚基发生反应,并且其与菠菜Rubisco大亚基的反应程度明显低于水芦和沙芦;(3)用与Rubisco大亚基抗体同样的制备方法,均未检测到水芦、沙芦Rubisco小亚基抗体的产生;(4)菠菜Rubisco全酶抗体可与菠菜、水芦、沙芦、水稻Rubisco大亚基均发生抗原交叉反应,但仅与其自身小亚基反应,且与菠菜Rubisco大亚基显色反应最深,水稻略浅,沙芦、水芦稍有反应.由此说明,水芦、沙芦Rubisco全酶蛋白及其大亚基免疫学特性差异较小,而与双子叶植物菠菜相比差异较大;水芦、沙芦Rubisco蛋白免疫化学决定簇的差异主要决定于小亚基上,且其小亚基不具有抗原活性或抗原活性较弱.     

二磷酸核酮糖羧化酶／加氧酶装配研究进展

本文对Rubis CO大、小亚基在叶绿体和大肠杆菌中的合成,装配,酶的体外重组以及亚基结合蛋白的性质和作用等进行了综述,并对这个领域的研究前景作了展望。     

各种因子对固定化烟草核酮糖1,5-二磷酸羧化酶/加氧酶解离作用的影响

pH,温度、离子强度及效应剂等对固定化烟草RuBP羧化酶在2.5mol/L尿素处理下的解离作用有各种不同的影响。在pH6.0时,仅小亚基从大亚基核(L_8)解离,当pH为中性偏碱时,大亚基核也解离。低温和低离子强度均促进酶的解离,而温度和离子强度对大亚基之间的解离的影响显著大于对大、小亚基之间的影响。这表明酶的亚基之间存在着不同的极性和疏水作用,而大亚基之间的疏水作用比大、小亚基之间的强。6-PG对大、小亚基之间解离的抑制作用表明大亚基上的催化位置与小亚基之间有一定的密切关系。     

烟草核酮糖-1,5-二磷酸羧化酶/加氧酶四级结构的研究(英文)

本文提出三种证据证明烟草核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)的大亚基伸展在小亚基的外面,小亚基排列在大亚基中间的概念。证据是:1.固定化胰蛋白酶在一定条件下可水解RubisCO的大亚基但不水解小亚基,而天然胰蛋白酶水解大亚基,也水解小亚基。2.固定化抗小亚基IgG-Sepharose可与游离的小亚基相结合,但不能与全酶结合。3.低浓度尿素处理可使固定化的RubisCO-Sepharose上的小亚基解离下来,而大亚基仍结合在载体上,这说明RubisCO是通过定位在分子表面上的大亚基的ε-氨基与Sepharose共价偶联的。当RubisCO中的小亚基全部被解离后,大亚基之间的结合进一步增强,这时解离大亚基所需的尿素浓度要比小亚基存在时高。任何RubisCO的四级结构模型都应将小亚基置于大亚基中间受保护的位置,一部份小亚基可暴露于全酶分子表面。     

低温锻炼对水稻幼苗叶片中Rubisco的影响

低温锻炼能提高水稻幼苗的抗冷力,低温锻炼虽不能明显提高Rubisoc活性,却提高了冷胁条件下Rubisoc的稳定性和增强了胁迫后正常生长条件下其活性的恢复能力。分别用火箭免疫电泳分析Rubisoc蛋白和SDS-PAGE分析大、小亚基量表明：低温锻炼未提高Rubisoc蛋白的合成能力,但增加了大、小亚基的合成量。经锻炼、冷胁迫以及恢复后Lsu／Ssu比值的变化主要是由于小亚基对温度变化更敏感所致。Rubisco酶特性分析表明,低温锻炼有减少水稻幼苗Rubisoc表面的SH数,并提高Rubisco蛋白在高、低温下的稳定性。     

转录后基因沉默系统研究烟草rbcS基因功能

初步建立了利用病毒载体诱导转录后基因沉默系统研究烟草(Nicotiana benthamiana)1,5-二磷酸核酮糖羧化酶／加氧酶小亚基(Ribulose-1,5-bisphosphate carboxylase／oxylase small subunit,rbcS)基因功能的模式。用携带与1,5-二磷酸核酮糖羧化酶／加氧酶小亚基基因同源的cDNA片段的烟草脆裂病毒载体(pTV．rbcS)侵染烟草(Nicotiana benthamiana),诱导内源rbcS基因沉默并在此基础上建立了研究rbcS基因功能的模式：初步进行了rbcS基因沉默后的表型分析、转录水平分析、蛋白质表达水平分析以及利用HPLC方法定量分析rbcS基因沉默后的光合色素变化。结果表明：病毒诱导基因沉默瞬时表达体系中烟草最佳侵染时期为苗龄21-24d,用于侵染的重组农杆菌的最佳浓度的OD值为1～1．5;烟草Rubisco小亚基的表达量可能调节Rubisco大亚基的表达量;烟草rbcS基因与光合作用中的光能收集无关。对rbcS基因沉默的烟草叶片及对照烟草叶片的部分重要光合作用指标分析表明,运用烟草脆裂病毒载体诱导转录后基因沉默系统研究烟草rbcS基因功能具有可行性,为进一步深入研究rbcS基因功能奠定了基础。     

蚕豆核酮糖1，5-磷酸羧化酶／加氧酶大亚基基因的分子克隆

核酮糖l,5－二磷酸羧化酶／加氧酶由大亚基(Ls)和小亚基组成。Ls由叶绿体DNA编码。蚕豆Ls的基因已被克隆到pBR322。应用几种限制性内切酶酶解以及Southern印迹法构建了该重组质粒的物理图谱。     

短命植物东方旱麦草Rubisco大亚基基因(rbcL)的克隆及表达分析

利用PCR、DNA重组、原核与真核生物表达等技术从新疆短命植物东方旱麦草(Eremopyrum orientale)基因组中扩增出Rubisco大亚基基因rbcL(GenBank登录号为FJ346562),并构建了原核与真核表达载体pGEX4T-1-rbcL和pcDNA3-rbcL,随后分别在原核宿主BL21(DE3)和小鼠中进行了表达,并利用真核表达载体和纯化蛋白制备的抗体,对表达产物的特异性进行了Western印迹检测。结果表明:东方旱麦草的rbcL基因可读区包含1431 bp,与旱麦草rbcL基因的同源性达99.86%,推测编码476个氨基酸,分子量56 kD。该基因在BL21中以融合蛋白GST-RBCL形式表达并存在于包含体中,融合蛋白大小约为82 kD。RT-PCR检测到该基因在小鼠肝脏中的表达。利用真核表达载体与纯化融合蛋白进行联合免疫制备的RBCL抗体可与RBCL特异性地结合,这为短命植物东方旱麦草基因后续的免疫定位检测奠定了基础。     

外源海藻糖对PEG渗透胁迫下小麦Rubisco及其活化酶的影响

以抗旱品种‘晋麦47’和干旱敏感品种‘郑引1号’为材料,通过室内水培试验研究了外源海藻糖对PEG渗透胁迫下小麦叶片净光合速率、1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco)和1,5-二磷酸核酮糖羧化酶/加氧酶活化酶(RCA)含量和相关基因表达特性的影响。结果表明:(1)外源海藻糖和渗透胁迫均能显著增加2个小麦品种叶片海藻糖含量。(2)渗透胁迫显著降低了2个品种小麦叶片的净光合速率,而外源海藻糖能显著缓解受胁迫小麦叶片净光合速率的降低幅度。(3)渗透胁迫仅使‘郑引1号’Rubisco大亚基基因(rbcL)相对表达量及相应蛋白含量显著降低;渗透胁迫显著降低了小麦RCAα和β亚基基因相对表达量,并显著降低RCA蛋白含量,而外源海藻糖不能缓解RCA蛋白含量的降低;渗透胁迫显著降低了Rubisco总活性、初始活性、活化状态及RCA活性,而外源海藻糖则能显著缓解上述酶活性的下降。(4)小麦叶片净光合速率与其rbcL、RCAα和β亚基基因相对表达量及Rubisco总活性、初始活性、活化状态及RCA活性均呈极显著正相关关系。研究发现,在渗透胁迫条件下,外源海藻糖主要从翻译后层面对小麦叶片Rubisco和RCA的活性发挥显著保护作用,从而缓解了小麦净光合速率的降低。     

Light and metabolite regulation of the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase and the corresponding mRNAs in the unicellular alga Chlorogonium

In the unicellular green alga Chlorogonium elongatum, the synthesis of the plastid enzyme ribulose bisphosphate carboxylase/oxygenase (RuBPCase) and its mRNAs is under the control of light and acetate. Acetate is the sole metabolizable organic carbon source for this organism. Light greatly promotes the synthesis of RuBPCase and the increase in the concentration of the mRNAs of both subunits of the enzyme while acetate has a strong inhibitory effect on this process. There is a good agreement between RuBPCase synthesis and the amount of translateable RuBPCase mRNA present in cells which are cultured under different conditions (autotrophic, heterotrophic, mixotrophic). During the transition period after transfer of the cells from heterotrophic to autotrophic growth conditions the amounts of the large and small subunits of the enzyme increase well coordinated. In contrast to the protein subunits the two subunit-mRNAs accumulate with different kinetics.Abbreviations LSU large subunit of RuBPCase - poly(A)^- RNA - poly(A)⁺RNA non-, poly-adenylated RNA - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase EC 4.1.1.39 - SSU small subunit of RuBPCase     

D-Ribulose-1,5-bisphosphate carboxylase/oxygenase from chemolithotrophically grown Rhizobium japonicum

Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified from chemolithotrophically grown Rhizobium japonicum SR and ribulose-5-phosphate kinase activity has also been detected in extracts of such cells. Electrophoretically homogeneous ribulosebisphosphate carboxylase/oxygenase purified in the presence of PMSF showed two types of large subunits of 55 000 and 53 000 daltons and small subunits of 14 200 daltons. The heterogeneity of large subunits was not observed when the enzyme was prepared in the presence of PMSF and DIFP. Ribulose-1,5-bisphosphate carboxylase from R. japonicum was inhibited by antibodies to this enzyme and a single precipitin band from the antibody-enzyme interaction was observed on double diffusion plates. Antibodies to R. japonicum enzyme did not cross-react on immunodiffusion plates with the ribulosebisphosphate carboxylase/oxygenases from wheat, spinach, soybean and tobacco.     

Phosphorylation of small subunit plays a crucial role in the regulation of RuBPCase in moss and spinach

RuBPCase has been purified to electrophoretic homogeneity from moss and spinach. On denaturing SDS-polyacrylamide gels the purified enzyme revealed two discrete bands, thereby indicating the presence of large and small subunits. The phosphoprotein nature of RuBPCase was proved by in vivo labelling of enzyme with [³²P]orthophosphate. Autoradiographic analysis of ³²P-labelled RuBPCase on SDS-PAG demonstrated that phosphorylation was restricted to the small subunit. Dephosphorylation of purified RuBPCase with alkaline phosphatase resulted in a dramatic decline (70% decrease) in the biological activity of the enzyme. Fractionation of the dephosphorylated enzyme on denaturing gels revealed only the presence of large subunits of RuBPCase. Thus it became evident that dephosphorylation of RuBPCase brings about the dissociation of small subunits from the catalytic large subunits (octamer). The dephosphorylated small subunits were isolated as dimers. These results clearly indicate that phosphorylation of small subunits is mandatory for the reconstitution of holoenzyme and hence crucial for the activation of RuBPCase.     

Immunocytological and chemical studies on the stromacentre-forming protein from Avena plastids

The stromacentre (SC), a particular structure in the plastids of Avena, was isolated from etioplasts of Avena sativa by density gradient centrifugation and then analyzed and compared with ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase) from A. sativa, with pyrenoids of Chlorella vulgaris and with the stromacentre of Opuntia. Purified SC-elements consisted of protein subunits with a relative molecular weight of 63 kDa, different from the isolated RuBPCase of A. sativa as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After peptide mapping, the proteolytic cleavage patterns of the 63-kDa protein were also found to be different from those of the RuBPCase. Antibodies against SC-elements, RuBPCase, and the large subunit of RuBPCase were produced. Ouchterlony double immunodiffusion tests did not give crossreactions between the SC-elements and RuBPCase or the large subunit of this enzyme. Immunogold labelling of ultrathin sections showed that antibodies against the SC-elements marked the stromacentre in Avena, but not the pyrenoids in Chlorella. Antibodies against the large subunit of RuBPCase, however, did not label the SC, but labelled the stroma of the plastids in Avena and the pyrenoids of Chlorella. In Opuntia, a comparable structure described as an SC was not labelled by any of the antisera. Immunoelectrophoretical investigations demonstrated a strong correlation between the presence of the 63-kDa protein and the occurrence of the SC in different Avena species with and without SC.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SC stromacentre - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethy;)-1,3-propanediol Dedicated to Professor Ludwig Bergmann on the occasion of his 60th birthday     

光和暗条件培养的眼虫藻(Euglena gracilis)内RuBP羧化酶的免疫定位

应用免疫金标记技术证明,在眼虫藻和其它藻类中RuBP羧化酶主要分布在蛋白核部位,这与高等植物中RuBP羧化酶分布不同,在眼虫藻叶绿体间质中有少量RuBP羧化酶存在,这与高等植物中RuBP羧化酶的分布也有相似之处。 暗中培养的眼虫藻不能形成类囊体,无RuBP羧化酶,无光合能力,只能进行异养代谢。     

Ribulose-1,5-bisphosphate carboxylase/oxygenase expression in melon plants infected with Colletotrichum lagenarium

Ribulose-1,5-bisphosphate carboxylase/oxygelase (RuBPCase) was studied in melon leaves infected by Colletotrichum lagenarium, a fungal pathogen of melons. Electrophoretic analysis of melon leaf proteins indicated a strong effect of infection on RuBPCase, the subunits of which gradually disappeared during the different stages of infection. Enzyme activity also declined 4 d after inoculation and its content, measured by immunoelectrophoresis, decreased to a similar extent. Synthesis of the large and small subunits of RuBPCase was followed by in-vivo pulse-labeling experiments. A drastic decrease in the rate of RuBPCase-subunit synthesis occurred 3 d after inoculation and preceded the appearance of disease symptoms. There was an apparent coordination of the synthesis of the two subunits under these conditions.Abbreviations LS (SS) Large (small) subunit of RuBPCase - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Serological characteristics within the genus Desulfovibrio

Antisera were prepared against one strain each of Desulfovibrio desulfuricans, D. vulgaris and D. salexigens. The antisera were tested for cross reactivity against 36 heterologous Desulfovibrio strains by both agglutination titration and by double immunodiffusion precipitin plates.Generally no cross-reaction was demonstrated by agglutination even between heterologous strains of the same species, suggesting that the surface antigens of Desulfovibrio are highly specific. In immunodiffusion plates a single apparently genus-specific surface antigen could be shown to be present in all but two of the strains tested. Although other common precipitin bands showed the presence of some antigens common between heterologous strains these appeared to be randomly distributed among the strains tested, with the exception of one band shown to be generally specific to strains of D. salexigens. With this exception no other precipitin band could be shown to be consistently specific to any other species, nor consistently common to more than one species.