高柠檬酸盐对固氮酶铁钼辅基重组活性的影响

用高柠檬酸铁、柠檬酸钠、ＡＴＰ和Ｎａ２ＭｏＯ４分别处理ＦｅＭｏｃｏ，然后与ＵＷ４５组份Ⅰ蛋白进行重组，结果发现，高柠檬铁和柠檬酸钠分别使ＦｅＭｏｃｏ重组体的Ｃ２Ｈ２还原活性提高６７％和５４％，Ｎ２还原活性分别提高１７０％和１３５％．ＦｅＭｏｃｏ与ＡＴＰ预作用后再分别与高柠檬酸铁、柠檬酸钠作用，其重组体的Ｃ２Ｈ２还原活性分别提高１２１％和１１９％，而Ｎ２还原活性分别提高３０３％和１３５％．而ＦｅＭｏｃｏ，ＦｅＭｏｃｏ－高柠檬酸铁体系及ＦｅＭｏｃｏ－ＡＴＰ－高柠檬酸铁体系与Ｎａ２ＭｏＯ４作用后，重组体的Ｃ２Ｈ２还原活性分别下降５％，１２％及２１％．ＦｅＭｏｃｏ－高柠檬酸铁体系在１４．６Ｋ下的ＥＰＲ谱，与单独ＦｅＭｏｃｏ的略有不同，而ＦｅＭｏｃｏ－ＡＴＰ－高柠檬酸铁体系的ＥＰＲ谱则与前者有明显的差异．研究结果表明，高柠檬酸可能是ＦｅＭｏｃｏ的有机组份，它可能结合在ＦｅＭｏｃｏ的Ｍｏ原子上，而这种结合是比较松散的．     

Identification of the V factor needed for synthesis of the iron-molybdenum cofactor of nitrogenase as homocitrate

Nitrogenase catalyses the ATP-dependent reduction of N2 to NH3, and is composed of two proteins, dinitrogenase (MoFe protein or component I) and dinitrogenase reductase (Fe protein or component II). Dinitrogenase contains a unique prosthetic group (iron-molybdenum cofactor, FeMoco) comprised of Fe, Mo and S, which has been proposed as the site of N2 reduction. Biochemical and genetic studies of Nif- (nitrogen fixation) mutants of Klebsiella pneumoniae which are defective in nitrogen fixation, have shown that the nifB, nifQ, nifN, nifE and nifV genes are required for the biosynthesis of FeMo-co. Recently, a system for in vitro synthesis of FeMoco was described. The assay requires at least the nifB, nifN and nifE gene products, and a low-molecular-weight factor (V factor) produced in the presence of the nifV gene product. We have used this system to study FeMoco biosynthesis. We report here the isolation of V factor and identify it as homocitric acid ([R]2-hydroxy-1,2,4-butanetricarboxylic acid).     

花生根瘤菌在自生条件下固氮和吸氢相关性

花生根瘤菌x_(-1)菌株在自生条件下和合适的培养基中,可诱导固氮酶及氢酶的活性,固氮酶反应产生的H_2(内源H_2)能直接诱导氢酶,氢酶活性表达的时间进程是在固氮反应之后,在外源H_2的存在下,固氮酶和氢酶则可同时表达,不同有机碳化合物对固氮酶与氢酶的影响不同,丙酮酸明显提高固氮活性,但对氨酶没有促进作用,蔗糖对固氮活性没有促进作用但对吸氢表现促进作用,分子H_2明显提高固氮活性,2.4-二硝基苯酚抑制需H_2的固氮活性,在外源H_2存在下其抑制作用更明显,铵抑制固氮酶的形成、固氮酶受铵抑制时氢酶也相应受到抑制。     

固氮鱼腥藻(Anabaena azotica Ley HB686)氮固定的能量和还原剂来源

某些外源碳水化合物提高固氮鱼腥藻的固氮能力.在光照和5%氧浓度条件下,分子氢促进固氮作用.光合抑制剂、呼吸链抑制剂和解偶联剂均抑制固氮酶活性,若同时加入葡萄糖,DCMU的抑制作用被解除,而对DBMIB和解偶联剂的作用影响甚微.固氮鱼腥藻固氮作用的还原剂与光合作用产生的碳化合物有关,能量主要由循环光合磷酸化供给.     

台湾毛豆根瘤吸氢及固氮特性的研究

测定了台湾毛豆根瘤在整个发育期吸氢和乙炔还原活性，发现该根瘤属于放氢型；根瘤的吸氢活力与根瘤的发育及固氮活力的高低相关，在根瘤固氮功能的旺盛期，表现较低的吸氢活性．花生根瘤菌变株Ｘ０２－４能与台湾毛豆建立共生关系，并提高根瘤的吸氢能力．     

Analysis of active sites for N2 and H^＋ reduction on FeMo-cofactor of nitrogenase

Dinitrogen (N2) and proton (H ),which act as physiological substrates of nitrogenase,are reduced on FeMo-co of the MoFe protein. However,researchers have different opinions about their exact reduction sites. Nitrogenases were purified from the wild type (WT) and five mutants of Azotobacter vinelandii (Av),including Qα191K,Hα195Q,nifV-,Qα191K/nifV- and Hα195Q/nifV-; and the activities of these en-zymes for N2 and H reduction were analyzed. Our results suggest that the Fe2 and Fe6,atoms closed to the central sulfur atom (S2B) within FeMo-co,are sites for N2 binding and reduction and the Mo atom of FeMo-co is the site for H reduction. Combining these data with further bioinformatical analysis,we propose that two parallel electron channels may exist between the 8Fe7S cluster and FeMo-co.     

The composition and distribution of metal clusters in the MoFe protein from a nifZ deletion strain （DJ 194） of Azotobacter vinelandii

Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, a nitrogenase MoFe protein (△nifZ Av1) was obtained from a nifZ deleted mutant of Azotobacter vinelandii (stain DJ194).The results of Western blotting after anoxic native electrophoresis and SDS-PAGE showed that △nifZ Av1 was similar to wild type MoFe protein (OP Av1) at the electrophoretic mobility, molecular weight and subunit composition. Furthermore, △nifZ Avl was also similar to OP Av1 at the molybdenum content, EPR signal (g≈4.3, 3.65 and 2.01), and the molar extinction coefficient (△ε) of circular dichroism (CD)at 660 nm region. All of these indicated that, besides having the same α2β2 composition as OP Av1, the △nifZ Av1 also contained equal amount of reductive FeMoco in the spin state of S=3/2 to OP Av1. However, the iron content and substrate (C2H2, H^ and N2)-reduction activity of △nifZ Av1 were 74% and 46%-50% of those of OP Av1, respectively. Furthermore, the △ε at around 450 nm, which reflects P-cluster in Av1, was obviously lower than that of OP Av1. It suggested that the difference between △nifZ Avl and OP Av1 resulted from P-cluster rather than FeMoco, and from the half number of P-cluster in △nifZ Av1, but the composition or redoxstate of P-cluster in △nifZ Av1 were not changed. Thus it could propose that △nifZ Av1 is composed of two different αβsubunit pairs. One is a FeMoco-and P-cluster-containing pair, and the other is a P-cluster-deficient but FeMoco-containing pair. Since the deletion of nifZ gene leads to the deficiency of only one of two P-clusters in a α2β2 tetramer, the assembly of P-cluster may not simply depend on one gene product, and so a possible mechanism of NifZ is supposed here.     

Isolation and characterization of a <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis> mutant that cannot utilize proline as the sole carbon and nitrogen source

Sinorhizobium fredii strain HN01 can use proline as the sole carbon and nitrogen source. A mutant strain GXHN100 unable to catabolize proline was screened from 6000 Tn5gusA5 random insertional mutants of S.fredii strain HN01. Sequencing analysis showed that an open reading frame, named pmrA (proline metabolic relative), was inserted by the Tn5gusA5. A positive clone, namedp GXHN100 which containing 3.3kb foreign DNA fragment of S.fredii strain HN01, was isolated from a partial gene library of S.fredii HN01 by colony in situ hybridization. Sequence analysis showed that pGXHN100 contained the entire pmrA gene. The 3.3kb DNA fragment of pGXHN100 was cloned into a broad-host-range cosmid vector pLAFR3 to form plasmid pGXHN200 which was subsequently introduced into GXHN100 to form a complemented strain GXHN200. Plant test showed that GXHN100 was effective and no obvious changes in nitrogenase activity comparing with parental strain. But GXHN100 nodulated 2 days later on soybean and its nodulation efficiency and competitiveness were decreased.The complemented strain GXHN200 restored the nodulation efficiency and competitiveness of GXHN100 to the wild type.     

Characterization of Arabidopsis calreticulin mutants in response to calcium and salinity stresses

As an important calcium-binding protein, calreticulin plays an important role in regulating calcium homeostasis in endoplasmic reticulum （ER） of plants. Here, we identified three loss-of-function mutants of calreticulin genes in Arabidopsis to demonstrate the function of calreticulin in response to calcium and salinity stresses. There are three genes encoding calreticulin in Arabidopsis, and they are named AtCRT1, 2, and 3, respectively. We found that both single mutant of crt3 and double mutant of crtl crt2 were more sensitive to low calcium environment than wild-type Arabidopsis. Moreover, crt3 mutant showed more sensitivity to salt treatment at germination stage, but tolerance to salt stress at later stage compared with wild-type plant. However, there was no obvious growth difference in the mutant crtl and crt2 compared with wild-type Arabidopsis under calcium and salt stresses. These results suggest that calreticulin functions in plant responses to calcium and salt stresses.     

Role for the nitrogenase MoFe protein alpha-subunit in FeMo-cofactor binding and catalysis

Two components constitute Mo-dependent nitrogenase (EC 1.18.6.1)--the Fe protein (a homodimer encoded by nifH) and the MoFe protein (an alpha 2 beta 2 tetramer encoded by nifDK). The MoFe protein provides the substrate-binding site and probably contains six prosthetic groups of two types--four Fe-S centres and two Fe- and Mo-containing cofactors. To determine the distribution and catalytic function of these metalloclusters, we and others are attempting to change the catalytic and spectroscopic features of nitrogenase by substituting specific amino acids targeted as potential metallocluster ligands, particularly those to the FeMo-cofactor, which is responsible for the biologically unique electron paramagnetic resonance signal (S = 3/2) of nitrogenase, and is believed to be the N2-reducing site. Here we describe mutant strains of Azotobacter vinelandii that have single amino-acid substitutions within the MoFe protein alpha-subunit. These substitutions alter both substrate-reduction properties and the unique electron paramagnetic resonance signal, indicating that the FeMo-cofactor is associated with both the alpha-subunit and the substrate-reducing site.     

Hydrogen production by draTGB hupL double mutant of Rhodospirillum rubrum under different light conditions

To increase H2 yield of Rhodospirillum rubrum in two-stage hydrogen production process, two deletion mutants were constructed. One is single mutant designated R. rubrum UR801 that deleted hupL gene encoding the large subunit of uptake hy- drogenase, and the other is a double mutant desig- nated R. rubrum UR805 lacked both draTGB encod- ing regulators for the activity of nitrogenase and hupL. Comparing H2 yields of two mutants with R. rubrum UR2 (wild type) and UR472 (ΔdraTGB) under differ- ent light conditions, the results showed that the H2 yield of R. rubrum UR801 under continuous light is the highest (5700 mL of H2 per liter culture), and it is 1.56, 2.24 and 2.32-fold that of R. rubrum UR2, UR472 and UR805, respectively. However, the total H2 yield of R. rubrum UR805 in two-stage hydrogen production process is the highest (4303 mL/L), and it is 1.35, 1.21 and 1.04-fold that of R. rubrum UR2, UR801 and UR472, respectively. Thus, R. rubrum UR805 might be a valuable strain to produce a large amount of hydrogen in two-stage hydrogen produc- tion process.     

Y-152和K-156在果蝇ADH催化中的作用

果蝇醇脱氢酶酪氨酸－１５２（Ｙ－１５２）和赖氨酸－１５６（Ｋ－１５６）处于同类脱氢酶的保守位点。经人工定点突变和酶动力学分析,前者的苯丙氨酸（Ｙ１５２Ｆ）、组氨酸（Ｙ１５２Ｈ）和谷氨酸突变体（Ｙ１５２Ｅ）及后者的异亮氨酸突变体（Ｋ１５６Ｉ）均丧失催化活性。而半胱氨酸－１５２（Ｙ１５２Ｃ）及精氨酸－１５６（Ｋ１５６Ｒ）突变体活性分别是对应野生型的０．２５％和２．２％。此外,Ｙ１５２Ｃ和Ｋ１５６Ｒ的Ｋ     

拟南芥CARK家族响应脱落酸信号的研究

植物激素脱落酸(Abscisic acid,ABA)在植物应对生物和非生物胁迫中起着重要作用.本研究利用以carks单基因突变体为亲本,构建双重突变体来检测CARKs在ABA信号途径中的功能.然后,分析多重突变体在ABA处理下,种子萌发率和子叶变绿的响应.结果显示:单基因突变体和双重变体与野生型相比,萌发率更高,双重突变体的子叶绿芽率高于单基因突变体.以上结果表明,CARKs家族基因在ABA信号途径中起正调控作用,而且它们的功能是冗余的.     

晚疫病过敏反应阻断突变体抗氧化酶与相关基因的变化分析

马铃薯抗晚疫病基因R3a和Avr3a互作符合基因对基因假说.为了解R3a和Avr3a基因互作后过敏反应(HR反应)发生机制,本实验利用一个在番茄中构建的MM-R3a-Avr3a系统,以两份筛选到的HR反应被阻断的突变体为材料,研究它们在喷施诱导剂地塞米松(DEX)诱导Avr3a基因表达后活性氧爆发、活性氧清除酶和抗氧化基因的变化情况.结果显示:MM-R3a-Avr3a在DEX处理后有O2-产生和H2O2累积并产生整株的HR反应并导致植株死亡,在突变体中也有O2-产生和H2O2累积却没有导致细胞死亡,说明突变基因与HR反应的发生关系密切;DEX处理后抗氧化酶基因SOD、PPO、CAT在转基因番茄MM-R3a-Avr3a和突变体中的变化有明显差异,由此推测番茄突变体中关键基因的突变导致活性氧清除酶和相关基因的表达发生变化.该研究为探索HR反应的发生机制及了解晚疫病抗病基因抗病机理的打下基础.     

Enhanced activity of yqhD oxidoreductase in synthesis of 1,3-propanediol by error-prone PCR

yqhD oxidoreductase was determined to be an NADP-dependent dehydrogenase,and was more active toward 3-HPA when compared to 1,3-propanediol oxidoreductase.To further improve enzyme activity towards 3-hydroxypropionaldehyde (3-HPA),error-prone PCR was implemented to mutant yqhD gene.Two mutants,D99QN147H and Q202A with increased catalytic and affinity efficiency,were obtained after one round of error-prone polymerase chain reaction.And the catalytic efficiency of the mutant D99QN147H was up to 4-fold greater than the wild enzyme (0.0375 min-1 mM-1 vs.0.0078 min-1 mM-1).The recombined strain containing pET28yqhD D99QNI47H yielded 28 g L-1 of 1,3-propanediol in the fed-batch LB cultures (1 L volume) with an initial 3-HPA concentration of 40 g L-1,which was higher than the 21 and 17 g L-1 of 1,3-propanediol from the mutant Q202A and the wild-type,respectively.Except for propionaldehyde,the optimal mutant D99QN147H also exhibited higher activity on a range of substituted aldehydes than the wild-type.     

黑腹果蝇杂合体中ADH杂二聚体的形成

研究了醇脱氢酶基因 (Adh)诱变体与正常基因相互作用后的部分显性现象 .所有 8个由乙基亚硝基尿素 (ENU)和 1个 X-射线诱变体仅为单碱基置换体 ,其余 3个 X-射线诱变体则为 9～2 1个碱基的缺失体 .这 1 2个诱变体 (除 1个外 )都能产生可测的突变肽 ,其中 7个不能与正常肽形成二聚体 ,杂合体酶活性约为正常纯合体的 1 /2 ;另 4个形成二聚体 .形成二聚体突变基因产物中所有氨基酸突变均发生在肽链 1 82～ 1 94氨基酸区域 ,可见该区域对于二聚化不是必需的 ,该序列可能是重要的催化表面功能区     

Mutational effect of the “- 35” element of sorghumpsbA gene promoter

Mutations of the first position T and the third position G in TTGACA, the " - 35" element of sorghum psbA gene promoter, were induced using chemically synthesized 20 nt oligonucleotide primer. Three mutants were produced: ATTACA, GTGACA, and ATGACA. Then the protein binding affinity of the mutants and the wild type sorghum psbA gene promoter was tested in a spinach chloroplast protein extract system. Gel retardation assay of the wild type showed a strong protein-binding band. On the other hand, the protein-binding band of the mutant resulting from single base mutation, ATGACA or GTGACA, showed reduced intensity, while that of the mutant resulting from double base mutation, ATTACA, showed increased intensity. It is thus shown that the " - 35" element plays an important role in controlling the binding between psbA gene promoter and the specific chloroplast proteins; mutation of a single base may exert a substantial influence on the binding affinity.     

两个水稻叶片反向卷曲突变体的表型及遗传分析

为了揭示水稻叶片卷曲的形态建成,分析了水稻Ac/Ds转座子插入突变体库中的2个叶片反向卷曲突变体的表型及组织形态.研究发现,与野生型平展叶片相比,内卷突变体和外卷突变体叶片中泡状细胞数目明显减少,这可能是造成水稻叶片卷曲的重要原因.另外,内卷突变体叶片主脉薄壁细胞少,薄壁细胞崩裂形成的气腔面积较大.对2个卷叶突变体的纤维素含量进行测定发现,内卷突变体叶片及茎杆中纤维素含量明显减少,而外卷突变体叶片及茎杆的纤维素含量与野生型没有显著差异,说明2个卷叶突变体叶片的卷曲可能由不同基因突变造成.分析了2个水稻卷叶突变体的遗传规律,结果表明,二者均由隐性单基因控制.