大豆叶片硝酸还原酶活性动态研究

试验采用框栽和砂培两种方法,框栽土壤为黑土,试验品种为东农42、东农47、龙选1号和秣食豆;砂培试验品种为东农47,分别对不同品种及不同施氮处理大豆叶片的硝酸还原酶(NR)活性进行了研究。结果表明,在大豆叶片中都检测到了NR活性,框栽与砂培试验测得的NR活性都呈单峰曲线,框栽大豆峰值出现在R2期(秣食豆为R1期),砂培大豆出现在R2和R4期;NR活性上部叶片明显高于中、下部;NR活性随氮水平提高而升高,施氮水平明显影响NR活性。     

Heat inactivation of the relaxing site of actomyosin: prevention and reversal with dithiothreitol

Adenosine triphosphate and magnesium (MgATP) inhibit contraction by binding to a specific relaxing site on natural actomyosin gel. This inhibitory control site is distinct from the active sites where MgATP causes contraction.In high concentrations of MgATP, calcium triggers contraction by releasing the protein from substrate inhibition, allowing the contractile reactions to occur. Heating the protein for 5 minutes at 43 degrees C selectively inactivates the relaxing site. After this treatment, actomyosin with MgATP contracts as well without calcium as with it. That this effect of heat is prevented and reversed by dithiothreitol (an agent that reduces disulfide bonds) indicates that the structure of the relaxing site depends on certain labile sulfhydryl groups, which may be those of tropomyosin. When these are oxidized to disulfide bonds, the site loses its activity; when the disulfide bonds are reduced, the site regains its activity.     

α?芋螺毒素LvIA第11位氨基酸的新突变体合成及其靶点活性

为了进一步探究第11位氨基酸的性质对LvIA靶点结合活性的影响，设计了LvIA的2个新型突变体[D11R]LvIA和[D11H]LvIA，即用2个碱性氨基酸?精氨酸（R）和组氨酸（H）分别替换原来的酸性氨基酸D。先人工合成了这2个新突变体的线性肽，然后采用2步氧化法进行折叠，以获得在第1位和第3位半胱氨酸（Cys 1～3）、第2位和第4位半胱氨酸（Cys 2～4）之间定点连接形成二硫键。经高效液相色谱分离纯化和质谱鉴定，合成了含有Cys(1～3, 2～4)二硫键连接方式的多肽，其分子质量正确，纯度在95%以上。利用双电极电压钳电生理学技术对这2种突变体与α3β2 nAChR的结合活性进行了检测。结果发现，当该位点的氨基酸性质由酸性转换为碱性后，对LvIA的活性影响巨大，直接导致对α3β2 nAChR的阻断活性丧失。[D11R]LvIA和[D11H]LvIA的活性与野生型LvIA相比分别降低了574.38%和408.62%。由此表明，第11位氨基酸的酸碱性对LvIA的活性至关重要。     

盆栽条件下土壤无机氮浓度对大豆结瘤、固氮和产量的影响

【目的】大豆结瘤固氮和产量对氮肥的反应不同,实际上是由于大豆共生固氮系统及其根系系统对土壤无机氮浓度的感知不同造成的。通过对不同土壤无机氮浓度下大豆结瘤、固氮及产量影响的研究,探索能提高大豆产量、结瘤和固氮的土壤无机氮浓度,即掌握土壤无机氮浓度与大豆共生固氮和产量的数量关系,为调控氮肥施用量及施用时期、预测氮肥对大豆共生固氮能力和产量的影响提供理论依据。【方法】采用盆栽土培试验方法,分别在第一片复叶充分生长（V2期）、始花期（R1期）、始荚期（R3期）和始粒粒（R5期）一次性施用不同量的氮肥,从而形成不同无机氮浓度的土壤。利用获得的不同无机氮浓度土壤为供试土壤,对各生育时期根瘤数量、干重和固氮酶活性及成熟期产量及其构成因子进行调查,明确大豆根瘤固氮和产量对土壤无机氮浓度的响应,掌握土壤无机氮浓度与氮肥及与大豆固氮和产量的数量关系。【结果】不同时期土壤无机氮浓度处理下的根瘤干重、数量和固氮酶活性均随着大豆生育时期的推进在R4期时达到最大值。R6期时大豆平均根瘤干重、数量和固氮酶活性均表现为：V2期＞R5期＞R3期＞R1期,较CK处理根瘤平均干重分别下降15%、18%、17%和32%;根瘤数量下降13%、18%、19%和20%;固氮酶活性下降19%、22%、23%和32%。不同生育时期土壤无机氮浓度与R6期大豆根瘤干重、数量和固氮酶活性间均具有显著的线性负相关关系,即土壤无机氮浓度越大对根瘤干重、数量和固氮酶活性的抑制作用越大。大豆干物质积累量和产量的变化趋势均表现为：R1期＞R3期＞V2期＞R5期。除R5期不同土壤无机氮浓度处理与CK处理间的生物量和产量差异不显著外,V2、R1和R3期不同土壤无机氮浓度处理,均显著地促进大豆生物量和产量的增加。不同生育时期处理均以N3和N4处理的生物量、株高、株荚数、株荚重、株粒重显著高于其它处理。V2期土壤无机氮浓度对大豆固氮能力和产量的影响最大,而R1期土壤无机氮浓度对大豆生长和产量的影响最大。不同生育时期不抑制大豆固氮同时还提高大豆产量的土壤无机氮浓度不同：V2期土壤无机氮浓度达到135.8 mg•kg -1;R1期土壤无机氮浓度为58-91 mg•kg-1;R3期土壤无机氮浓度为29.4-62.8 mg•kg -1;在R5期土壤无机氮浓度达到102.3 mg•kg -1。【结论】大豆对氮肥的反应主要取决于土壤无机氮浓度的大小,而土壤无机氮浓度大小的调节,除了与氮肥施用量有关外还与大豆的生育时期有关系,可以根据农业生产和科学试验的需要进行调节。其中V2期土壤无机氮浓度对大豆根瘤数量、干重和固氮酶活性的影响大于其它生育时期土壤无机氮浓度处理;而R1期土壤无机氮浓度对大豆生物量和产量的影响大于其它生育时期土壤无机氮浓度处理。     

Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I

Site-directed mutagenesis of the large fragment of DNA polymerase I (Klenow fragment) yielded two mutant proteins lacking 3',5'-exonuclease activity but having normal polymerase activity. Crystallographic analysis of the mutant proteins showed that neither had any alteration in protein structure other than the expected changes at the mutation sites. These results confirmed the presumed location of the exonuclease active site on the small domain of Klenow fragment and its physical separation from the polymerase active site. An anomalous scattering difference Fourier of a complex of the wild-type enzyme with divalent manganese ion and deoxythymidine monophosphate showed that the exonuclease active site has binding sites for two divalent metal ions. The properties of the mutant proteins suggest that one metal ion plays a role in substrate binding while the other is involved in catalysis of the exonuclease reaction.     

Design and synthesis of a peptide having chymotrypsin-like esterase activity

A peptide having enzyme-like catalytic activity has been designed and synthesized. Computer modeling was used to design a bundle of four short parallel amphipathic helical peptides bearing the serine protease catalytic site residues serine, histidine, and aspartic acid at the amino end of the bundle in the same spatial arrangement as in chymotrypsin (ChTr). The necessary "oxyanion hole" and substrate binding pocket for acetyltyrosine ethyl ester, a classical ChTr substrate, were included in the design. The four chains were linked covalently at their carboxyl ends. The peptide has affinity for ChTr ester substrates similar to that of ChTr and hydrolyzes them at rates approximately 0.01 that of ChTr; total turnovers greater than 100 have been observed. The peptide is inhibited by ChTr specific inhibitors and is inactive toward benzoyl arginine ethyl ester, a trypsin substrate. The peptide is inactivated by heating above 60 degrees C, but recovers full catalytic activity upon cooling and lyophilization from acetic acid.     

The radical site in chlamydial ribonucleotide reductase defines a new R2 subclass

Ribonucleotide reductase (RNR) synthesizes the deoxyribonucleotides for DNA synthesis. The R2 protein of normal class I ribonucleotide reductases contains a diiron site that produces a stable tyrosyl free radical, essential for enzymatic activity. Structural and electron paramagnetic resonance studies of R2 from Chlamydia trachomatis reveal a protein lacking a tyrosyl radical site. Instead, the protein yields an iron-coupled radical upon reconstitution. The coordinating structure of the diiron site is similar to that of diiron oxidases/monoxygenases and supports a role for this radical in the RNR mechanism. The specific ligand pattern in the C. trachomatis R2 metal site characterizes a new group of R2 proteins that so far has been found in eight organisms, three of which are human pathogens.     

叶片老化对甘薯品种光合特性与产量的影响

以3个不同甘薯品种为试验材料,对3个品种的光合特性和产量进行了比较研究。结果表明:徐薯18较其他两个品种JS008和JS004的叶片净光合速率、蒸腾速率、气孔导度等光合特性有关指标随叶片的老化下降速度慢,叶片老化速度慢,T/R值小,其光合成产物分配到地下部块根的比例大,产量高。     

尼罗罗非鱼P2X4R基因克隆及原核表达分析

[目的]克隆尼罗罗非鱼P2X4R基因,并构建原核表达载体进行诱导表达,为深入研究P2X4R在鱼类中的生物学功能打下基础.[方法]利用PCR克隆尼罗罗非鱼P2X4R基因的3个片段(G1、G2和G3),拼接获得目的基因后连接pCold II载体构建pCold II-P2X4R重组质粒,再转化大肠杆菌BL21(DE3)感受态细胞,以IPTG进行诱导表达.分别采用SDS-PAGE和Western blotting检测分析重组蛋白P2X4R的表达情况,并运用生物信息学在线分析软件对其理化性质、糖基化位点、跨膜区域、亚细胞定位及信号肽等进行预测分析.[结果]克隆获得的尼罗罗非鱼P2X4R基因大小为1108 bp,与pCold II载体重组后转化BL21(DE3)感受态细胞获得的原核表达载体经IPTG诱导可表达获得目的蛋白,在IPTG 1.0 mmol/L、37℃诱导4 h的条件下重组蛋白表达量高于在IPTG 0.1 mmol/L、16℃过夜诱导的表达量.重组蛋白P2X4R的分子量约43.0 kD,其氨基酸数量为354个,理论等电点(pI)为6.78,不稳定指数为37.62,属于稳定蛋白,脂肪族指数为74.35;重组蛋白P2X4R具有3个N-糖基化位点和1个O-糖基位点;该蛋白未见跨膜区,其蛋白几乎100%位于细胞膜内,不含信号肽.[结论]诱导表达获得的尼罗罗非鱼P2X4R蛋白具有3个N-糖基化位点和1个O-糖基化位点,推测其存在糖基化现象,可制备相应抗体用于揭示罗非鱼巨噬细胞的抗原呈递作用机制.     

有机肥基质对辣椒苗期生长及病害发生的影响

以鸡粪、食用菌下脚料、草炭、蛭石为供试材料,按体积比2：3：2：3和2：3：O：5分别复配成基质A和基质B,以1：5的草炭和蛭石作为对照1（CK1）,以菜园土为对照2（CK2）,通过人工接种发病的方式,研究了盆栽条件下不同基质对辣椒苗期生长与发病的影响。结果表明：基质A的辣椒出苗率为99．7％,与CK1无明显差异,但显著高于CK2。盆栽30d后辣椒的干物重、根活性等以基质A为最高,基质B次之。基质A和基质B对辣椒苗期猝倒病均具明显的抑制作用,发病率均显著低于两个对照。     

叶片老化对甘薯品种光合特性与产量的影响*

 以3个不同甘薯品种为试验材料,对3个品种的光合特性和产量进行了比较研究。结果表明:徐薯18较其他两个品种JS008和JS004的叶片净光合速率、蒸腾速率、气孔导度等光合特性有关指标随叶片的老化下降速度慢,叶片老化速度慢,T/R值小,其光合成产物分配到地下部块根的比例大,产量高。     

通过正交实验设计突变体组合提高甲基对硫磷水解酶OPHC2对乙基对硫磷的降解能力

分别自假产碱假单胞菌(Pseudomonas pseudoalcaligenes C2-1)和假单胞菌（Pseudomonas sp. 1-7）中克隆得到甲基对硫磷水解酶基因ophc2和ophc3,其编码甲基对硫磷水解酶OPHC2和OPHC3的氨基酸序列相似性达98.5%,仅5个氨基酸残基不同,但酶学性质差异很大,特别是对不同底物降解特性有很大差异。OPHC2对甲基对硫磷的催化效率是OPHC3的2.75倍,对乙基对硫磷的催化效率仅为OPHC3的12.9%。为了提高OPHC2对乙基对硫磷的降解活性,以OPHC3的序列为依据,针对5个不同的氨基酸设计正交实验,对OPHC2进行组合突变,最终获得了一个突变体P165S/A169V,对乙基对硫磷的催化效率是OPHC2的1.19倍,对甲基对硫磷的催化效率是OPHC2的1.36倍。     

A manganese(IV)/iron(III) cofactor in Chlamydia trachomatis ribonucleotide reductase

In a conventional class I ribonucleotide reductase (RNR), a diiron(II/II) cofactor in the R2 subunit reacts with oxygen to produce a diiron(III/IV) intermediate, which generates a stable tyrosyl radical (Y*). The Y* reversibly oxidizes a cysteine residue in the R1 subunit to a cysteinyl radical (C*), which abstracts the 3'-hydrogen of the substrate to initiate its reduction. The RNR from Chlamydia trachomatis lacks the Y*, and it had been proposed that the diiron(III/IV) complex in R2 directly generates the C* in R1. By enzyme activity measurements and spectroscopic methods, we show that this RNR actually uses a previously unknown stable manganese(IV)/iron(III) cofactor for radical initiation.     

羊栖菜多酚氧化酶特性

对羊栖菜(Sargassumfusiforme)多酚氧化酶(PPO)进行浸提和硫酸铵分级盐析粗提,并测定了酶的特性.结果表明:在供试反应体系中,当邻苯二酚量为0.3-1.2mL时,酶活力呈直线增长,底物含量太高会产生抑制作用,当底物含量∶酶含量=2∶1时,酶活力表现最高;其酶活力的最适温度为40-45℃,在35-60℃范围内酶均能保持较高的活力,但超过60℃时,其活力则迅速下降;在50-100℃下水浴处理1h,酶活力丧失50%-75%;酶活力的适宜pH相对偏碱,在pH9.1时表现为一个活力高峰,在pH11.0和12.0时酶活力仍很高.提取的羊栖菜多酚氧化酶,存在不同特性的同功酶.5种供试抑制剂对该酶均有一定的抑制效果,其中NaHSO3和L-半胱氨酸的抑制作用最强,在浓度为50μmol·L-1时,抑制率达100%.     

不同根瘤菌、大豆品种、土壤类型对固氮酶活性的影响

本研究结合黑龙江省生态环境,采用盆栽试验方法分别在黑土和草甸土中对大豆品种V1和V2接种根瘤菌株R1、R2和R3,在盛花期通过乙炔还原法测定离体根瘤的固氮酶活性。单因素效应对固氮酶活性的影响高于因素间的作用,菌株与品种两因素间的交互作用大于其它交互作用。结果表明,处理R1V2S2和R1V1S1的固氮酶活性最高,二者都极显著高于两种阴性处理CK。     

The cellular src gene product regulates junctional cell-to-cell communication

Overexpression of the cellular src gene in NIH 3T3 cells causes reduction of cell-to-cell transmission of molecules in the 400- to 700-dalton range. This down-regulation of gap junctional communication correlates with the activity of the gene product, the protein tyrosine kinase pp60c-src. The down-regulation was enhanced by point mutation of Tyr527 (a site that is phosphorylated in pp60c-src and that inhibits kinase activity) or by substitution of the viral-src for the cellular-src carboxyl-terminal coding region. Mutation of Tyr416 (a site phosphorylated upon Tyr527 mutation) suppresses both the down-regulation of communication by Tyr527 mutation and that by gene overexpression. The regulation of communication by src may be important in the control of embryonic development and cellular growth.     

发酵柠条粉混配基质对辣椒幼苗生长发育的影响

以发酵柠条粉(替代草炭基质)、珍珠岩和蛭石为基质材料,按照不同比例混配形成柠条粉复合基质,对混配基质物理性状和在辣椒育苗中的应用效果进行了研究。结果表明,添加发酵柠条粉基质,提高了混配基质的总孔隙度和通气孔隙度,降低了基质的持水孔隙度,部分混配基质完全符合育苗基质要求,且育苗效果明显优于对照(壮苗二号育苗基质);发酵柠条粉体积百分比在55%至60%之间,总孔隙度在70%至90%之间,通气孔隙度在10%至11%之间育苗效果更佳。通过测定辣椒幼苗根系活力和壮苗指数,确定用于辣椒育苗的发酵柠条粉混配基质中柠条粉∶珍珠岩∶蛭石的最佳比例为3∶1∶1或4∶1∶2。     

外源6-BA对不同生育时期淹水花生根系生长和荚果产量的影响

【目的】土柱栽培条件下,研究外源细胞分裂素(6-BA)对花生不同生育时期淹水胁迫下根系呼吸酶活性、内源激素含量及荚果产量的影响,为提高淹水胁迫下花生抗性及采用外源激素调控花生生长提供理论依据。【方法】选用山花108为试验材料,以全生育时期正常水分管理(CK)为对照,设置苗期(V3)、花针期(R3)、结荚期(R5)、饱果期(R7)4个淹水时期,于淹水10 d后喷施15 mg·L^-1的6-苄基腺嘌呤(6-BA),共9个喷施组合处理,即全生育时期正常水分管理(CK)、苗期淹水(V3-W)、苗期淹水后喷施外源细胞分裂素(V3-S)、花针期淹水(R3-W)、花针期淹水后喷施外源细胞分裂素(R3-S)、结荚期淹水(R5-W)、结荚淹水后喷施外源细胞分裂素(R5-S)、饱果期淹水(R7-W)、饱果期淹水后喷施外源细胞分裂素(R7-S)。处理后每隔5 d,取样测定根系无氧呼吸酶、有氧呼吸酶活性,内源激素含量,根系干重及根长密度等指标。【结果】淹水显著降低了20—60 cm土层的根系干重及根长密度。淹水结束后,V3-W处理20—40 cm土层无根系。2018与2019生长季,R3-...