Phylogenetic analysis and heterologous expression of surface layer protein SlpC of Bacillus sphaericus C3-41

The surface layer protein encoding genes from five mosquito-pathogenic Bacillus sphaericus isolates were amplified and sequenced. Negative staining of the S-layer protein extracted from the cell wall of wild-type B. sphaericus C3-41 was prepared. It showed a flat-sheet crystal lattice structure. Two genes encoding the entire and N-terminally truncated S-layer protein (slpC and DeltaslpC respectively), were ligated into plasmid pET28a and expressed in Escherichia coli. SDS-PAGE revealed that about 130 KD and 110 KD proteins could be expressed in the cytoplasm of recombinant E. coli BL21(pET28a/slpC) and E. coli BL21(pET28a/DeltaslpC) respectively. Furthermore, an intracellular sheet-like or fingerprint-shape structure was investigated in two recombinant strains, which expressed SlpC and DeltaSlpC protein respectively, by ultrathin microscopy study, but bioassay results suggested that the S-layer protein of wild B. sphaericus C3-41 and recombinant E. coli BL21 (pET28a/slpC) have no direct toxicity against mosquito larvae. These results should provide information for further understanding of the function of S-layer protein of pathogenic B. sphaericus.     

Mixed Infection of Two Begomoviruses in Malvastrum coromandelianum in Fujian, China

Virus isolates were obtained from three Malvastrum coromandelianum plants showing vein thickening symptoms in Fujian Province, China. A fragment of approximately 500 bp was amplified from all the samples by PCR using the special degenerate primer pair PA/PB for begomoviruses. Sequence differences among the partial DNA-A fragments revealed that all three samples contained two virus isolates. Isolate I and isolate II share the highest nucleotide sequence identity (98–99%), respectively, with Malvastrum leaf curl Guangdong virus (MLCuGdV) and Ageratum yellow vein virus (AYVV). The complete nucleotide sequences of Fs1 and Fs2 isolates representing each virus were determined to be 2741 and 2756 nucleotides, respectively. Alignment and phylogenetic analysis showed that the complete DNA-A sequences of Fs1 and Fs2 were most closely to those of MLCuGdV (AM503104) and AYVV (AB100305), with 90.4% and 93.3% nucleotide sequence identity, respectively. Fs1 and Fs2 are considered therefore to be isolates of MLCuGdV and AYVV, respectively. This is the first report of AYVV in M. coromandelianum.     

青鱼生长激素的重组表达及其多克隆抗体的制备

以含有的青鱼生长激素编码区cDNA的重组质粒pbcGHc为模板,高保真PCR扩增青鱼生长激素（GH）成熟肽cDNA序列,定向插入原核表达载体pET-28a,构建青鱼GH原核表达质粒pET-bcGH。将pET-bcGH转化大肠杆菌BL21(DE3),IPTG诱导青鱼GH基因在大肠杆菌中的融合表达,SDS-PAGE凝胶电泳结果显示一条23 kDa的诱导表达重组青鱼GH带。以草鱼GH多克隆抗体为一抗,Western Blot证明,该重组青鱼GH具有免疫学活性。将经过亲和层析、透析纯化后的重组青鱼GH作为抗原,采用改进的方法对家兔进行皮下免疫注射,获得青鱼GH多克隆抗血清。以该多抗为一抗,Western Blot 可以检测出4 ng的抗原量;并且在青鱼垂体组织抽提液中和血清中检测到一种能与该抗血清作用的大小为21 kDa的蛋白质。这些结果表明本研究得到的青鱼GH多克隆抗血清具有较好的免疫特性。     

大球盖菇硒多糖体内抗氧化能力检测

采用液体发酵的方法对大球盖菇菌丝进行富硒培养,提取硒多糖进行抗氧化能力测定.结果表明,大球盖菇硒多糖能明显提高小鼠血中SOD、GSH-Px的含量,能在一定程度上显著降低血中MDA的含量,具有显著的抗氧化功能.     

盐离子对次壶腹腺丝蛋白重组模块的影响

为研究不同生理环境对蛛丝蛋白组装及成丝的影响,首次以MiSp序列为对象,研究其NTR1SR2CT重组模块在不同种类(浓度)盐离子条件下的聚集和成纤维特性及其在成纤维过程中二级结构的变化。基于大腹园蛛MiSp全长序列构建NTR1SR2CT模块,并在大肠杆菌Escherichia coli BL21中成功表达。借助8 mol/L尿素裂解包涵体进行变性纯化得到NTR1SR2CT重组蛋白。NTR1SR2CT重组蛋白二级结构主要为无规则卷曲(Random coil)或α螺旋(Helix),在自然成丝及冻干过程中部分random coil或helix转变为β折叠(β-sheet),甲醇能促进该转变过程。另外,钾离子和磷酸根离子有利于NTR1SR2CT重组蛋白聚集从而促进丝纤维的形成。研究结果为成丝机理研究奠定了基础,同时也为工业化生产高品质的蛛丝纤维提供了条件。     

模拟酸雨胁迫对青冈幼苗光合特性和叶绿素荧光参数的影响

选择亚热带常绿阔叶林优势树种青冈幼苗为研究对象,设置3个酸雨处理：pH 2.5、pH 4.0、pH 5.6(CK),研究不同强度的模拟酸雨对青冈幼苗光合特性、叶绿素荧光参数和叶绿素含量的影响.结果表明：经过2年酸雨处理,青冈的净光合速率随着酸雨强度的增加而显著上升.pH 2.5、pH 4.0处理的酸雨增大了青冈的气孔导度和蒸腾速率,且对pH 2.5处理的影响更为显著.胞间CO₂浓度的大小顺序是pH 2.5>pH 5.6>pH 4.0.pH 2.5、pH 4.0处理青冈的最大净光合速率、光补偿点、光饱和点、暗呼吸速率显著高于对照,表观量子效率对酸雨胁迫不敏感.pH 2.5、pH 4.0处理青冈的PSⅡ原初光能转化效率和PSⅡ的潜在活性显著高于对照.青冈的叶绿素相对含量大小顺序是pH 2.5>pH 5.6>pH 4.0,且pH 2.5与pH 4.0之间有显著差异.说明青冈幼苗的光合参数、叶绿素荧光参数指标在pH 2.5和pH 4.0的酸雨处理下有所增加,且在pH 2.5强度下增加更为明显.     

Identification of catalytically important residues in the active site of Escherichia coli transaldolase.

The roles of invariant residues at the active site of transaldolase B from Escherichia coli have been probed by site-directed mutagenesis. The mutant enzymes D17A, N35A, E96A, T156A, and S176A were purified from a talB-deficient host and analyzed with respect to their 3D structure and kinetic behavior. X-ray analysis showed that side chain replacement did not induce unanticipated structural changes in the mutant enzymes. Three mutations, N35A, E96A, and T156A resulted mainly in an effect on apparent kcat, with little changes in apparent Km values for the substrates. Residues N35 and T156 are involved in the positioning of a catalytic water molecule at the active site and the side chain of E96 participates in concert with this water molecule in proton transfer during catalysis. Substitution of Ser176 by alanine resulted in a mutant enzyme with 2.5% residual activity. The apparent Km value for the donor substrate, fructose 6-phosphate, was increased nearly fivefold while the apparent Km value for the acceptor substrate, erythrose 4-phosphate remained unchanged, consistent with a function for S176 in the binding of the C1 hydroxyl group of the donor substrate. The mutant D17A showed a 300-fold decrease in kcat, and a fivefold increase in the apparent Km value for the acceptor substrate erythrose 4-phosphate, suggesting a role of this residue in carbon-carbon bond cleavage and stabilization of the carbanion/enamine intermediate.