重组L-天冬氨酸-β-脱羧酶融合蛋白复性条件的研究

以本实验室构建保存的天冬氨酸脱羧酶工程菌为研究材料,通过基因表达,对所得到的重组胞内融合表达型天冬氨酸脱羧酶蛋白进行体外复性研究,考察温度、复性液的pH、复性时间、复性液种类等因素对重组天冬氨酸脱羧酶体外复性的影响。结果表明:最佳复性液为pH8.7的Tris-HCl缓冲液,在保温时间2h,温度40℃下,复性效果最好。另外一些pH能达到8.7的缓冲液复性效果都不如Tris-HCl缓冲液。     

中国对虾继饥饿后的补偿生长研

1999年7至8月份,在25.0±0.5℃条件下对中国对虾（湿重,1.454±0.150g）进行了不同时间的饥饿处理后再供食的恢复生长实验。对照组C连续饱食投喂32d;处理组S4、S8和S12分别饥饿4、8和12d后再饱食投喂28、24和20d。主要结果如下饥饿结束时各处理组的干重和湿重显著低于对照组（P<0.05）;实验结束时S4组和对照组间的干重和湿重差异不显著（P>0.05）,而S8和S12两组的干重和湿重仍显著低于对照组（P<0.05）;恢复生长后各处理组的湿重摄食率显著高于对照组（P<0.05）。实验结果表明,中国对虾继饥饿后再恢复喂食出现完全或部分补偿生长效应,且这种补偿生长效应主要是通过恢复生长阶段食欲增大,摄食水平提高实现的。     

对虾-罗非鱼-缢蛏封闭式综合养殖的水质研究

研究了对虾、罗非鱼和缢蛏封闭式综合养殖的环境状况.结果表明,混养各组环境状况总体要优于单养组.实验期间混养组DO的波动幅度略小于单养组,最低值则高于单养组;各混养组COD值和水层细菌数量相互差异不显著,但各混养组均显著低于单养组(t检验,α<0.05),混养围隔水体有机质含量明显低于单养;混养各围隔组中浮游生物生物量、Chl.a均低于单养组,滤食性动物对浮游生物的压制作用明显.在底泥中,混养组N、P的积累量比单养组低39.76%和51.26%,混养组细菌总数则比单养组低7.63%.本研究表明,封闭式综合养殖可以大大减少注入近海的养殖污水的排放量,从而降低对近海水质的污染.     

Identification of the Third Binding Site of Arsenic in Human Arsenic (III) Methyltransferase

Arsenic (III) methyltransferase (AS3MT) catalyzes the process of arsenic methylation. Each arsenite (iAs³⁺) binds to three cysteine residues, methylarsenite (MMA³⁺) binds to two, and dimethylarsenite (DMA³⁺) binds to one. However, only two As-binding sites (Cys156 and Cys206) have been confirmed on human AS3MT (hAS3MT). The third As-binding site is still undefined. Residue Cys72 in Cyanidioschyzon merolae arsenite S-adenosylmethyltransferase (CmArsM) may be the third As-binding site. The corresponding residue in hAS3MT is Cys61. Functions of Cys32, Cys61, and Cys85 in hAS3MT are unclear though Cys32, Cys61, and Cys85 in rat AS3MT have no effect on the enzyme activity. This is why the functions of Cys32, Cys61, and Cys85 in hAS3MT merit investigation. Here, three mutants were designed, C32S, C61S, and C85S. Their catalytic activities and conformations were determined, and the catalytic capacities of C156S and C206S were studied. Unlike C85S, mutants C32S and C61S were completely inactive in the methylation of iAs³⁺ and active in the methylation of MMA³⁺. The catalytic activity of C85S was also less pronounced than that of WT-hAS3MT. All these findings suggest that Cys32 and Cys61 markedly influence the catalytic activity of hAS3MT. Cys32 and Cys61 are necessary to the first step of methylation but not to the second. Cys156 and Cys206 are required for both the first and second steps of methylation. The S^C32 is located far from arsenic in the WT-hAS3MT-SAM-As model. The distances between S^C61 and arsenic in WT-hAS3MT-As and WT-hAS3MT-SAM-As models are 7.5 Å and 4.1 Å, respectively. This indicates that SAM-binding to hAS3MT shortens the distance between S^C61 and arsenic and promotes As-binding to hAS3MT. This is consistent with the fact that SAM is the first substrate to bind to hAS3MT and iAs is the second. Model of WT-hAS3MT-SAM-As and the experimental results indicate that Cys61 is the third As-binding site.     

Functional analysis of the seven in absentia ubiquitin ligase family in tomato

Seven in absentia (SINA) protein is one subgroup of ubiquitin ligases possessing an N‐terminal cysteine‐rich really interesting new gene (RING) domain, two zinc‐finger motifs, and a C‐terminal domain responsible for substrate‐binding and dimerization. In tomato (Solanum lycopersicum), the SINA gene family has six members, and we characterize in this study all tomato SINA (SlSINA) genes and the gene products. Our results show that SlSINA genes are differentially regulated in leaf, bud, stem, flower, and root. All SlSINA proteins possess RING‐dependent E3 ubiquitin ligase activity, exhibiting similar specificity towards the E2 ubiquitin‐conjugating enzyme. SlSINA1/3/4/5/6 are localized in both cytoplasm and nucleus, whereas SlSINA2 is exclusively localized in the nucleus. Moreover, all SlSINAs can interact with each other for homo‐ or hetero‐dimerization. The functionality of SlSINA proteins has been investigated. SlSINA4 plays a positive role in defense signalling, as manifested by elicitation of E3‐dependent hypersensitive response‐like cell death; the other SlSINAs are negative regulator and capable to suppress hypersensitive response cell death. Transgenic tomato plants overexpressing SlSINA2 exhibit pale‐green leaf phenotype, suggesting SlSINA2 regulates chlorophyll level in plant cells, whereas transgenic tomato plants overexpressing SlSINA5 have altered floral structure with exserted stigma, implicating SlSINA5 plays a role in flower development.     

限氧自养硝化-反硝化生物脱氮新技术

限氧自养硝化—反硝化是部分硝化与厌氧氨氧化相耦联的生物脱氮反应过程,通过严格控制溶解氧在0．1～0．3mg·L^-1,实现硝化反应控制在亚硝酸阶段,然后以硝化阶段剩余的NH4^+作为电子供体,在厌氧条件下实现反硝化,该反应过程是完全的自养硝化—反硝化过程,具有能耗低、脱氮效率高、反应系统占地面积小等优点,适用于处理COD／NH4^+—N低的废水,是一种非常有应用前景的生物脱氮技术,文中详细介绍了限氧自养硝化—反硝化生物脱氮反应过程的研究进展,讨论了其微生物学机理及应用前景。     

黑曲霉固态发酵木聚糖酶中试条件的研究

通过对黑曲霉在曲盘中进行固态发酵木聚糖酶中试条件进行优化研究,结果表明最适的产酶培养基中麸皮与玉米芯比例为6:4,添加硫酸铵可以促进黑曲霉菌丝生长,对酶活没有显著影响;最适的培养基初始pH值为4.0,培养基厚度为4cm;在28℃下培养60～84h,木聚糖酶活稳定在最高峰。     

五氯酚厌氧生物降解体系中细菌的多样性分析

为了解五氯酚(PCP)降解过程中参与PCP降解的微生物多样性,本文应用16S rRNA基因克隆文库方法对PCP厌氧生物降解体系中细菌群落的组成和相对丰度进行了研究.结果表明,TM7类群的微生物在整个细菌群落中占有最大丰度(48.6%),检测到的序列与在三氯乙烯污染的地下水中检测的克隆子有一定的序列相似性(93.6%).丰度位居第二的微生物类群为β-变形菌纲(Betaproteobacteria)细菌,其中的一些克隆子(10.8%)与脱氯微生物Dechlorosoma suillum具有极高的序列同缘性(99.7%).此外,也检测到少数Clostridium属[厚壁菌门(Firmicutes)类群]的微生物.克隆文库中发现许多序列(占整个克隆文库的51.3%)与GenBank中已报道的序列具有较远的同源性(小于93.4%),它们可能代表新的微生物.本研究进一步拓宽了对PCP降解微生物多样性的认识.