嗜热脂肪土芽孢杆菌木聚糖酶基因的合成及其在大肠杆菌中的表达

来自嗜热脂肪土芽孢杆菌的木聚糖内切酶XT6在工业上有着重要的应用,已经成功应用于工业规模的生产试验.本文作者在合成XT6基因全序列的同时对其密码子进行了优化,且构建重组质粒在大肠杆菌中高表达.通过优化表达条件,功能正常的XT6基因在大肠杆菌中成功过量表达,蛋白表达量占细胞中总蛋白的65%.重组表达的木聚糖内切酶XT6特性和天然酶相似,以桦木木聚糖为底物测定细胞提取物中木聚糖酶活性,最大活性高达3 030 U/mL.本文首次报道了来自嗜热脂肪土芽孢杆菌中木聚糖酶基因全序列的合成和在大肠杆菌中成功过量表达.图6表1参19     

抗柑桔溃疡病菌可溶性单链抗体的表达及鉴定

前期采用核糖体展示技术筛选了抗柑桔溃疡病菌(Xanthomonas axonopodis pv.citri,Xac)高亲和力单链抗体(Single chain variable fragment,scFv)GX13、GX44和GX95,为高效表达具有生物活性的单链抗体,本研究将前期筛选的高亲和力单链抗体基因转入大肠杆菌HB2151中进行可溶性表达,点印迹和Western印迹检测可溶性单链抗体的表达水平,亲和层析纯化表达的单链抗体,酶联免疫吸附法(ELISA)检测纯化单链抗体的抗原结合活性.结果显示,3株抗柑桔溃疡病菌可溶性单链抗体在大肠杆菌HB2151中均获得成功表达,表达产物分子量(Mr)约为32.0×103,主要集中于细菌周质腔中.ELISA检测纯化的单链抗体都具备抗原结合活性,可进一步应用于柑桔溃疡病菌的免疫诊断和病害防治.图4参14     

营养失衡下的分层分质EPS对异型膨胀污泥及其沉降性差异影响

为探索EPS对膨胀污泥沉降性的影响,通过减少进水P源、N源的方式培养异型膨胀活性污泥,分析不同层、不同组分EPS对异型膨胀污泥沉降性影响.结果表明：异型膨胀（非丝状菌粘性膨胀、丝状菌膨胀）活性污泥EPS多糖组分Total-PS、中层组分（L-PS）含量均高于正常污泥.非丝状菌粘性膨胀污泥EPS各层多糖含量高于丝状菌膨胀污泥,而各层蛋白质（PN）含量均低于丝状菌膨胀污泥.非丝状菌粘性膨胀污泥Total-EPS（（229±94）mg/g MLSS）、PS/PN值不仅显著高于正常活性污泥（（86±16）mg/g MLSS）,也高于丝状菌膨胀污泥（（108±30）mg/g MLSS）.非丝状菌粘性膨胀污泥EPS多糖总量Total-PS、蛋白质总量Total-PN越高,SVI值越大,污泥沉降性越差,越易膨胀,且EPS蛋白质组分对污泥膨胀作用大于多糖;从不同层EPS看,松散外层（S层）EPS对非丝状菌粘性膨胀作用最大（S-PN与SVI值相关性最大,r为0.881,P<0.05）,是主要影响因子.丝状菌膨胀污泥,除了内层紧密型T-PN对SVI值影响较大外,其他各层、各组分EPS含量与SVI均呈微弱负相关.     

Estrogenic Compounds Downstream From Three Small Cities in Eastern Nebraska: Occurrence and Biological Effect1

Abstract: Recent studies have detected estrogenic compounds in surface waters in North America and Europe. Furthermore, the presence of estrogenic compounds in surface waters has been attributed, in some cases, to the discharge of wastewater treatment plant (WWTP) effluent. The primary objective of the current study was to determine if WWTP effluent contributes estrogens to the surface waters of Nebraska. A second objective of this study was to determine if estrogens were found in concentrations sufficient enough to manifest feminizing effects on fish. These objectives were satisfied by deploying polar organic chemical integrative samplers (POCIS) and caged fathead minnows at eight field sites. Deployment sites included: three reference sites (Pawnee Creek, the Little Blue River, and the Middle Loup River), two sites upstream of the WWTPs at Grand Island and Columbus, and three sites downstream of the WWTPs at Grand Island, Columbus, and Hastings. Following the seven day deployments, POCIS extracts were analyzed for estrone, 17β‐estradiol, estriol and 17α‐ethinylestradiol using liquid chromatography tandem mass spectrometry (LC/MS/MS). 17β‐estradiol was detected in POCIS from six of the eight field sites with the greatest quantities recovered in POCIS deployed downstream from the Grand Island and Hastings WWTPs. Estrone was detected only in the POCIS deployed downstream from the Grand Island and Hastings WWTPs. Estrogenic effects were detected in caged minnows analyzed for the hepatic mRNA expression of two estrogen‐responsive genes, vitellogenin (vg1) and estrogen receptor α (ERα). Fish deployed at the site where the greatest quantities of estrogens were recovered (Hastings) had significantly higher expression of both vg1 and ERα than fish deployed at any of the other sites. These results confirm that WWTP effluent contributes biologically significant levels of estrogens to Nebraska surface waters.     

甘蔗蔗糖磷酸合成酶SPSⅡ cDNA片段克隆与表达分析

蔗糖磷酸合成酶(Sucrose phosphate synthase,SPS)是蔗糖合成途径的关键限速酶,在蔗糖积累和碳分配中具有重要作用.在茎组织中SPSⅡ表达量占SPS转录总量的40%,表明SPSⅡ cDNA的克隆与表达对蔗茎中蔗糖的积累有着重要的影响.通过RT-PCR技术克隆分离SPSⅡ基因cDNA片段.序列分析表明该cDNA片段包含1个3 183 bp的开放读码框,可编码1 060个氨基酸.GenBank登录号为EU269038.通过实时荧光定量PCR检测SPSⅡ基因在甘蔗糖分积累的不同时期、不同组织部位的相对表达量.结果表明,在糖分积累初期蔗茎中的SPSⅡ相对表达量最大,在糖分积累中期蔗叶中的SPSⅡ相对表达量达到最高峰;同组织部位中,糖分积累初期SPSⅡ相对表达量高于糖分积累中期、后期.图6参11     

水稻精细胞优势表达基因RSSG58的启动子克隆和功能鉴定

利用本实验室已经克隆的水稻精细胞优势表达基因RSSG58的cDNA序列与NCBI中的粳稻基因组文库进行比对、定位,结合生物信息学方法分析预测基因上游的启动子序列.在此基础上设计引物,以粳稻Oryzasativa(japonicacultivar-group)日本晴黄化苗总DNA为模板,采用DNA聚合酶链式反应(PCR)的方法扩增出基因上游1093bp和462bp两个不同长度的启动子缺失片段(分别命名为JP58B和JP58S),测序结果显示,其具有大多数高等植物启动子的保守元件.进一步构建启动子片段的植物表达载体pBI121-JP58B和pBI121-JP58S,瞬时表达结果显示,两个启动子片段对报告基因GUS均具有启动活性.图4参20     

碳中和的缘起、实现路径与关键科学问题：气候变化与可持续城市化

碳中和是我国应对气候变化的新目标,提出以来引起国内外广泛关注。碳中和目标实现是一个复杂系统科学问题,涉及大气圈、岩石圈、水圈、生物圈、人类圈等多圈层耦合过程与作用机制,需要跨学科交叉、综合与协同研究。简要梳理碳中和提出的气候变化和温控目标缘起,归纳碳中和实现的碳源、碳汇的多元路径,强调从碳源的需求侧推动可持续城市化对于碳中和目标实现的重要作用,并且可能是成本低、潜力大、更直接有效的方案途径。提出适应与减缓气候变化下碳中和与可持续城市化研究领域8个方面的科学问题：气候变化—碳中和—可持续城市化关联关系、中国及各地区碳中和实现的路线图与时间表、城市区域尺度碳排放的精准核算、绿色生活方式与低碳社会、绿色生产方式与低碳经济、能源结构优化、风险与不确定性和碳中和长效机制的治理创新等,以及该领域当前研究面临的机遇和挑战。     

DHDPS的细胞表达、提纯和结晶

ＤＨＤＰＳ是赖氢酸合成通路中第一步的合成酶。将中国春小麦的ＤＨＤＰＳ基因质粒移植入ＲＤＡ８中，得到ＲＤＡ８／ｐＤＢ２６菌株，本研究通过细胞培养、层析提纯和结晶条件的探索，给出了一个较好的技术路线，并为开展义衍射分析蛋白结构创造了条件，通过该研究，对中国春小麦ＤＨＤＰＳ和野种大肠杆菌的ＤＨＤＰＳ差异有了一定的了解，并且对细胞培养的ＭＭ介质处理，ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ，Ｐｈｅｎｙｌ－Ｓｅｐｈａｒｏｓｅ和ＭｏｎｏＱ层析的方法给出具体实验条仲。酶活的检测和蛋白浓度测定都是采取高灵敏度的方法。结晶的ＳＣｒｅｅｎｉｎｇ条件对于二种ＤＨＤＰＳ有很大的差异。对野种大肠杆茵的ＤＨＤＰＳ，需要表面活性剂Ｎ－ｏｃｔｙｌ－Ｄ－ｇｌｕｃｏｐｙｒａｎｏｓｉｄｅ，ｐＨ１０．０～１０．５，对于中国春小麦一ＤＨＤＰＳ则未找到较好的表面活性剂，ｐＨ６．８～７．６。中国春小麦一ＤＨＤＰＳ晶体培养条件在以往文献中未见报导。     

Polycyclic aromatic hydrocarbons (PAH) in different forest humus types

The behavior of 20 PAH in the organic layers of a L mull, an Of mull and a mor was assessed by a combined approach of a soil profile study, and the analysis of particle-size separates. Increasing PAH concentrations with depth in the mor profile (L, 866 μg kg⁻³; Of, 2902 μg kg⁻¹; Oh, 10489 ug kg⁻¹) were assigned to selective enrichment during organic matter decomposition. PAH were further highly enriched within the finer separates. For the L horizons, significant positive correlations were established between the enrichment of individual PAH (as observed from the decomposition gradient between the >2-mm fraction and the < 0.05-mm fraction), and the K_OW for each compound. The slope of the regression line, m, described the degree of differentiation between low- and high-molecular PAH during litter decomposition. Since m was greatest in the most biologically active humus type (L mull, 0.33) and smallest in the most inactive (mor, 0.20), microbial breakdown was assumed as the dominating process for this differentiation. The results also indicated that decomposition processes had already taken place in the L horizons, leading to morphological and chemical changes of organic matter, and to an enrichment of high molecular PAH.