邻苯二甲酸二(2-乙基己基)酯对罗非鱼肝脏转录组影响研究

邻苯二甲酸二（2-乙基己基）酯（DEHP）作为生产量和使用量最多的一种邻苯二甲酸酯类化合物,其对水生生物具有多种毒性.本研究选用中国南方地区最重要的经济鱼类尼罗罗非鱼（Oreochromis niloticus）作为实验对象,研究DEHP（50mg/kg bw）作用下罗非鱼肝脏组织内转录组的响应特征.利用IlluminaTM HiSeq 4000测序平台对DEHP处理组和对照组尼罗罗非鱼肝脏RNA样品分别进行转录组测序.对照组和DEHP暴露组分别获得30.10Mb和30.16Mb待分析数据（clean reads）,对转录组数据进行组装并去冗余后得到58,585个Unigene.将DEHP处理组和对照组的转录组数据进行比较一共获得5,008个差异表达基因,其中上调表达基因和下调表达基因分别为3,217个和1,791个.通过GO和KEGG功能分析后发现这些差异表达基因主要为机体免疫、生殖内分泌以及脂类代谢相关基因,这表明DEHP对尼罗罗非鱼肝脏毒性主要表现为免疫毒性、生殖毒性和脂类代谢紊乱.另外,实时荧光定量PCR验证结果发现转录组分析数据基本可靠.上述结果为在转录组水平筛选DEHP生物标志物,解析DEHP对罗非鱼毒性作用的分子机制提供了科学参考.     

Gene expression changes in blood RNA after swimming in a chlorinated pool

Exposure to disinfection by-products(DBP) such as trihalomethanes(THM) in swimming pools has been linked to adverse health effects in humans, but their biological mechanisms are unclear. We evaluated short-term changes in blood gene expression of adult recreational swimmers after swimming in a chlorinated pool. Volunteers swam 40 min in an indoor chlorinated pool. Blood samples were drawn and four THM(chloroform,bromodichloromethane, dibromochloromethane and bromoform) were measured in exhaled breath before and after swimming. Intensity of physical activity was measured as metabolic equivalents(METs). Gene expression in whole blood m RNA was evaluated using Illumina Human HT-12v3 Expression-Bead Chip. Linear mixed models were used to evaluate the relationship between gene expression changes and THM exposure. Thirty-seven before-after pairs were analyzed. The median increase from baseline to after swimming were: 0.7 to 2.3 for MET, and 1.4 to 7.1 μg/m~3 for exhaled total THM(sum of the four THM).Exhaled THM increased on average 0.94 μg/m~3 per 1 MET. While 1643 probes were differentially expressed post-exposure. Of them, 189 were also associated with exhaled levels of individual/total THM or MET after False Discovery Rate. The observed associations with the exhaled THM were low to moderate(Log-fold change range:-0.17 to 0.15). In conclusion, we identified short-term gene expression changes associated with swimming in a pool that were minor in magnitude and their biological meaning was unspecific. The high collinearity between exhaled THM levels and intensity of physical activity precluded mutually adjusted models with both covariates. These exploratory results should be validated in future studies.     

苯丙氨酸解氨酶的分子生物学研究进展

苯丙氨酸解氨酶 (Ｐｈｅｎｙｌａｌａｎｉｎｅａｍｍｏｎｉａｌｙａｓｅ ,ＰＡＬＥ .Ｃ .4.3 .1.5 )是一种只存在于植物及微生物细胞内的酶 ,主要分布于高等植物如小麦、大豆、玉米、土豆及酵母[1] 、真菌[2 ] 、链霉菌[3]中 .在 ｐＨ 8.8时 ,ＰＡＬ催化苯丙氨酸脱氨生成肉桂酸和氨 ,当过量的氨存在 ,如 ｐＨ 10时 ,ＰＡＬ催化肉桂酸和氨 ,氨化加成生成Ｌ 苯丙氨酸 (Ｌ ｐｈｅｎｙｌａｌａｎｉｎｅ) .利用ＰＡＬ这种逆向催化特性转化肉桂酸生产Ｌ Ｐｈｅ ,已在国内外投入商业规模开发 ,成为当前生物化工领域研究与开发的热点 .Ｌ Ｐｈｅ是人…     

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     

豆豉溶栓酶基因在毕赤酵母中的表达及其产物的纯化

将含有和不含前肽的豆豉溶栓酶编码序列在毕赤酵母中分别进行表达研究,发现在含有前肽的豆豉溶栓酶基因转化的毕赤酵母工程菌用甲醇诱导时,其培养液中可检测到较强的溶栓酶活性;而在不含前肽的豆豉溶栓酶基因转化的毕赤酵母菌用甲醇诱导时,培养液中未检测到溶栓酶活性.对毕赤酵母表达和分泌的豆豉溶栓酶进行了分离纯化,并对其纯化产物进行溶栓酶活性、SDS-PAGE电泳、抑制剂作用及免疫印迹分析.结果表明,分泌到培养液中的豆豉溶栓酶已经过剪切加工,其前肽已被切除,重组与天然的豆豉溶栓酶具有相同的溶栓酶活性,其所测定的各理化指标均一致.本研究实现了豆豉溶栓酶在毕赤酵母菌中成功表达,并首次直接证明了前肽对豆豉溶栓酶在毕赤酵母中分泌表达是必须的.图3表1参20     

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。     

细胞分裂素基因（T－cyt）在转基因烟草中的表达及其对生长发育的影响

将编码细胞分裂素合成关键酶──异戊烯基转移酶的Ｔ－ｃｙｔ基因分别置于ＣａＭＶ３５Ｓ启动子，ｒｂｃＳ启动子和Ｔ－ｃｙｔ基因自身启动子的调控下，构建成不同的嵌合质粒用于转化烟草，通过ＰＣＲ检测，Ｓｏｕｔｈｅｒｎ杂交和ＮＰＴⅡ的酶活检测对获得的转基因烟草进行了鉴定．Ｎｏｒｔｈｅｒｎ分析表明：ＣａＭＶ３５Ｓ启动子驱动下的Ｔ－ｃｙｔ基因在转基因烟草的根、茎、叶中均有ｍＲＮＡ的积累；ｒｂｃＳ启动子指导下Ｔ－ｃｙｔ基因在叶中转录较强，茎中次之，在根中几乎未表达．以根据抗原决定簇进行人工合成的复合抗原免疫家兔得到异戊烯基转移酶抗体，ＥＬＩＳＡ结果表明转基因烟草根中异戊烯基转移酶含量较高．Ｔ－ｃｙｔ基因的表达对转基因烟草的生长发育有明显影响：其叶绿素ａ、ｂ含量明显增加，叶衰老迟缓；顶端优势受到抑制，侧芽生长旺盛；与对照相比，其根系不发达．     

中药青蒿鲨烯合酶的大肠杆菌表达、纯化与功能鉴定

利用PCR方法,将经RACE方法克隆到的中药青蒿鲨烯合酶cDNA(AF302464)开放阅读框的3′末端截短99bp,插入到原核表达载体pET30a( )的NcoⅠ和BamHⅠ酶切位点之间,构建N端和C端均携带有HIS6表达标签的鲨烯合酶重组表达载体pETSSA.将pETSSA转入大肠杆菌BL21(DE3),0.5mmol/LIPTG(isopropyl-beta-D-thiogalactoside)28℃诱导重组鲨烯合酶的表达.表达产物经镍琼脂糖柱纯化.纯化蛋白加入酶促反应体系(含FPP和NADPH),GC-MS分析酶促反应体系的正己烷萃取物,结果显示重组鲨烯合酶可以催化FPP向鲨烯的转化.青蒿鲨烯合酶的功能鉴定为进一步利用反义或RNAi技术限制甾类生物合成,从而提高青蒿中的青蒿素含量提供了基础.图5参15     

Cd2+、Cu2+ 和Zn2+ 对5种单细胞藻酯酶基因表达调控的影响

在Cu^2 ,Cd^2 和Zn^2 的作用下，三角褐指藻（Phaeodactylum tricornutum Bohlin)，湛江叉鞭藻（Dicrateria Zhanjiangensis Hu)，绿色巴夫藻（Pavlova viridis Tseng,Chen et Zhang sp.nov.)青岛大扁藻（Platymonas helgolandica var.Tsingtaoensis Tseng et T.J.Chang) ,小球藻（Chlorella sp.) 等单细胞藻酯酶安生明显的变化，受高浓度的Cu^2 胁迫时三角褐指藻的Est-3座位的a基因，受中等浓度Cd^2 胁迫时的湛江叉鞭藻的Est-3座位的b基因，受低等浓度Cd^2 胁迫时的绿色巴夫藻的Est-1座位的a,b,两个,高浓度Zn^2 胁迫时绿色巴夫藻Est-1座位的b基因，受高浓度Cd^2 胁迫时青岛大扁藻的Est-2座位的c基因，受Cu^2 胁迫时小球藻的Est-1座位的b基因，受中低浓度的Cd^2 ,Zn^2 和低浓度的Cu^2 胁迫时小球藻的Est-2座位的b基因等基因的表达明显增强，而绝大部分位点的基因表达受3种离子的抑制。图5表5参9     

Molecular toxicity of earthworms induced by cadmium contaminated soil and biomarkers screening

Earthworms (Eisenia fetida) were used to study the impact of low-dose cadmium in treated artificial soil (0, 0.6, 3, 6, 15, 30 mg/kg) and contaminated natural soil (1.46 mg/kg). The changes of earthworms' physiological related gene expressions of metallothionein (MT), annetocin, calreticulin and antimicrobial peptides were detected using real-time PCR after a 70-day incubation period. The results showed that low doses of cadmium could up regulate earthworms' MT and down regulate annetocin gene expression and show a significant positive and negative correlation respectively. The expression of two other genes, calreticulin and anti-microbial peptides, was induced at low doses of cadmium (highest gene expression at 0.6 mg/kg for calreticulin and 6 mg/kg for anti-microbial peptides) and inhibited at high doses. No significant correlation was found for these two genes. This study shows that MT and annetocin genes expression found in earthworms in contaminated soil have the potential to be developed as biomarkers of soil cadmium pollution.