五氯酚诱导斑马鱼p53基因点突变的nested PCR-RFLP分析

将斑马鱼经50μg/L五氯酚(PCP)体内暴露染毒10d,提取肝脏基因组DNA,通过巢式PCR(nestedPCR)扩增包含斑马鱼p53基因外显子2,3,4及其间内含子2,3的片段,扩增产物分别用限制性内切酶NcoI、SacI、ScaI、BclI、NsiI、RsaI进行限制性片段长度多态性(RFLP)分析,以检测目标片段在酶切位点上点突变的发生.检测了20条斑马鱼上述6个酶切位点上的共680个碱基,但均未检测到点突变.结果表明,如果PCP能够诱导斑马鱼p53基因点突变,则其碱基突变率小于1/680.     

湖泊中军团菌的巢式PCR-MPN检测法

为了直接检测环境水体中军团菌的存在,文章将巢式PCR与倍比稀释法相结合(巢式PCR-MPN法),采用两对引物扩增环境样品基因组DNA。将检测到含有军团菌的样品DNA模板进行十倍梯度稀释,由最低检测的稀释度,计算出样品中军团菌的细胞数,同时确定方法检测的灵敏度。并用克隆建库、构建系统树的方法来验证该文研究方法的特异性。研究结果表明,巢式PCR第二步后获得的序列均为军团菌序列,该方法军团菌DNA的检出限可以低至2.74 fg/μL。该研究建立的水体军团菌检测方法具有简便快捷、特异性好、灵敏度高的特点。     

显微分光光度计对水体有机污染物诱导的转化细胞DNA含量的分析

在水源水及自来水有机提取物诱导人胚肺（BKM）细胞恶性转化基础上,运用MPV－SP型显微分光光度计对Ⅲ型转化灶中细胞的DNA含量进行了分析。结果表明：水样在实验剂量范围内可不同程度地诱导细胞转化灶的形成,并有明显的剂量反应关系。与源水相比,自来水有较强的潜在致癌性,从Ⅲ型转化灶克隆中挑取的细胞DNA含量增高,与阴性对照比有显著性差异,说明转化细胞形态学改变与细胞DNA含量的改变有一定的关系。     

二氧化硫体内衍生物诱发CHL细胞染色体畸变效应

研究了SO2在体内的衍生物一亚硫酸氢钠和亚硫酸钠对中国仓鼠肺纤维细胞（CHL）细胞染色体畸变（CA）的诱发作用研究结果表明,SO₂的衍生物不论是否有S₉混合物的存在情况下,均可诱发CHL细胞的CA频率显著增高,且呈明确的剂量一效应关系．这说明SO₂不需要体内代谢转化的直接的细胞染色体断裂剂和基因毒性因子研究亦发现,SO₂产低浓度下主要诱发染色单体型畸变,在高浓度下既可引起染色单体型畸变,又可引起染色体型畸变研究还发现,SO₂衍生物处理细胞时间愈长,引起细胞遗传损伤所需的最低浓度就越低．提示：长期接触环境低浓度SO₂污染,有引起接触人群体内细胞遗传物质损伤的潜在危险。     

沼泽红假单胞菌phbC-hupL双突变株构建及产氢测定

利用湖底淤泥分离的沼泽红假单胞菌(Rhodopseudomonas palustris)CQU01和该菌株的吸氢酶基因hupL缺失菌株CQU012作为出发菌株,分别构建聚羟基丁酸酯合成酶基因phbC单突变株及聚羟基丁酸酯合成酶基因phbC与吸氢酶基因hupL双突变株,以提高其在光照培养条件下的产氢量.以同源重组双交换方法构建含有Em抗性基因的自杀载体,通过接合转移转化R. palustris CQU01菌株,经PCR扩增以及测序验证,成功获得了沼泽红假单胞菌phbC单突变株R. palustris CQU013及phbC-hupL双突变株R. palustris CQU014.相同条件下测定突变菌株与野生菌株的生长和产氢特性,结果显示,突变菌株生长曲线与野生菌株有明显差异,两株突变菌株的产氢量分别是原始菌株的1.31和1.76倍,达到454mL/L和604mL/L.双突变菌株产氢能力较phbC基因和hupL基因单突变菌株的产氢能力有明显提高,说明phbC和hupL基因对菌株R. palustris 的产氢代谢有着显著的影响.     

Phaeocystis globosa核糖体大亚基(28SrDNA)的序列分析与分类学意义

对一株Phaeocystisglobosa的28SrDNA基因序列测定,共得到碱基大小为1795bp的两个序列片段,其中序列一的长度为970bp,序列二的长度为825bp。将Ph.globosa与Ph.antarctica和Prymnesiumpatelliferum同源序列进行对比,发现Ph.globosa与Ph.antarctica仅在序列二中有一个碱基插入/缺失,同源性为99.99%,而与Pr.patelliferum相比,有7处插入/缺失,碱基总变异率为6.13%;研究发现序列一中有一个比较明显的高度保守区和两个高变区;序列二中有两个比较明显的高变区、一个保守区和一个高度保守区。对28SrDNA基因RNA二级结构分析发现,DNA序列保守区在RNA二级结构上也非常保守,与DNA序列分析结果一致,都证明28SrDNA基因只适用于种以上水平的分类研究,不宜用于种间和种下水平的研究。     

诺卡氏菌株C-14-1中腈水解酶基因的鉴定、测序及分析

从腈纶废水中分离到高效降解多种污染物的诺卡氏菌株C-14—1,并对该菌中腈水解酶的基因进行鉴定和测序．利用红球菌中腈水解酶氨基酸保守区设计核苷酸引物,以菌株C-14—1的总DNA为模板,PCR扩增发现一条预期大小的DNA带．Southern杂交显示基因组中存在一个腈水解酶基因．进一步构建基因文库和菌落原位杂交,克隆到一个约4．5kb的DNA片段．DNA序列测定和分析表明,该DNA片段携带长度为1143bp的腈水解酶基因．比对分析表明,该基因与国际上发表的红球菌和诺卡氏菌中的腈水解酶基因高度相似．     

养殖场周边环境污染对畜禽养殖质量影响研究——以基于DNA条形码技术的黄牛肉质检测物研究为例

当前养殖场周边环境严重影响畜禽类的养殖质量,以基于DNA条形码技术的黄牛肉质检测物研究为例,对黄牛肉的真伪进行了检测。对黄牛种类特异性基因COI基因(NC_006853.1)序列进行设计,利用DNA提取、DNA纯度和浓度测定、PCR扩增、电泳检测、DNA纯化和回收、DNA克隆,完成肉质真伪的检测。得到电泳检测结果,真正黄牛肉扩增片段长度为534 bp,其余肉品样本不能扩增出534 bp。实验结果表明,该检测方法操作方便,检测时间短,应用前景较好,可以满足市场肉类监督检测需要。     

二氯乙腈对大肠杆菌基因表达的影响

为探究二氯乙腈（DCAN）对大肠杆菌（E.coli）基因表达的影响及相应的毒性作用,采用自组织映射（SOM）聚类及剂量效应关系分析方法考察了E.coli在不同剂量DCAN暴露120min过程中其基因表达状况.结果表明：随时间及浓度改变E.coli基因表达具有动态性;在DCAN浓度为1.429×10^-3mg/L时,多个参与应急反应（SOS response）调节、氧化还原应激及普通应激的基因启动子活性发生改变,导致DNA损伤、氧化应激加剧,细胞生物化学和物理稳态可能受到干扰;此外,毒性终点结果表明DNA损伤是DCAN主要的毒性作用模式.     

离子色谱法测定地下水中碘离子

建立毛细管离子色谱-积分安培检测法测定地下水中碘离子的方法.样品经0.20 μm滤膜过滤后注入离子色谱仪,采用IonPac AS16阴离子交换分离柱分离,62.5 mmol/L NaOH溶液梯度洗脱,由安培检测器积分安培检测模式定量检测.方法检出限为0.20 μg/L,线性范围5～100μg/L,相关系数0.9999.重现性良好,相对标准偏差小于0.6％(n=9).加标回收率为91.2％～106.0％.     

氧弹燃烧-离子色谱法测定高分子聚合物的卤素

随着欧盟RoHS标准的实施,对塑料制品中卤素的检测也越来越受到重视。文章建立了一种采用氧弹燃烧法对塑料样品进行燃烧,以碳酸钠为吸收液进行吸收,离子色谱法分离测定微量卤素离子的方法。选择的色谱条件是:IonPac AS22离子交换柱,0.48mmol/L碳酸钠和0.1 mmo/L碳酸氢钠混合液作淋洗液,流速为1.0 mL/L,抑制型电导检测。氟、氯、溴、碘四种卤素的检测限(3倍噪声比)分别为0.007、0.009、0.027和0.033.相对标准偏差分别为1.19%、1.61%、1.02%和1.28%,回收率为78.2%~119.8%。方法具有简便、准确、灵敏等特点,可较方便地应用于常规的质量检测和控制。     

镍钴转运酶NiCoT基因的克隆表达及基因工程菌对镍离子的富集

利用PCR技术从Staphylococcus aureus/ ATCC6538基因组中扩增出大小为1?053 bp的镍钴转运酶基因NiCoT gene,将其连接到pET-3c载体上构建重组质粒,并转化至E.coli BL21.筛选阳性菌并经酶切分析和PCR扩增双重鉴定.核苷酸序列测定及分析结果与GenBank中报道的同类基因相似性高达97%以上,表明其具有正确的NiCoT基因核苷酸序列.重组菌的SDS-PAGE结果图谱中,在相对分子量为39?000附近有特异性蛋白条带,大小符合预测值,表明NiCoT基因在E.coli BL21中成功表达.基因工程菌在IPTG用量为1.00 mmol·L^-1,诱导时间为4 h的条件下培养对镍离子的富集能力最高.在不同镍离子浓度时,基因工程菌对溶液中Ni²⁺的平衡富集量为11.33 mg·g^-1,与原始宿主菌相比提高了3倍.对基因工程菌吸附镍和钴的实验表明,Staphylococcus aureus ATCC6538的NiCoT对镍具有较高的特异性和富集容量,属于第Ⅲ类镍钴转运酶.     

Rapid molecular prenatal diagnosis of ataxia-telangiectasia by direct mutational analysis

Mutations of the ataxia-telangiectasia-mutated (ATM) gene are responsible for the autosomal recessive disorder ataxia-telangiectasia (A-T). This study reports the first A-T prenatal diagnosis performed in Spain by direct molecular analysis. The pregnant woman had a previous child suffering from A-T due to a deletion in the ATM gene. The ATM coding region was sequenced in the A-T patient and her parents. Then, a specific polymerase chain reaction (PCR) to detect the deletion was performed for prenatal diagnosis. Additionally, polymorphic HLA loci were examined in order to exclude the possible contamination by maternal DNA. In this family of Gypsy origin, we carried out a rapid molecular diagnosis of A-T. Then, a prenatal diagnosis was carried out, identifying the deletion in the fetal DNA. Additionally, we performed a population study in unrelated Spanish Gypsies and in unrelated controls, showing that the deletion described could be a hotspot in the Spanish Gypsy population. The size of the coding region and the genomic structure, together with the absence of hotspots, make the mutation screening of the ATM gene difficult. The ability to identify ATM mutations provides a tool that can be applied in confirmatory diagnosis, genetic counselling, carrier prediction and prenatal diagnosis. Copyright © 2007 John Wiley & Sons, Ltd.     

Allele-specific amplification for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy

We have developed a new allele-specific amplification method for the preimplantation genetic diagnosis (PGD) of spinal muscular atrophy (SMA; Werdnig-Hoffmann disease) from a single cell. This method is based on the detection of the deletion of exon 7 of the telomeric copy of the survival motor neurone (SMN^t) gene. An oligonucleotide was designed to be specific to the SMN^t nucleotidic sequence with exonic mismatch G (for SMN^t)→A (for SMN^c) at its 3′ end. This test produces reliable PCR products in 95% of single lymphoblasts (85/88) tested as well as in 16/16 blastomeres from normal controls. Specificity analysis showed that we were able to detect homozygous deletion of the SMN^t gene in 99% of single lymphoblasts (103/104) from a SMA patient. No contamination was detected in 68 blanks tested. Multiple cell and DNA dilution analysis revealed that the test is accurate and specific up to 100 pg DNA and should thus also be suitable for PGD at the blastocyst stage. This rapid procedure requires a single round of fluorescent PCR and no restriction digestion, while previously described single cell methods include nested PCR followed by restriction enzyme digestion. Two PGD cycles for SMA using this procedure were performed in our centre. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal diagnosis for arginase deficiency by second-trimester fetal erythrocyte arginase assay and first-trimester ARG1 mutation analysis

Hyperargininemia is a progressive neurometabolic disorder caused by deficiency of hepatic cytosolic arginase I, resulting from mutations in the ARG1 gene. We diagnosed arginase deficiency in a three-year-old male child of first-cousin Palestinian Arab parents. Prenatal diagnosis of an unaffected fetus was achieved in the second trimester of a subsequent pregnancy by cordocentesis and analysis of arginase activity in fetal erythrocytes. ARG1 mutation analysis in the proband revealed homozygosity for a deletion of 10 753 bp extending from the first intron to beyond the poly (A) site of the gene. This is the first gross deletion in the ARG1 gene to be identified and the first mutation to be described in an arginase-deficient patient of this ethnic origin. The identification of the ARG1 deletion in this family enabled first-trimester prenatal diagnosis in a subsequent pregnancy by multiplex PCR analysis performed on chorionic villous DNA. Copyright © 2004 John Wiley & Sons, Ltd.     

Case report: birth after preimplantation genetic diagnosis of a subtle mutation in SMN1 gene

Spinal muscular atrophy (SMA) preimplantation genetic diagnosis (PGD) has been available since 1998. Protocols are based on the detection of the homozygous deletion of exon 7, which are present in 90–98% of SMA patients. A couple where the woman was a heterozygous carrier of the usual SMN1 Del7 mutation and the man was a heterozygous carrier of pMet263Arg substitution in exon 6 of SMN1 gene was referred for PGD. The usual PGD test being unsuitable for this couple, we developed a novel duplex polymerase chain reaction (PCR)-based PGD test for the detection of the mutation pMet263Arg by allele specific amplification, combined with the amplification of D5S641 extragenic polymorphic marker. PCR conditions were established using single control lymphoblasts and lymphocytes from the pMet263Arg substitution carrier. Amplification was obtained in 100% of the 86 single cells tested, amplification refractory mutation system (ARMS) PCR was specific in 100% of single cells tested and a complete genotype (mutation plus D5S641) was achieved in 88% of them. A PGD cycle was performed successfully and a pregnancy was obtained. An unaffected girl was born and postnatal diagnosis confirmed PGD results. This is the first PGD described for SMA because of another mutation than the major homozygous exon 7 deletion of SMN1. In the future, a similar strategy could be adopted for other subtle mutations of this gene. Copyright © 2006 John Wiley & Sons, Ltd.     

大豆过敏原P34蛋白基因检测技术与环境污染的关系研究

环境污染易引起大豆种植区域出现过敏原P34蛋白基因变异情况,当前蛋白基因检测方法不能满足检测需求,提出基于探针引物的大豆过敏原P34蛋白基因荧光PCR定量检测技术,针对大豆过敏原特异性基因P34蛋白基因序列进行设计;通过DNA提取、浓度测定,荧光定量PCR检测,实现大豆过敏原P34蛋白基因检测。实验结果表明,所提方法能够准确检测出包括环境污染区域在内的大豆食品过敏原P34蛋白基因成分,具有很好的应用前景,满足市场监督检测的需要。     

一株稻田土壤梭状芽孢杆菌的分离及其固氮产氢能力研究

为研究水稻土壤中协同固氮产氢菌的固氮产氢特性,拓展协同固氮产氢菌菌种资源,利用厌氧微生物富集培养、亨盖特厌氧滚管等技术,从华南稻田土壤中筛选到一株同时具有产氢和固氮能力的菌株BZ-1. 经形态学观察及16S rRNA基因序列系统发育分析,鉴定菌株BZ-1属于梭状芽孢杆菌属(Clostridium sp.). 通过测定产氢量、生物量、发酵产物及固氮酶酶活等,对菌株BZ-1的产氢能力及协同固氮产氢特性进行分析. 结果表明:菌株BZ-1发酵34.55 mmol/L葡萄糖可产生42.19 mmol/L H₂,主要副产物为丁酸(15.96 mmol/L)、乙酸(7.14 mmol/L)和乳酸(5.09 mmol/L);菌株BZ-1具有固氮酶活性,能够以N₂为唯一氮源进行生长;菌株BZ-1固氮产氢时,相比于添加7 mmol/L氯化铵的试验组,产氢量提高了14.71%,最大生物量降低了33.33%,乙酸产量提升了61.49%. 研究显示,菌株BZ-1在协同固氮产氢条件下固氮能力的提升、生物量的降低以及核心碳代谢途径的改变可能是其产氢量提升的原因. 固氮产氢菌株BZ-1的获得将为提高土壤肥力以及缓解铅、镉等重金属对农作物的胁迫作用提供新的思路.      

一株好氧反硝化菌的分离及特性研究

利用富集培养的方法从城镇污水处理厂的活性污泥中分离到1株好氧反硝化菌,命名为CW。通过形态、生理生化特征和16SrRNA基因序列分析,初步鉴定为睾丸酮丛毛单胞菌(Comamonas testosteroni)。生长特性的研究表明最佳碳源是乙酸钠,最佳碳氮比10:1,最佳DO4.0～6.0mg/L,在此条件下培养24h后,对初始浓度108mg/L的硝酸盐降到27mg/L,去除率达到75%,亚硝酸盐积累仅为4mg/L。以组合载体为填料,挂膜后对污水进行处理,HRT48h,DO5.0mg/L左右,氨氮去除率达到95%左右,最终的NO3-N和NO2-N分别由起始的40～52mg/L和21～28mg/L降为--12～15mg/L和7～9mg/L。