应用牙釉质基因鉴定绒山羊早期胚胎的性别

采用酚-氯仿法和煮沸法从南疆绒山羊血液和早期胚胎中提取基因组DNA,分别以雄羊和雌羊的血液基因组DNA和早期胚胎基因组DNA(约20～30胚胎细胞)为模板,以AMEL引物进行PCR扩增和电泳分析.结果表明:绒山羊5ng量和10pg量血液基因组DNA经扩增后雌性只得到1条非特异性带,雄性得到1条非特异性带和1条特异性带;超微量血液基因组DNA样本(10pg)经巢式扩增和电泳分析能够鉴定绒山羊性别;32枚绒山羊胚胎鉴定结果,雄雌性别比17/15;移植胚胎产羔雄雌比15/14.采用牙釉质基因(AMEL),经巢式扩增和电泳分析能够鉴定绒山羊性别,并对胚胎无损伤;南疆绒山羊早期胚胎性别鉴定结果与胚胎移植后产羔性别结果对比,雌雄性别比率差异不显著(P>0.05).     

山羊TGFβ2部分cDNA的扩增和序列分析

在人和小鼠等的 TGFβ2 基因序列基础上 ,选用 1对同源性引物 ,采用反转录 -聚合酶链反应 (RT-PCR)技术从山羊卵泡总 RNA中扩增出 2 73bp的 TGFβ2 c DNA片段 ,进行序列测定 ,由此 c DNA序列推出了相应的 TGFβ2 蛋白分子的氨基酸排列。比较了山羊与人、小鼠、大鼠的 TGFβ2 部分 c DNA核苷酸的同源性和氨基酸的相似性 ,核苷酸同源性分别为 91.9% ,87.5 %和 88.6 % ;氨基酸相似性分别为 97.8% ,96 .7%和 96 .7%。     

Identification of DNA sequence responsible for 5-bromodeoxyuridine-induced gene amplification

Bromodeoxyuridine (BrdUrd) treatment of the prolactin nonproducing subclone of GH cells (rat pituitary tumor cells) induces amplification of a 20-kilobase DNA fragment including all of the prolactin gene coding sequences. This amplified DNA segment, which is flanked by two unamplified regions, thus designates a unit of BrdUrd-induced amplified sequence. Cloned DNA segments, 10.3 kilobases long, from the 5' end of the rat prolactin gene of BrdUrd-responsive and -nonresponsive cells, were ligated to the thymidine kinase gene of herpes simplex virus type 1 (HSV1TK), and the hybrid DNA was transferred to thymidine kinase-deficient mouse fibroblast cells by transfection. The HSV1TK gene and the rat prolactin gene were amplified together in drug-treated transfectants carrying the hybrid DNA HSV1TK gene and rat prolactin gene of BrdUrd-responsive GH cells. These results suggest that the 10.3-kilobase DNA segment at the 5' end of the rat prolactin gene of BrdUrd-responsive GH cells carries the information for drug-induced gene amplification (amplicon) and that another gene, such as the HSV1TK gene, is also amplified when the latter is placed adjacent to this segment.     

农杆菌介导法获得转可溶性淀粉合成酶基因籼稻

采用农杆菌介导法将胚乳特异性表达谷蛋白1(Glutelinl，Gtl)基因控制下的可溶性淀粉合成酶基因sss导入籼稻恢复系明恢86，共获得53个独立转化再生植株。PCR检测表明，sss基因已整合进水稻的基因组中。直链淀粉含量测定结果表明，转基因植株后代直链淀粉含量较对照有较大幅度的下降。     

Reduction of arsenic bioavailability by amending seven inorganic materials in arsenic contaminated soil

Seven inorganic amendment materials were added into arsenic(As) contaminated soil at a rate of 0.5%(w/w); the materials used were sepiolite, red mud, iron grit, phosphogypsum, ferrihydrite, iron phosphate, and layered double oxides(LDO). Plant growth trials using rape(edible rape, Brassia campestris L.) as a bio-indicator are commonly used to assess As bioavailability in soils. In this study, B. campestris was grown in a contaminated soil for 50 days. All of the inorganic amendments significantly inhibited the uptake of As by B. campestris. Following soil treatment with the seven aforementioned inorganic ammendments, the As concentrations in the edible parts of B. campestris were reduced by 28.6, 10.5, 8.7, 31.0, 47.4, 25.3, and 28.8%, respectively, as compared with the plants grown in control soil. The most effective amendment was ferrihydrite, which reduced As concentration in B. campestris from 1.84 to 0.97 mg kg–1, compared to control. Furthermore, ferrihydrite-treated soils had a remarkable decrease in both non-specifically sorbed As and available-As by 67 and 20%, respectively, comparing to control. Phosphogypsum was the most cost-effective amendment and it showed excellent performance in reducing the water soluble As in soils by 31% and inhibiting As uptake in B. campestris by 21% comparing to control. Additionally, obvious differences in As transfer rates were observed in the various amendments. The seven amendment materials used in this study all showed potential reduction of As bioavailability and influence on plant growth and other biological processes still need to be further explored in the long term.     

锯缘青蟹Sox基因的PCR扩增(英文)

SRY基因(sex-determining region of the Y chromosome)是人类及哺乳动物睾丸决定因子TDF(testisdetermining factor)的最佳候选基因。SRY蛋白含有204个氨基酸,其中1个约79个氨基酸区域即HMG盒(high mobility group box)是SRY基因编码蛋白质的唯一功能区。由于SRY的发现,人们进而发现了1个庞大的与性别决定有关的SRY盒-Sox(SRY-related HMG box gene)基因家族。锯缘青蟹是我国重要的海洋经济蟹类之一,其性染色体和性别决定机制仍处于进化的早期阶段,在分子水平上探讨其性别决定机制尚不多见。通过采用PCR技术,以特异扩增人SRY基因HMG盒保守区的1对兼并引物,扩增了锯缘青蟹基因组的Sox基因。结果表明锯缘青蟹雌雄个体与人一样,均能扩增出1条大小约为216bp左右的基因片段,显示出该基因在进化上的高度保守性,为探索锯缘青蟹的性别决定机制及Sox基因的进化提供了分子资料。     

锯缘青蟹Sox基因的PCR扩增

SRY基因(sex-determining region of the Y chromosome)是人类及哺乳动物睾丸决定因子TDF(testisdetermining factor)的最佳候选基因。SRY蛋白含有204个氨基酸,其中1个约79个氨基酸区域即HMG盒(high mobility group box)是SRY基因编码蛋白质的唯一功能区。由于SRY的发现,人们进而发现了1个庞大的与性别决定有关的SRY盒-Sox(SRY-related HMG box gene)基因家族。锯缘青蟹是我国重要的海洋经济蟹类之一,其性染色体和性别决定机制仍处于进化的早期阶段,在分子水平上探讨其性别决定机制尚不多见。通过采用PCR技术,以特异扩增人SRY基因HMG盒保守区的1对兼并引物,扩增了锯缘青蟹基因组的Sox基因。结果表明锯缘青蟹雌雄个体与人一样,均能扩增出1条大小约为216bp左右的基因片段,显示出该基因在进化上的高度保守性,为探索锯缘青蟹的性别决定机制及Sox基因的进化提供了分子资料。     

鸡传染性支气管炎病毒N基因片段的RT-PCR扩增及限制性内切酶分析

根据鸡传染性支气管炎病毒 (IBV)的已报道基因序列设计了 1对可扩增 N基因片段的引物 ,并得到了预期的 438bp扩增产物 ;将目的条带纯化并回收后 ,克隆到 PMD18- T质粒载体中 ,对插入片段进行序列测定 ,并与已发表的 IBV N基因序列进行了比较 ,证实扩增和克隆的产物是正确的 ;根据扩增的 N基因的序列 ,选择了 Bst X 酶对 IBV进行分析 ,发现 M41和澳大利亚 T株 Bst X 酶切图谱存在明显的差异 ,从而建立了一种新的鉴定 IBV病毒的方法。     

环介导等温扩增法（LAMP）检测最短尾短体线虫

为了快速、简便、准确地检测重要植物病原线虫—最短尾短体线虫（ Pratylenchus brachyurus ）,依据GenBank中短体线虫的线粒体DNA细胞色素氧化酶I（mtDNA COI）序列,设计了扩增最短尾短体线虫的环介导等温扩增（loop-mediated isothermal amplification,LAMP）引物,并对LAMP反应条件进行优化,成功建立了一种可特异、快速检测最短尾短体线虫的LAMP体系。该体系在64℃下反应60 min,扩增效率最高。用琼脂糖凝胶电泳、酶切分析和SYBR Green I染色均能特异检测到最短尾短体线虫的扩增产物。所建立的LAMP体系能从供试的9种短体线虫和另外10种植物线虫中特异检测出最短尾短体线虫,其灵敏度可检测到1/200条线虫DNA,是常规PCR的10倍。表明本研究建立的最短尾短体线虫的LAMP检测体系,可用于最短尾短体线虫的快速检测。     

山羊TGFβ_2部分cDNA的扩增和序列分析

在人和小鼠等的 TGFβ2 基因序列基础上 ,选用 1对同源性引物 ,采用反转录 -聚合酶链反应 (RT-PCR)技术从山羊卵泡总 RNA中扩增出 2 73bp的 TGFβ2 c DNA片段 ,进行序列测定 ,由此 c DNA序列推出了相应的 TGFβ2 蛋白分子的氨基酸排列。比较了山羊与人、小鼠、大鼠的 TGFβ2 部分 c DNA核苷酸的同源性和氨基酸的相似性 ,核苷酸同源性分别为 91.9% ,87.5 %和 88.6 % ;氨基酸相似性分别为 97.8% ,96 .7%和 96 .7%。     

人工湿地砷污染去除研究进展

砷在环境中普遍存在,因其高毒性,世界卫生组织将砷污染列为重大公共卫生问题。因此,科学家们在不断寻求科学有效的砷污染去除方法。人工湿地砷污染去除系统自上世纪七十年代推出以来,因其环境友好、成本低廉的特性得到蓬勃发展,快速成为绿色环保修复技术之一。人工湿地被认为是理想的砷富集去除场所,其对砷污染的去除方式主要通过植物和微生物相互作用及腐殖酸强化修复来实现。本文通过砷污染治理历史及技术发展、人工湿地的兴起及砷在湿地生态系统中的赋存状态以及人工湿地生态系统砷污染去除三个方面综述了人工湿地砷污染去除的研究进展。通过大量文献综述分析,我们认为在人工湿地砷污染去除的后续研究工作中,应着重研究砷在湿地生态系统中的扩散迁移及演变规律,构建湿地生态系统沉水-浮水-挺水植物立体结构,并不断探索植物-微生物及腐殖酸强化修复对砷污染的协同阻滞及界面作用。     

外源磷输入对香蒲生长及砷积累的影响

[目的]探究不同水平外源磷(P)输入对挺水植物香蒲在不同梯度砷(As)污染底泥中的生长指标及其对砷累积与迁移的影响,为砷污染湿地生态恢复区的建设和日常管理提供科学参考.[方法]通过试验模拟0 mg/L(PCK)、0.2 mg/L(P0.2)、2 mg/L(P2)和20 mg/L(P20)4种不同水平的外源磷输入0 mg/kg(As0)、150 mg/kg(As150)和600 mg/kg(As600)3种不同程度砷胁迫生境,分析香蒲的生长指标和其对砷的积累特征.[结果]底泥砷处理为As0时,P0.2处理下香蒲有最大生物量积累(95.57 g/株),根部耐性指数(TI)和香蒲单位面积砷迁移总量(W)最高,分别为110.18%和2.68 g/ha;P20处理下香蒲PSⅡ光合系统最大光合潜力(Fv/Fm)、PSⅡ光合系统电子转运效率(ETo/RC)和植株间转运系数(TF)最大,分别为0.81、1.16和0.50,此时香蒲对底泥中砷去除率(Rc)、固定率(Rs)及提取量(EX)最高,分别为32.96%、33.12%和717.06μg.底泥砷处理为As150时,P0.2处理下生长于污染底泥中的香蒲生物量积累、TI和TF均最高,分别为100.20 g/株、120.16%和0.08,此时Rc、Rs和EX也最高,分别为32.45%、35.35%和1921.98μg;P2处理下Fv/Fm、ETo/RC和W最大,分别为0.84、1.25和8.29 g/ha.底泥砷处理为As600时,P0.2处理下香蒲Fv/Fm、Rc和Rs最大,分别为0.80、56.99%和52.73%;P2处理下香蒲生物量积累、ETo/RC和W最高,分别为96.23 g/株、1.07和20.43 g/ha;P20处理下香蒲地上部对砷BCF和TF最大,分别为0.58和0.24.[结论]当底泥处于中重度砷污染(<150 mg/kg)时,较高磷水平(0.2 mg/L)处理可获得更好的香蒲生态修复砷污染效果;当底泥处于严重砷污染(150~600 mg/kg)时,高浓度磷水平(2 mg/L)处理使香蒲对砷生态修复效果最优.实际应用中适当提高供磷水平可改善香蒲生态修复砷污染湿地的效果.     

不同改良剂和水分管理对水稻吸收积累砷的影响

为筛选出有效治理As污染农田土壤的修复模式,采用盆栽试验的方法,比较了不同混合改良剂和水分管理对As污染土壤中水稻吸收积累As的影响。结果表明,土壤以及水稻籽粒、茎叶和根系中的As含量在不淹水条件下较低,在淹水条件下较高,在孕穗期至灌浆期淹水条件下处于前两者之间。不同水分管理方式下,添加Si+Mo使籽粒As含量降低4.8%～19.9%,茎叶As含量降低16.9%～56.3%;添加Fe+Ca使籽粒As含量降低26.6%～50.6%,茎叶As含量降低40.3%～81.2%。添加改良剂还能降低水稻根系向茎叶和籽粒转运As的能力。综合而言,不淹水+Fe+Ca处理降低水稻As含量的效果最明显。     

Rapid detection of Pseudomonas aeruginosa by cross priming amplification

Pseudomonas aeruginosa(PA) is an opportunistic pathogen of humans and animals and a common source of nosocomial infections especially of the respiratory tract. Pseudomonas aeruginosa is also a major bacterial disease of poultry and in particular, eggs and newly hatched chicks. In this study, we developed a simple, accurate and rapid molecular detection method using cross priming amplification(CPA) with a nucleic acid test strip to detect P. aeruginosa. The assay efficiently amplified the target gene within 45 min at 62°C only using a simple water bath. The detection limit of the method was 1.18×10~2 copies μL~(–1) for plasmid DNA and 4.4 CFU mL~(–1) for bacteria in pure culture, and was 100 times more sensitive than conventional PCR. We screened 83 clinical samples from yellow-feather broiler breeder chickens and hospitalized/treated dogs and cats using CPA, PCR and traditional culture methods. The positive-sample ratios were 15.3%(13/83) by CPA, 13.3%(11/83) by PCR and 12.1%(10/83) by the culture method. The established CPA method has significant advantages for detecting P. aeruginosa. The method is easy to use and possesses high specificity and sensitivity without the requirements of complicated experimental equipment. The PA-CPA assay is especially fit for outdoor and primary medical units and is an ideal system for the rapid detection and monitoring of P. aeruginosa.     

鲨鱼弧菌LAMP检测方法的建立

采用环介导等温扩增技术(LAMP)成功构建了一种鲨鱼弧菌的检测方法。根据鲨鱼弧菌的rpoA基因序列设计4条引物(内引物F3和B3,外引物FIP和BIP),并对LAMP扩增反应的各物质浓度和反应条件进行优化,确定最佳反应温度为65°C,最佳反应时间为55 min。该法对鲨鱼弧菌纯菌培养物的检测浓度约为2.4×102CFU/mL,结果比PCR法具有相似或更高的灵敏度,而且操作简单,反应后体系变浑浊,肉眼可辨;加入SRYB Gold荧光染料,阳性反应为绿色,阴性对照为浅橘色,更便于观察,有望发展成为快速检测鲨鱼弧菌的一种有效方法。     

无性系良种茶园建设的难点及其克服方法

通过田间调查 ,认识了扦插苗与实生苗的差别及无性系茶园建设的难点 ,分类阐明了扦插苗移栽死亡的原因 ,结合栽培实践 ,提出了克服苗难栽的方法     

Human multidrug-resistant cell lines: increased mdr1 expression can precede gene amplification

The development of simultaneous resistance to multiple structurally unrelated drugs is a major impediment to cancer chemotherapy. Multidrug resistance in human KB carcinoma cells selected in colchicine, vinblastine, or Adriamycin is associated with amplification of specific DNA sequences (the multidrug resistance locus, mdr1). During colchicine selection resistance is initially accompanied by elevated expression of a 4.5-kilobase mdr1 messenger RNA (mRNA) without amplification of the corresponding genomic sequences. During selection for increased levels of resistance, expression of this mRNA is increased simultaneously with amplification of mdr1 DNA. Increased expression and amplification of mdr1 sequences were also found in multidrug-resistant sublines of human leukemia and ovarian carcinoma cells. These results suggest that increased expression of mdr1 mRNA is a common mechanism for multidrug resistance in human cells. Activation of the mdr1 gene by mutations or epigenetic changes may precede its amplification during the development of resistance.     

Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome

In tumor cells from virtually all patients with chronic myelogenous leukemia, the Philadelphia chromosome, a fusion of chromosomes 9 and 22, directs the synthesis of the P210bcr/abl protein. The protein-tyrosine kinase activity and hybrid structure of P210bcr/abl are similar to the oncogene product of the Abelson murine leukemia virus, P160gag/v-abl, which induces acute lymphomas. To determine whether P210bcr/abl can induce chronic myelogenous leukemia, murine bone marrow was infected with a retrovirus encoding P210bcr/abl and transplanted into irradiated syngeneic recipients. Transplant recipients developed several hematologic malignancies; prominent among them was a myeloproliferative syndrome closely resembling the chronic phase of human chronic myelogenous leukemia. Tumor tissue from diseased mice harbored the provirus encoding P210bcr/abl. These results demonstrate that P210bcr/abl expression can induce chronic myelogenous leukemia. Retrovirus-mediated expression of the protein provides a murine model system for further analysis of the disease.     

Alleviation of arsenic toxicity by phosphate is associated with its regulation of detoxification,defense, and transport gene expression in barley

Arsenic(As) contamination in soils has posed a severe threat to safe crop production. The previous studies showed the antagonism between phosphorus(P) and As in plant growth and As uptake, while the mechanisms of alleviating As toxicity by P is not completely clear. Due to the limiting P condition, it is imperative to understand how low P addition can be used to suppress arsenate As(V) uptake and the subsequent mechanisms involved. Thus in this study we investigated the effect of P addition on As uptake, anti-oxidative enzyme activity, and anti-oxidant content, and the relative expression of transport, defense, and detoxification genes using two barley genotypes differing in As toxicity tolerance. P addition significantly reduced As concentration in plant tissues, and caused the great changes in activities of catalase and superoxide dismutase, glutathione content, and the relative expression of examined genes when the plants of the two barley genotypes were exposed to 100 μmol L~(–1) As, with ZDB160(As-tolerant) being much more affected than ZDB475(As-sensitive). The current results show that P addition can alleviate As toxicity by regulating the expression of As transport, defense, and detoxification genes to a greater extent in As tolerance of barley, suggesting the possibility of controlling As uptake and toxicity by applying low amount of P fertilizers in the As-contaminated soils.