尖孢镰刀菌古巴专化型4号生理小种中FoGAS1基因的克隆与序列分析

采用PCR和RT-PCR的方法,从尖孢镰刀菌古巴专化型4号生理小种中克隆得到了FoGAS1基因的DNA及cDNA序列,并运用生物信息学的方法对该基因所编码的氨基酸序列进行分析,预测其编码蛋白质的结构和功能。结果表明,FoGAS1基因全长1 672 bp,其中开放阅读框全长1 623 bp,编码含有540个氨基酸残基的蛋白质,该蛋白分子量为58398.1,等电点pI=4.83,为性质稳定的亲水性蛋白。FoGAS1蛋白为跨膜蛋白,含有22个丝氨酸磷酸化位点,2个苏氨酸磷酸化位点,8个酪氨酸磷酸化位点。该蛋白在C端有一个信号肽结构,为一种分泌蛋白。通过系统进化树分析,该蛋白在不同种属镰刀菌中高度保守。     

绵羊甘露聚糖结合凝集素基因(MBL)全长DNA序列克隆及生物信息学分析

绵羊甘露聚糖结合凝集素(MBL)是肌体天然免疫系统的重要组成部分。根据人(Homo sapiens)和牛(Bos taurus)的MBL基因序列的同源保守区域,分段设计9对特异性引物,以中国美利奴羊(Ovis aries)血液基因组DNA为模板,经扩增、测序、拼接,首次克隆到长为4462bp的绵羊MBL基因组DNA序列,包括了该基因的启动子区、4个外显子和3个内含子,编码249个氨基酸;BLAST分析其与牛MBL-C编码的氨基酸同源性最高为96.39%,判定其为MBL-C型基因,并将该序列提交至GenBank(Accession No.FJ977629);通过Expasy网站预测其蛋白结构功能特征,1～19氨基酸为绵羊MBL基因信号肽区域,外显子1编码N-端富含胱氨酸区和8个Gly-X-Y重复序列的胶原样区,外显子2含有12个Gly-X-Y的胶原样区,外显子4编码的碳水化合物识别域(CRD)具有C型凝集素CRD的共同特征。本研究为国内首次报道绵羊MBL基因比较完整基因组DNA序列,为深入研究MBL的结构与功能以及MBL缺损的分子机制提供基础数据。     

甜高粱SUT1基因克隆、表达及生物信息学分析

为获知甜高粱蔗糖转运蛋白基因SUT1及其功能,采用同源克隆方法结合RACE技术克隆甜高粱SUT1基因,并对其进行生物信息学分析。克隆得到甜高粱蔗糖转运蛋白基因SUT1 c DNA全长为2 472bp,包括1 560bp编码区序列,编码含有519个氨基酸的蛋白。该蛋白是1个疏水性膜蛋白,分子量约为55k Da,理论等电点p I为8.86;无信号肽剪切位点,含有12个明显的跨膜螺旋拓扑结构,预测含有6个丝氨酸激酶磷酸化位点、4个苏氨酸激酶磷酸化位点和2个酪氨酸激酶磷酸化位点;包含1段低复杂度序列和12个保守的跨膜螺旋结构域。在亚细胞水平,SUT1主要定位于叶绿体类囊体膜、质膜、高尔基体及内质网膜上;二级结构主要以α-螺旋为主,其中α-螺旋占43.35%,无规则卷曲占34.68%,延伸链占19.08%,β-转角占2.89%;预测SUT1蛋白主要在运载结合中发挥重要作用。SUT1基因表达分析结果表明,SUT1基因在各组织中均有表达,叶中表达量最高,其次分别为叶鞘、茎和根,穗中表达量最低。SUT1基因的克隆及其结构、性质与功能的初步分析,可为研究该基因在甜高粱源库互作关系中的功能机制奠定基础。     

钝齿棒杆菌精氨酸生物合成相关基因簇argCJBDFR的扩增及序列分析

采用PCR方法扩增钝齿棒杆菌精氨酸生物合成基因簇argCJBDFR,其序列长为6080bp,其中含有argJ、argB、argD、argF和argR5个完整的开放阅读框。序列和结构分析表明这5个结构基因编码的氨基酸序列与谷氨酸棒杆菌的相应基因编码的氨基酸高度同源,同源性分别为92%、95%、96%、97.5%和100%。     

陆地棉GhMYB9基因克隆及结合特性分析

MYB转录因子蛋白普遍存在于植物中,广泛参与植物的生长发育和代谢调控。为研究GhMYB9基因对棉纤维的调控机制,使用PCR方法,从棉花中克隆1个R2R3-MYB基因GhMYB9(Gen Bank登录号:AF336286.1);利用生物信息学方法分析其氨基酸序列;构建酵母表达载体。结果表明,该基因c DNA全长1 145 bp,开放阅读框长795 bp,编码264个氨基酸,预测其蛋白分子量约为29.624 k D,等电点为9.13。推测的氨基酸序列中含有2个高度保守的SANT结构域。GhMYB9基因组包含2个外显子和1个内含子。进化树分析表明,GhMYB9和Gh MYB聚在同一分支(HQ234875.1)。酵母单杂交试验结果表明,GhMYB9转录因子能够特异结合AC顺式作用元件。本研究结果为进一步阐明GhMYB9的生物学功能奠定了基础。     

苎麻α-amylase基因的克隆与表达

α-amylase基因不仅参与植物糖代谢与生物能量的调动,而且与植物抗逆功能相关。为了克隆Bn-α-amylase基因,分析其序列及表达特征,本研究以湘苎3号苎麻(Boehmeria nivea)转录组文库中Unigene4746序列为基础,采用RT-PCR技术克隆该基因的全长编码序列,并进行生物信息学分析;利用Real-time PCR分析该基因在不同组织和不同胁迫条件下的表达特征。研究结果表明,苎麻Bn-α-amylase的编码区序列长2 295 bp,编码765个氨基酸(GenBank登录号:KF860891),推导该蛋白质的等电点和分子量分别为5.685和86.11 kD,该蛋白在羧基端含有两个保守的结构域,亚细胞定位预测显示该蛋白位于细胞质中,不存在信号肽和跨膜结构域。与苹果(Malus×domestic)、拟南芥(Arabidopsis thaliana)、猕猴桃(Actinidia chinensis)、蓖麻(Ricinus communis)、大豆(Glycine max)的α-amylase基因的核苷酸序列相似性分别为80%、76%、76%、73%和67%,与之编码的氨基酸序列相似性分别为68%、65%、65%、69%和51%。进化树分析表明Bn-α-amylase与大豆、葡萄、猕猴桃、蓖麻的α-amylase基因亲缘关系较近,与拟南芥、水稻、高粱的α-amylase基因亲缘关系次之,与卷柏的α-amylase基因亲缘关系较远。实时荧光定量PCR分析表明,Bn-α-amylase在苎麻根、茎皮、茎木质部、茎尖、叶片中均有表达,其中,在叶中表达量最高,在根中表达量最低,且受干旱、高盐胁迫表达增强,受ABA胁迫表达降低。本研究获得了Bn-α-amylase基因的编码区序列,其编码的蛋白具有植物α-amylase典型的结构域,且该基因响应ABA、干旱和高盐逆境胁迫,提示该基因可能与苎麻抗逆境机制密切相关。     

草地螟中肠运载蛋白LstiSLC25基因的克隆及表达

利用甜菜夜蛾Spodoptera exigua（Hübner）围食膜多克隆抗体免疫筛选草地螟（Loxostege sticticalis L．）中肠cDNA表达文库,得到编码运载蛋白LstiSLC25的全长cDNA克隆。该cDNA克隆全长1399bp(GenBank登录号EU924506),开放阅读框长897 bp,编码299个氨基酸,预测分子量和等电点分别为32.1kD和9.46。序列含有三个典型的溶质运转重复结构,具有溶质运载蛋白家族的典型特征。将该基因与pET30载体重组后,经IPTG诱导,蛋白在大肠杆菌中获得了表达。     

青稞OMT1基因克隆及原核表达分析

类黄酮O-甲基转移酶(FOMT)是一种在植物甲基化黄酮代谢合成中有着重要作用的酶,但在中国青藏高原的特色作物青稞中研究较少。为了更好了解该酶及编码基因在青稞中的生理功能及作用,以高总黄酮青稞品系94-19-1为材料,通过RT-PCR扩增、克隆得到青稞OMT1基因的ORF序列。结果表明,该基因ORF长1 071 bp,编码356个氨基酸,预测蛋白分子量为38.65 KDa,等电点为5.33。序列比对分析发现,所克隆的基因与NCBI数据库收录的大麦Hv OMT1基因有99.44%的一致性,氨基酸序列存在5个残基差异,编码的蛋白序列具有OMT蛋白家族典型的保守结构域。进一步将该基因连接到原核表达载体转化大肠杆菌,最终成功将该基因所编码的蛋白在大肠杆菌诱导表达,并利用亲和层析柱分离技术,将该表达蛋白进行了纯化。这为进一步研究该酶蛋白功能和选育高品质青稞品种奠定了基础。     

玉米Ubi-1启动子驱动挪威鼠Hspa4基因植物表达载体的构建

根据Genbank中挪威鼠（Rattus norvegicus）Hspa4基因序列(登录号：NM 153629)和玉米Ubi-1启动子序列(登录号：DQ141598 )设计引物,采用RT-PCR和PCR技术分别进行克隆。测序及同源性分析结果表明：Hspa4基因片段含有2530bp,编码840个氨基酸;获得的启动子Ubi-1序列大小为1992bp;克隆的序列与相应原序列同源性分别为99.96％和99.55%。以植物表达载体pCAMBIA1301质粒为基础,成功构建了植物表达载体pCAMBIA1301-Ubi-Hspa4,为利用Hspa4基因改良植物抗逆性打下了基础。     

草地螟中肠肉碱-脂酰肉碱转位酶基因(LstiSLC25)的克隆及表达

利用甜菜夜蛾(Spodoptera exigua)围食膜多克隆抗体免疫筛选草螟(Loxostege sticticalis)中肠cDNA表达文库,得到编码肉碱-脂酰肉碱转位酶(LstiSLC25)基因的全长cDNA克隆.该cDNA克隆全长1 474 bp(GenBank登录号EU924506),开放阅读框长897 bp,编码299个氨基酸,预测分子量和等电点分别为32.1 kD和9.46.序列含有3个典型的溶质运转重复结构,具有溶质运载蛋白家族的典型特征.将该基因与pET30载体重组后,经IPTG诱导,蛋白在大肠杆菌(Escherichia coli获得了表达.     

Degradation of endogenous and exogenous genes of roundup-ready soybean during food processing

Roundup-Ready soybeans have been genetically modified to resist the effects of the herbicidal glyphosate and have become the most prevalent transgenic crop in the world. In this work, Roundup-Ready soybeans were used as raw material to study the effects of critical processing procedures such as grinding, cooking, blending, homogenization, sterilization, and spray-drying on the length of DNA fragments of an endogenous gene (lectin) and an exogenous gene (epsps) examined in material from three soybean foods of bean curd, soy milk, and soy powder and from samples taken during their processing. The results showed that various processing procedures caused degradations of both the endogenous and exogenous genes to different degrees. In the grinding procedure, endogenous gene DNA was degraded from 1883 to approximately 836 bp, and exogenous gene DNA was degraded from 1512 to approximately 408 bp. In the blending and squeeze-molding procedures, exogenous gene DNA was also degraded from about 408 to 190 bp, but there was no obvious action on the endogenous gene. After the endogenous and exogenous genes had been degraded to some degree, such as 836 and 408 bp, respectively, they were not evidently affected by cooking procedure at 100 degrees C for 15 min. However, the endogenous gene was further considerably degraded from around 836 to 162 bp in the sterilization procedure at 121 degrees C for 30 s. The effect of the homogenization step on endogenous and exogenous genes was similar to that of the cooking procedure. The coagulation procedure, principally a biochemical reaction, did not greatly affect the exogenous gene but did affect endogenous gene, reducing DNA size from about 836 to 407 bp. Furthermore, the spray-drying procedure, a process of physical shearing, high temperature, and sudden high pressure, distinctly caused degradation of both the lectin and epsps genes, rapidly decreasing the sizes from about 836 to 162 bp for the endogenous gene and from about 408 to 190 bp for the exogenous gene.     

The influence of transient phenomena on the biodegradation of nitrilotriacetic acid in the activated sludge process I: Variations in hydraulic loading and sewage strength

The removal of nitrilotriacetic acid (NTA) following acclimatization in an activated sludge pilot plant has been studied during transient changes in operating conditions. These changes included increases in hydraulic loading and influent chemical oxygen demand (COD) and such phenomena in combination with transient temperature reductions. Short-term increases in hydraulic loading from 1 to 2 dry weather flow (dwf) had a very limited effect on NTA removal. Increasing the influent COD had a slight effect on NTA removal. Short term increases in hydraulic loading combined with transient reductions in temperature significantly reduced NTA removal. The effect of these combined changes on NTA removal was synergistic, rather than additive. Increases in influent COD combined with transient temperature reductions also caused a significant reduction of NTA removal. The effect of such combined changes on NTA removal was at most only additive.     

Mobilization of Cu and Zn in contaminated soil by nitrilotriacetic acid

Batch and upflow column leaching experiments were used to evaluate the nature and extent of Cu and Zn solubilization from contaminated soil by nitrilotriacetic acid (NTA) in 0.025 M NaClO₄. In batch soil suspensions, NTA levels of 10^?5 to 10^?3 M substantially promoted Cu and Zn release from the metal-enriched soil. The ability of NTA to enhance Cu and Zn solubility decreased with increasing solution acidity probably due to competitive binding of NTA by protons and Fe released by hydrous oxide dissolution. However, in the pH range typically encountered in northeastern U.S. soils, soluble metal levels were nearly constant for a given NTA concentration. Leaching soil columns with NTA solutions enhanced Cu release more than Zn, as the enrichment ratio (cumulative metal leached by NTA compared to the 0.025 M NaClO₄ control leachate) after 85 pore volumes displacements was 23.6 and 4.3 for Cu and Zn, respectively. While Cu release by 0.01 M CaCl₂ differed little from the control, 0.01 M CaCl₂ was substantially more effective than 10^?5 M NTA in displacing bound Zn. The data reflect different retention mechanisms for Cu and Zn in this soil.     

Investigations into the fate of nitrilotriacetic acid in sewage sludge applied to agricultural land

The mobility of nitrilotriacetic acid (NTA) in agricultural soil was assessed in a soil/sewage sludge admixture and following surface applications using laboratory scale soil columns. Rates of application were based on the guidelines published by the Department of the Environment (United Kingdom). Nitrilotriacetic acid concentrations were based on expected sewage sludge concentrations were NTA to be introduced as a replacement builder in detergent formulations. Admixtures were found to retain NTA more effectively in the topsoil (0 to 200 mm) than surface applications, with up to 9.5 % of the applied NTA passing through the topsoil following a surface application, when irrigated at average rainfall. Even at higher rates of irrigation a great proportion of the NTA was retained in a soil column containing topsoil and subsoil (200 to 300 mm). The adsorption capacity of the soil was determined in batch experiments and was found to be 17.5 μNTA g^?1 soil.     

农杆菌介导的蓝色基因转化中国水仙

从矮牵牛(Petunia hybrida)花瓣中分离得到编码蓝色基因F3'5'H(类黄酮3',5'羟基化酶)的Hf2基因(1527 bp),从牵牛(Ipomoea nil)花瓣中分离得到dfr基因(二氢黄酮醇4-还原酶,1212 bp),将正向Hf2片段和反向dfr片段连接到pBRLys双元载体质粒上,得到具有Ubi(ubiquitin)组成型启动子的植物表达载体pU-bi-Hf2-dfr.利用冻融法将双价重组质粒pUbi-Hf2一afr导入农杆菌(Agrobacterium tumefaciens)菌株EHA105中,菌落PCR鉴定农杆菌转化子.以中国水仙(Narcissus tazetta var.chinensis)愈伤组织为材料,进行遗传转化.CTAB法提取130株中国水仙遗传转化再生苗的基因组DNA,进行PCR和PCR-Southem blot检测,获得3株阳性转基因植株.     

Desorption-complexation-dissolution characteristics of adsorbed cadmium from kaolin by chelators

In order to provide a sound experimental background for the remediation of metal-contaminated soil by chelators, the desorption/complexation/dissolution characteristics of Cd from kaolin as a representative soil component by four chelators (NTA, EDTA, EGTA and DCyTA) have been investigated as a function of solution pH. For all chelating agents under examination, the ratio of Cd (desorbed from kaolin) to chelator was found to be 1:1. The chelation/dissolution of Cd was strongly dependent on the solution pH for NTA and EDTA. In the NTA system, 100% Cd dissolution occurred only at a pH = 8 and pH < 3.2; under weakly acidic conditions only 45% of the Cd on kaolin was dissolved due to readsorption of CdNTA- complex on kaolin. At a pH ≥ 10, Cd dissolution decreased, due to Cd hydroxide precipitation. Only 85% of the total Cd on kaolin desorbed under weakly acidic conditions in the EDTA system, indicating metal complex readsorption similar to that found in the Cd-NTA system. Zeta potential measurements showed that the surface charge of Cd-loaded kaolin became more negative after addition of EDTA and NTA with a shift in the pH at the point of zero charge to a lower value. As compared to the EDTA and NTA systems, DCyTA and EGTA complexed strongly with Cd (100% Cd dissolution) over a wide pH range (2.5–12.0). The zeta potential of kaolin did not change and no Cd readsorption was found after addition of EGTA and DCyTA. The capacity of the four chelators for removing Cd from kaolin was found to be in the order DCyTA > EGTA > EDTA > NTA.     

A method for estimating organic‐bound iron and aluminum contents in soils

Abstract

Estimation of organic‐bound iron (Fe) and aluminum (Al) is an important diagnostic technique in pedology. The commonly used sodium pyro‐phosphate method yields somewhat ambiguous results and is inconvenient if an ultracentrifuge is not available in the laboratory. This study showed that 0.1M sodium nitrilotriacetate (NTA) could be used instead of pyrophosphate. The Fe and Al extracted by 0.1 M NTA were highly correlated to that by sodium pyrophosphate with R² = 0.993 for Fe and R² = 0.992 for Al, while the dissolution effects on standard mineral samples in NTA was kept at a minimum. NTA has the advantage of not requiring ultrafiltration, ultracentrifugation, or the addition of a flocculating agent as is the case for the pyrophosphate method.     

Nitrilotriacetic acid removal in septic tanks: A field study

The removal of nitrilotriacetic acid (NTA) dosed to two full-scale septic tanks each connected to a single dwelling was monitored weekly over a period of 75 weeks. Nitrilotriacetic acid in the septic tank effluent was detected in samples diluted at least 25 times by a differential pulse polarographic technique. After an initial period of stabilization, concentrations of NTA in the effluent at both sites were always > 10 mg L^?1. Mean five-weekly estimated removals of NTA during the last 50 weeks of the study ranged from 33 to 52%. Removal of NTA was probably by solids adsorption only as no acclimatization period or overall increase in removal during the investigation was apparent.     

Hot NTA Application Enhanced Metal Phytoextraction from Contaminated Soil

To increase the phytoextraction efficiency of heavy metals and to reduce the potential negative effects of mobilized metals on the surrounding environment are the two major objectives in a chemically enhanced phytoextraction process. In the present study, a biodegradable chelating agent, NTA, was added in a hot solution at 90°C to soil in which beans (Phaseolus vulgaris L., white bean) were growing. The concentrations of Cu, Zn and Cd, and the total phytoextraction of metals by the shoots of the plant from a 1 mmol kg^?1 hot NTA application exceeded those in the shoots of plants treated with 5 mmol kg^?1 normal NTA and EDTA solutions (without heating treatment). A significant correlation was found between the concentrations of metals in the shoots of beans and the relative electrolyte leakage rate of root cells, indicating that the root damage resulting from the application of a hot solution might play an important role in the process of chelate-enhanced metal uptake in plants. The application of hot NTA solutions did not significantly increase metal solubilization in soil in comparison with a normal application of solution of the same dosage. Therefore, the application of a hot NTA solution may provide a more efficient alternative in chemical-enhanced phytoextraction, although further studies of techniques of application in fields are sill required.     

Decomposition of nitrilotriacetate (NTA) in soils

The trisodium salt of nitrilotriacetic acid (NTA) has been proposed as a substitute for phosphate in detergents. The decomposition of this chelating agent in soils was studied by performing analyses for NTA and inorganic nitrogen after incubation of NTA-treated soils for various times. The results showed that NTA is readily decomposed by soil micro-organisms under aerobic or anaerobic conditions and that NTA-nitrogen is converted to nitrate under aerobic conditions. The latter finding indicates that use of detergents containing NTA may lead to nitrate enrichment of water resources.