降解2，4—二氯酚假单孢菌GT141—2的特性及3，5—二氯儿茶酚1，2—双加氧酶基因定位

从2，4－二氯酚生产厂排污口污泥中分离出一株2，4－二氯酚降解细菌GT141－2，经鉴定确定为假单胞菌属，在最适条件下菌株GT141－2能在36h内将90mg/L的2，4－二氯酚降解62％。GC／MS分析表明，该菌株能将2，4－二氯酚彻底降解，未积累中间代谢产物。菌株GT141－2有三4条大质粒带，Southern杂交显示其3，5－二氯儿茶酚1，2－双加氧酶基因定位于其中的最小的质粒上。     

源于Cupriavidus campinensis BJ71的cctfdA基因的遗传转化提高烟草2,4-D抗性

高浓度2,4-二氯苯氧乙酸(2,4-Dichlorophenoxyacetic acid,2,4-D)是一种选择性除草剂,被广泛应用于防除阔叶杂草。为了避免2,4-D喷施过程中的喷雾和蒸发漂移,有必要提高敏感植物的2,4-D抗性。来源于微生物的2,4-D降解基因编码蛋白可以将其代谢而解除毒性。本研究以前期从2,4-D降解菌Cupriavidus campinensis BJ71菌株中克隆得到的2,4-D降解基因cctfdA为模板进行适合于烟草(Nicotiana tabacum)表达的密码子优化,利用改造后的基因Nt-cctfdA构建植物表达载体pSH737-Nt-cctfdA遗传转化烟草。结果表明,与野生型烟草及转化空载体的烟草相比,转Nt-cctfdA基因烟草的离体叶片及丛生芽的2,4-D抗性明显高于对照;转基因烟草植株可以抗10 000 mg/L浓度的2,4-D,而对照植株经350 mg/L浓度处理后死亡,表明转基因烟草的2,4-D抗性显著高于对照烟草30倍以上,且可以对抗10倍的2,4-D田间使用剂量。转基因烟草在2,4-D喷施前后,叶绿素含量基本无变化,而对照烟草的叶绿素含量明显降低。研究结果表明转基因烟草的2,4-D抗性显著提高,为进一步阐明转基因烟草的2,4-D抗性机理及2,4-D抗性转基因烟草的研发提供了基础资料。     

Cupriavidus campinensis BJ71菌株2,4-D降解基因tfdA生物信息学分析

本文以2,4-二氯苯氧乙酸高效降解菌Cupriavidus campinensis BJ71菌株中克隆的tfdA基因全序列为研究对象,利用生物信息学方法分析该基因及其推测出的编码蛋白理化性质、蛋白的亲水性或疏水性及亚细胞定位、预测信号肽、跨膜结构域、蛋白二级结构及模体、三级结构等特性。结果表明,BJ71菌株的tfdA基因开放阅读框全长864 bp,系统发育树表明其属于Ⅰ族tfdA基因。TfdA蛋白包含287个氨基酸,分子量为32 kD、理论等电点为6.01,是一种不含信号肽、定位于细胞质中的可溶性亲水蛋白。α螺旋和无规卷曲是该蛋白主要的二级结构元件。用RaptorX进行三级结构建模、Ramachandran评估显示获得了合理可靠的TfdA蛋白结构模型。     

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。     

Sphingobium sp. JZ-1对菊酯类农药的降解特性研究

研究了菊酯降解菌株Sphingobium sp.JZ-1及其粗酶液对菊酯类农药的降解特性。结果表明,该菌能降解目前使用的各种菊酯类农药,降解速率顺序为:氯菊酯>甲氰菊酯≈氯氰菊酯>功夫菊酯>氰戊菊酯>溴氰菊酯>联苯菊酯;Sphingobium sp.JZ-1对菊酯的降解没有手性选择性;高效液相色谱检测结果表明Sphingobium sp.JZ-1对氯氰菊酯降解的初始反应是在菊酯水解酶的催化下羧酸酯键断裂生成二氯菊酸和3-苯氧基苯甲醛,3-苯氧基苯甲醛进一步氧化成苯氧基苯甲酸。Sphingobium sp.JZ-1细胞中菊酯水解酶酶活不需要菊酯的诱导。     

植物吸收硝态氮的分子生物学进展

植物吸收NO3-分为高亲和力(HATS,high.affinitynitratetransportsystem)和低亲和力(LATS,low.affinitynitratetransportsystem)转运系统。这两个系统的编码基因均已被克隆,其中HATS由NRT2基因家族和NAR2基因家族共同编码,LATS由NRT1基因家族编码。本文比较详细地介绍了这些转运蛋白的结构和功能以及在这方面的最新进展,概要性地介绍了这些基因的表达调控;同时摘要点评了该研究领域中还没有解决的一些问题。     

苏云金芽孢杆菌cry1Aa14基因的分离、克隆及其表达

Bt25是中国自行分离的对小菜蛾(Plutella xylotella)具有高毒力的苏云金芽孢杆菌(Bacillus thuringiensis),经PCR-RFLP鉴定含有cry1Aa基因。以全长基因PCR产物的粘端定向克隆的方法,设计1对特异引物,以Bt25质粒DNA为模板扩增cry1Aa全长基因。序列测定结果表明,该基因编码区为3552bp,编码1183个氨基酸,分子量为133．7kD,pI4．755。该基因序列已在GenBank注册,登记号为AY197341,并获得正式命名cry1Aa14。在氨基酸序列918～1180间,和已知的11种cry1Aa存在22-23个氨基酸的差异(其中1094～1097的4个氨基酸无对应序列),而这段区域和Cry1Ab氨基酸序列的对应区域无差异。cry1Aa14全长基因插入Bt表达载体,获得了重组表达质粒pBYB1,转化Bt无晶体突变株HD73cry-,经过抗性筛选、DNA酶切分析和PCR检测,证实转化成功。SDS-PAGE分析表明,该基因在上述受体中能正常表达133kD蛋白。杀虫生物测定结果表明,cry1Aa14表达产物对小菜蛾幼虫具有显著的毒杀作用,与cry1Aa12进行比较,毒力无明显差异。这种单基因菌株的发现及其基因的获得,为害虫抗性研究和高效工程菌的构建提供了重要实验材料。     

荔枝铜锌超氧物歧化酶基因的克隆与序列分析

以"三月红"荔枝基因组DNA为模板,用Cu*ZnSOD基因特异寡聚核苷酸为引物,进行PCR扩增,得到特异基因片段,将其克隆到pGEMT载体上,转化感受态大肠杆菌TG1中,对转化子中重组pGEMT上的Cu*ZnSOD基因片段的PCR扩增检测和序列分析,表明克隆成功;该基因片段有478个核苷酸,由4个外显子和3个内含子组成;外显子由234个核苷酸组成,编码78个氨基酸;该基因片段编码的氨基酸序列与水稻、玉米、番茄、大白菜和松树的Cu*ZnSOD基因编码的氨基酸序列的同源性分别为79.5%、73.1%、73.1%、71.8%和75.7%.     

养殖水体中氯酚残留特征及其与环境因子关系初探

采用固相微萃取（SPME）与气-质联用方法,测定分析了3种养殖模式水体中氯酚化合物（CPs）的污染特征。结果表明,19种CPs类化合物在一般四大家鱼养殖水体（A）、猪-鱼综合养殖模式水体（B）以及鸭-鱼综合养殖模式水体（C）表层水中的分布特征相似。总CPs及10种CPs化合物在不同养殖方式水体中浓度由高到低的顺序为A〉B〉C。表层水中残留浓度比较高的一氯酚、二氯酚、三氯酚和四氯酚分别是3-CP和4-CP、2,4-DCP、2,4,6-TCP、2,3,4,6-TCP。回归分析表明,铁、锰总含量与总CPs和PCP含量存在显著负相关。     

哈茨木霉A25-2纤维二糖水解酶I基因的克隆及其编码催化功能域的序列在绿色木酶HP35-3中的表达

本研究从哈茨木霉（rrichoderma harzianMm）A25—2总RNA中利用RT—PCR的方法扩增到其纤维二糖水解酶I基因的cDNA序列,并对该基因编码的氨基酸序列进行分析,得到cbhI基因中编码催化功能域的序列。将催化功能域编码序列克隆到表达载体pCP—GH中,用PEG—CaCl2介导的原生质体转化方法将重组质粒转化到绿色木霉（Trichoderma viride）HP35—3中,筛选得到12个转化子。以P-NPC为底物,测定了该12个转化子的酶活力,获得比活力最高的转化子Tv／CDHI-CBM-5,其纤维二糖水解酶活力是HP35—3的3．8倍。SDS—PAGE分析表明,绿色木霉表达了导入的含A25—2纤维二糖水解酶I催化功能域的编码序列。     

Importance of 2,4-DAPG in the biological control of brown patch by Pseudomonas fluorescens HP72 and newly identified genes involved in 2,4-DAPG biosynthesis

The Pseudomonas fluorescens strain HP72 used as biocontrol agent was isolated from the roots of creeping bentgrass on brown patch-suppressive soil. This strain can suppress brown patch disease caused by Rhizoctonia solani. The analysis of secondary metabolites from strain HP72 revealed that it produced known antifungal compounds, 2,4-diacetylphloroglucinol (2,4-DA-PG), HCN, and a fluorescent siderophore. In the present study, the Tn5inserted mutants of strain HP72, which did not show any antifungal activity, were selected. None of the mutants produced 2,4-DA-PG but they produced a fluorescent siderophore, while some strains produced HCN. Therefore, it is suggested that 2,4-DA-PG plays a major role in the biological control of brown patch disease caused by R. solani. In the genomic region where Tn5 was inserted, two open reading frames (ORFs A and B), which are not included in the 2,4-DAPG gene cluster of HP72, were detected. It was demonstrated that ORFs A and flare involved in the regulation of 2,4-DAPG biosynthesis.     

蜡样芽孢杆菌ATCC14579毒力基因plcR缺失株的构建及其一般特性

蜡样芽孢杆菌是土壤中的优势菌,具有作为益生菌的潜在能力,同时它也是条件致病菌,能引起食物中毒等。蜡样芽孢杆菌的多种毒力因子受到多效性调控子（pleiotropic regulator,plcR）的调控,在其条件致病性作用中起着重要作用。真养产碱杆菌JMP34（Alcaligenes eutrophus）质粒上的2,4-二氯苯氧乙酸（2,4-dichlorophenoxyacetic acid,2,4-D）单加氧酶（tfdA）基因可以降解2,4-D。本研究利用同源重组技术使tfdA基因置换掉蜡样芽孢杆菌的plcR基因,构建了蜡样芽孢杆菌ATCC14579突变株B.cereus△plcRΩtf-dA,并对其毒性、一般生理生化特性进行分析。研究结果表明,突变株B.cereus△plcRΩtfdA的毒性显著减弱;生理生化实验结果显示突变株与野生株并没有明显区别,且突变株并没有表现出tfdA酶活性。所有的结果表明plcR控制着蜡样芽孢杆菌ATCC14579的致病性,同时剔除plcR并不破坏其酶系统。本研究为今后构建蜡样芽孢杆菌工程菌提供了新的思路和依据。     

Substrate specificity of chlorophenoxyalkanoic acid-degrading bacteria is not dependent upon phylogenetically related tfdA gene types

The phenoxyalkanoic acid herbicides constitute a group of chemically related molecules that have been widely used for over 50 years. A range of bacteria have been selected from various locations for their ability to degrade these compounds. Previously reported strains able to utilise 2,4-dichlorophenoxyacetic acid (2,4-D) include, Ralstonia eutropha JMP134, Burkholderia sp. RASC and Variovorax paradoxus TV1 and Sphingomonas sp. AW5 able to utilise 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). In addition a novel set of mecoprop-degrading strains including Alcaligenes denitrificans, Alcaligenes sp. CS1 and Ralstonia sp. CS2 are here described. It has been reported recently that TfdA enzymes, initially reported to have a role in 2,4-D catabolism are also involved in the first-step cleavage of related phenoxyalkanoate herbicides. However, a diversity of tfdA gene sequences have been reported. We relate the tfdA gene type to the metabolic ability of these strains. The tfdA-like genes were investigated by polymerase chain reaction amplification using a set of specific tfdA primers. Degradation ability was observed via phenol production from a range of unsubstituted and substituted phenoxyalkanoics including, 2,4-D, 2-methyl 4-chlorophenoxyacetic acid (MCPA), racemic mecoprop, (R)-mecoprop, 2-(2,4-dichlorophenoxy) propionic acid (racemic 2,4-DP), 2,4,5-T, 2,4-dichlorophenoxybutyric acid (2,4-DB), 4-chloro-2-methylphenoxybutyric acid (MCPB) and phenoxyacetate. Mecoprop-degrading strains showed partial tfdA sequences identical to the one described for V. paradoxus TV1 (a strain isolated on 2,4-D). However, substrate specificity was not identical as V. paradoxus exhibited greatest activity towards 2,4-D and MCPA only, whereas the mecoprop-degrading strains showed intense activity towards 2,4-D, MCPA, racemic mecoprop and (R)-mecoprop as substrates. However, Sphingomonas sp. AW5 which has been shown to carry a very different tfdA-like gene was the only strain to utilise the phenoxybutyric acid MCPB as a sole carbon source. In this study, we thus demonstrate that sequence diversity is not related to substrate specificity within the tfdA-like gene family. However, phylogenetically unrelated sequences may govern substrate specific activity.     

2,4-Dichlorophenoxyacetic acid metabolism in transgenic tolerant cotton (Gossypium hirsutum)

The metabolic fate of 2,4-dichlorophenoxyacetic acid (2,4-D) was studied in leaves of transgenic 2,4-D-tolerant cotton (Gossypium hirsutum), which is obtained by transfer of the tfdA gene from the bacterium Alcaligenes eutrophus. The tdfA gene codes for a dioxygenase catalyzing the degradation of 2,4-D to 2, 4-dichlorophenol (2,4-DCP). [phenyl-(14)C]-2,4-D was administered by petiolar absorption followed by an 18 h water chase or converted to the isopropyl ester and sprayed onto the leaf surface; the leaves were harvested 48 h later. The herbicide was degraded to 2,4-DCP by the bacterial enzyme expressed in the plants. 2,4-DCP was rapidly converted to more polar metabolites and was never found in detectable amounts. Metabolite structures were deduced from enzymatic hydrolysis studies and mass spectrometric analyses. The first metabolite was the glucoside conjugate of 2,4-DCP (2, 4-DCP-beta-O-glucoside). The major terminal metabolites were two more complex glucosides: 2,4-DCP-(6-O-malonyl)glucoside and 2, 4-DCP-(6-O-sulfate)glucoside.     

Enhanced bioremediation of soil containing 2,4-dinitrotoluene by a genetically modified Sinorhizobium meliloti

The symbiotic nitrogen-fixing soil bacterium, Sinorhizobium meliloti, is well known for its ability to interact with the leguminous plant Medicago sativa L. It has, however, not been reported that this species possesses the capability to degrade toxic nitroaromatic compounds, such as 2,4-dinitrotoluene (DNT) which is commonly associated with the degradation of the explosive trinitrotoluene (TNT). In this study, the pJS1 DNT-biodegradative plasmid was genetically transferred to S. meliloti strain USDA 1936, which was confirmed by plasmid profile analysis. Several standard analytical and chemical tests including high performance liquid chromatography (HPLC), nitrite (NO₂) release assays, rhizosphere population and plant greenhouse studies were conducted to test the ability of S. meliloti to degrade 2,4-DNT. The possible presence of 2,4-DNT remaining in the treated soil was tested, and no 2,4-DNT had been absorbed by the soil. The pJS1-carrying recombinant strain DHK1 produced ‘ARC’ alfalfa plants that were almost 2-fold higher in shoot dry weight than that produced by the parent strain on soil containing 0.14 mM 2,4-DNT. The transconjugant strain DHK1 reduced significantly one-third more 2,4-DNT in both 0.14 and 0.28 mM contaminated soil, and in 0.55 mM contaminated soil it degraded 94% of the 2,4-DNT present. In liquid cultures, however, only about 4% reduction in 2,4-DNT concentrations was obtained in 10 days. We interpret the results as clearly establishing that genetic modification was successfully used, for the first time, to improve the capability of the symbiotic nitrogen-fixing soil bacterium S. meliloti DHK1 to bioremediate in situ 2,4-DNT-contaminated soil in the presence of alfalfa plants.     

枯草杆菌β-甘露聚糖酶基因的克隆及表达

以能水解魔芋葡甘聚糖的野生筛选菌种枯草杆菌A33为材料，通过PCR技术从A33基因组中扩增出β-甘露聚糖酶基因编码序列。经过克隆、测序及BLAST比对分析，证实该基因编码β-甘露聚糖酶，属于β-甘露聚糖酶家族中的一员。该基因已注册GenBank（GenBank注册号：DQ269473）。将该基因构建到大肠杆菌表达载体pET-32a并转入大肠杆菌表达系统BL21（DE3），经过诱导获得了此酶的高效表达.     

Suppression of root-knot disease by Pseudomonas fluorescens CHA0 in tomato: importance of bacterial secondary metabolite, 2,4-diacetylpholoroglucinol

The antimicrobial metabolites 2,4-diacetylphloroglucinol (2,4-DAPG) and pyoluteorin contribute to the ability of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogens. P. fluorescens strain CHA0 and its derivatives CHA89 (antibiotics-deficient) and CHA0/pME3424 (antibiotics overproducing) were investigated as potential biocontrol agents against Meloidogyne javanica the root-knot nematode. Exposure of root-knot nematode to culture filtrates of P. fluorescens under in vitro conditions significantly reduced egg hatch and caused substantial mortality of M. javanica juveniles. Nutrient broth yeast extract (NBY) medium amended with 2% (w/v) glucose or 1 mM EDTA markedly repressed hatch inhibition activity of the strain CHA0 but not that of CHA0/pME3424 or CHA89. On the other hand, NBY medium amended with glucose significantly enhanced nematicidal activity of the strain CHA0/pME3424. Neither glucose nor EDTA had an influence on the nematicidal activity of the strains CHA0 and CHA89. Under in vitro conditions, antibiotic overproducing strain CHA0/pME3424 and CHA0 expressed phl‘-’lacZ reporter gene but strain CHA89 did not. Expression of the reporter gene reflects actual production of DAPG. In general, CHA0/pME3424 expressed reporter gene to a greater extent compared to its wild type counterpart CHA0. Regardless of the bacterial strains, reporter gene expression was markedly enhanced when NBY medium was amended with glucose but EDTA had no such effect. A positive correlation between the degree of juvenile mortality and extent of phl‘-’lacZ reporter gene expression was also observed in vitro. Strain CHA0 produced zones of 4-6 mm on MM medium containing gelatin while strain CHA0/pME3424 and CHA89 did not. When MM medium containing gelatin was amended with 2% glucose of 1 mM EDTA size of haloes produced by the strain CHA0 reduced to 2 mm. Under glasshouse conditions aqueous cell suspension of the strains CHA0 or CHA0/pME3424 at various inoculum levels (10⁷, 10⁸ or 10⁹ cfu ml⁻¹) significantly reduced root-knot development. CHA89 caused significant reduction in galling when applied at 10⁹ cfu ml⁻¹. To better understand the mechanism of nematode suppression, split root bioassay was performed. Split-root experiments, that guarantee a spatial separation of inducing agent and a challenging pathogen, showed that soil treatment of one half of the root system with cell suspension of CHA0 or CHA0/pME3424 resulted in a significant systemic induced resistance leading to reduction of M. javanica infection of tomato roots in the non-baterized nematode treated half. The results clearly suggest that the antibiotic 2,4-DAPG from P. fluorescens CHA0 act as the inducing agents of systemic resistance in tomato roots. Populations of CHA0 and its derivatives declined progressively by 10-fold between first and fourth harvests (0-21 days after inoculation). However, bacterial populations increased at final harvest (28 days after application).     

Inheritance and physiological basis for 2,4-D resistance in prickly lettuce (Lactuca serriola L.)

Experiments were conducted to determine the inheritance and physiological basis for resistance to the synthetic auxinic herbicide (2,4-dichlorophenoxy)acetic acid (2,4-D) in a prickly lettuce biotype. Inheritance of 2,4-D resistance in prickly lettuce is governed by a single codominant gene. Absorption and translocation were conducted using (14)C-2,4-D applied to 2,4-D-resistant and -susceptible biotypes. At 96 h after treatment (HAT), the resistant biotype absorbed less applied 2,4-D and retained more 2,4-D in the treated portion of the leaf compared to the susceptible biotype. The resistant biotype translocated less applied 2,4-D to leaves above the treated leaf and crown at 96 HAT compared to the susceptible biotype. No difference in the rate of metabolism of 2,4-D was observed between the two biotypes. Resistance to 2,4-D appears to originate from a reduced growth deregulatory and overstimulation response compared to the susceptible biotype, resulting in lower translocation of 2,4-D in the resistant prickly lettuce biotype.     

根癌农杆菌介导的抗除草剂转基因甘薯植株的获得

以甘薯品种栗子香的胚性悬浮细胞为受体材料, 用根癌农杆菌介导法,获得了表达bar基因的抗除草剂转基因甘薯植株。农杆菌菌株LBA4404携带含有bar基因的双元载体pCAMBIA3300。共计450个胚性细胞团用于遗传转化。在添加2 mg/L 2,4－D、100 mg/L Carb和0.5 mg/L PPT 的固体MS培养基上选择培养8周后,得到了19个PPT抗性愈伤组织。将这19个抗性愈伤组织转移到添加1 mg/L ABA、100 mg/L Carb和0.5 mg/L PPT 的固体MS 培养基上,其中的10个抗性愈伤组织共再生出103株拟转基因植株。PCR分析表明,其中的69株为转基因植株。Southern blot分析表明,bar基因稳定整合到转基因植株的基因组中。除草剂喷洒试验结果表明,转基因植株具有高度除草剂抗性。