柴油食烷菌B-5及其卤代烷烃脱卤酶DadA对卤代化合物的降解

【目的】柴油食烷菌(Alcanivorax dieselolei)B-5是重要的石油降解菌。为研究其对卤代化合物的降解范围和降解机制,【方法】以不同的卤代化合物作为唯一碳源,观察菌株B-5在其中的生长情况;通过多重序列比对、系统发育分析和三维结构同源建模,分析该菌株基因组内一个假定的卤代烷烃脱卤酶(Haloalkane dehalogenase,HLD)DadA;利用大肠杆菌异源表达、纯化DadA,并测定了其对46个卤代底物的酶活。【结果】菌株B-5能够利用C3-C18链长范围的多种卤代化合物为唯一碳源生长;在系统进化树中,DadA相对独立于其他HLD-II亚家族成员,但具有典型的HLD-II亚家族的催化五联体残基;DadA确实具有脱卤活性,但该酶特异性高,底物范围明显小于其他已鉴定的HLDs,仅对1,2,3-三溴丙烷、1,2-二溴-3-氯丙烷和2,3-二氯-1-丙烯有脱卤酶活。【结论】因为DadA对很多B-5菌株可以利用做碳源的卤代底物没有脱卤酶活,所以推测B-5菌中可能还有其他脱卤酶参与了卤代烷烃的降解。菌株B-5及其卤代烷烃脱卤酶DadA在卤代烷烃污染物的生物降解方面具有应用潜力。     

植物转基因沉默与消除

植物基因工程研究是希望获得高稳定表达的转基因植株,而转基因沉默现象却限制了转基因植物的应用前景。基因沉默的机制是多方面的,包括转基因多拷贝之间的异位配对,转基因序列的甲基化,插入位点在染色体结构上的改变及转录后的衰退调控等。研究外源基因的失活原因及寻找相应的策略控制失活,对于植物基因工程的发展有着重要的意义。     

植物转基因沉默与消除

植物基因工程研究是希望获得高稳定表达的转基因植株,而转基因沉默现象却限制了转基因植物的应用前景,基因沉默的机制是多方面的,包括转基因多拷贝之间的异位配对,转基因序列的甲基化,插入位点在染色体结构上的改变及转录后的衰退调控等,研究外源基因的失活原因及寻找相应的策略控制失活,对于植物基因工程的发展有着重要的意义。     

细菌卤代烷烃脱卤酶基因在拟南芥菜中的高效表达

报道了细菌Xanthobacter autotrophicus编码卤代烷烃脱卤酶基因在拟南芥菜中的高效表达。以土壤农杆菌介导将该基因整合到拟南芥菜基因组中,经数代筛选得到了转基因纯合种子,Northern印迹和气相色谱检测表明,转基因的表达程度很高,酶量占细胞总可溶性蛋白的8%,酶活力达7.8mU·ml^-1提取物。转基因植株在含二氯乙烷的培养基上不能生长。     

微生物卤代烷烃脱卤酶研究进展

卤代烷烃脱卤酶是降解卤代脂肪族化合物的关键酶类,在各种地理环境中的不同微生物中广泛存在,在生物降解和工业生产等方面具有重要的应用价值。目前已经生化鉴定了20个卤代烷烃脱卤酶。近些年来对这些酶的酶学特征、蛋白质结构和系统进化进行了详细的研究。同时,为满足应用实践的需求还对卤代烷烃脱卤酶进行了蛋白质工程改造研究。本文将对卤代烷烃脱卤酶研究的一些新的进展进行综述。     

植物转基因的失活与沉默

植物外源基因转化研究揭示有相当比例的植物转基因表现失活与沉默。这一现象涉及转基因重复拷贝之间的异源配对,基因顺序的甲基化,转录后的衰退调控以及插入位点染色质高级结构的改变,并与外源基因的随机整合有关。克服转基因失活的方法包括单拷贝转基因子代筛选,构建细胞核基质支架附着区载体以及采用具有特殊功能的启动子与增强子。转基因失活问题的发生提示人们,外源基因的转化与整合存在一些特有的遗传机制。     

R-2-卤代酸脱卤酶的同源模建及底物对映体选择性研究

对来自假单胞菌ZJU26中的R-2-卤代酸脱卤酶(DehI-R)进行同源模建,分析其与底物的相互作用,为解析酶的底物对映体选择性提供理论依据.采用Sybyl中的APM模块首次构建并优化了R-2-卤代酸脱卤酶的三维结构,并用Procheck 验证结构模型的合理性.使用Suflex-Docking模块将结构模型与底物分别进行对接,分析相互之间的作用.序列比对结果显示,R-2-卤代酸脱卤酶与恶臭假单胞菌PP3中DehI的相似性达23.71％.Deh-R模建后的结构与模板很好的吻合.模型比对分析DehI-R中参与催化的残基,除Asn2.03外大部分都比较保守.分子对接结果表明,R-2-氯丙酸和S-2-氯丙酸都可以结合到活性位点上,决定其选择性的是值点Asn203,在RS-2-卤代酸脱卤酶所对应位点的残基为Ala,相比之下,Aan具备较大的空间位阻,从而阻止了S-2-氯丙酸的反应.利用Sybyl中的Biopolymer模块对R-2-卤代酸脱卤酶中的Asn203突变成具有不同空间位阻的Ala、Gly和Gln.突变酶与底物对接结果进一步证实了Asn203位点对R-2-卤代酸脱卤酶的底物对映体选择性作用.     

植物转基因的失活与沉默

植物外源基因转化研究揭示有相当比例的植物转基因表现失活与沉默。这一现象涉及转基因重复拷贝之间的异源配对,基因顺序的甲基化,转录后的衰退调控以及插入位点染色质高级结构的改变,并与外源基因的随机整合有关。克服转基因失活的方法包括单拷贝转基因子代筛选,构建细胞核基质支架附着区栽体以及采用具有特殊功能的启动子与增强子。转基因失活问题的发生提示人们．外源基因的转化与整合存在一些特有的遗传机制。     

转基因植物中外源基因沉默机制及防止对策

系统论述了诱发转基因沉默的因素、植物转基因沉默发生水平并提出了基因沉默的防止对策,基因沉默现象是导致外源基因不能在转化植物中正常表达的重要原因。甲基化、重复序列、反式失活和共抑制是基因沉默的主要诱因。其作用水平主要有三种：位置效应、转录水平的基因沉默和转录后水平的基因沉默,避免基因间的同源性、避免重复序列的出现、消除甲基化的影响、使用MAR以及使用诱导型启动子,可以提高外源基因的表达水平,有效防止基因沉默。     

GUS基因拷贝数对转基因在受体植物烟草中表达的影响

对农杆菌介导法获得的转 β_葡糖醛酸酶 (β_glucuronidase ,GUS)基因 (uidA)烟草 (NicotianatabacumL .)进行GUS表达分析 ,发现部分转基因植株无GUS活性。进一步Southern杂交结果发现 ,GUS基因失活植株的基因组中整合了多个uidA拷贝 ,而GUS活性高的转基因植株多为uidA单拷贝整合 ,表明uidA基因失活与基因多拷贝整合有关。Northern杂交结果显示 ,失活植株无特异uidARNA杂交带 ,而GUS活性高的植株可检测到明显的杂交信号 ,说明多拷贝引起的基因失活发生在RNA水平。     

Engineering a catabolic pathway in plants for the degradation of 1,2-dichloroethane

Plants are increasingly being employed to clean up environmental pollutants such as heavy metals; however, a major limitation of phytoremediation is the inability of plants to mineralize most organic pollutants. A key component of organic pollutants is halogenated aliphatic compounds that include 1,2-dichloroethane (1,2-DCA). Although plants lack the enzymatic activity required to metabolize this compound, two bacterial enzymes, haloalkane dehalogenase (DhlA) and haloacid dehalogenase (DhlB) from the bacterium Xanthobacter autotrophicus GJ10, have the ability to dehalogenate a range of halogenated aliphatics, including 1,2-DCA. We have engineered the dhlA and dhlB genes into tobacco (Nicotiana tabacum 'Xanthi') plants and used 1,2-DCA as a model substrate to demonstrate the ability of the transgenic tobacco to remediate a range of halogenated, aliphatic hydrocarbons. DhlA converts 1,2-DCA to 2-chloroethanol, which is then metabolized to the phytotoxic 2-chloroacetaldehyde, then chloroacetic acid, by endogenous plant alcohol dehydrogenase and aldehyde dehydrogenase activities, respectively. Chloroacetic acid is dehalogenated by DhlB to produce the glyoxylate cycle intermediate glycolate. Plants expressing only DhlA produced phytotoxic levels of chlorinated intermediates and died, while plants expressing DhlA together with DhlB thrived at levels of 1,2-DCA that were toxic to DhlA-expressing plants. This represents a significant advance in the development of a low-cost phytoremediation approach toward the clean-up of halogenated organic pollutants from contaminated soil and groundwater.     

A bacterial haloalkane dehalogenase gene as a negative selectable marker in Arabidopsis

The dhlA gene of Xanthobacter autotrophicus GJ10 encodes a dehalogenase which hydrolyzes dihalo- alkanes, such as 1, 2-dichloroethane (DCE), to a halogenated alcohol and an inorganic halide (Janssen et al. 1994, Annu. Rev. Microbiol. 48, 163-191). In Xanthobacter, these alcohols are further catabolized by alcohol and aldehyde dehydrogenase activities, and by the product of the dhlB gene to a second halide and a hydroxyacid. The intermediate halogenated alcohols and, in particular, the aldehydes are more toxic than the haloalkane substrates or the pathway products. We show here that plants, including Arabidopsis, tobacco, oil seed rape and rice, do not express detectable haloalkane dehalogenase activities, and that wild-type Arabidopsis grows in the presence of DCE. In contrast, DCE applied as a volatile can be used to select on plates or in soil transgenic Arabidopsis which express dhlA. The dhlA marker therefore provides haloalkane dehalogenase reporter activity and substrate dependent negative selection in transgenic plants.     

Characterization of resistance mechanism in transgenic Nicotiana benthamiana containing Turnip crinkle virus coat protein

Two transgenic lines, of Nicotiana benthamiana expressing Turnip crinkle virus (TCV)-coat protein (CP) gene with contrasting phenotype, the highest (#3) and the lowest (#18) CP expressers, were selected and challenged with the homologous TCV. The former, the highest expresser, showed nearly five times more CP expression than the latter. Progenies of #3 and #18 lines showed 30 and 100% infection rates, respectively. The infected progenies of #3 line showed mild and delayed symptom with TCV. This is a coat protein-mediated resistance (CP-MR), and its resistance level is directly proportional to CP transgene expression. However, CP-MR of the transgenic plants was specific only for TCV but not for heterologous viruses. Newly growing leaves of those infected progenies of #3 line did not show any visible symptoms at 4-week post-inoculation (wpi) with TCV, suggesting a reversal from infection. This was confirmed by RT-PCR analysis with the disappearance of the target at 4 wpi. This is a case of RNA-mediated resistance, and a threshold level of transgene expression may be needed to achieve the silent state. To confirm the RNA silencing, we infiltrated Agrobacterium carrying TCV-CP into leaves of progenies of #3 and performed RT-PCR analysis. The results indicate that TCV-CP’s suppressor activity against RNA silencing itself can be silenced by the homologous expression of TCV-CP in the transgenic plants. The transgenic plants containing TCV-CP seem to be a model system to study viral protection mediated by a combination of protein and RNA silencing. Ayyappan Vasudevan and Tae-Kyun Oh have contributed equally in this study.     

A test for transvection in plants: DNA pairing may lead to trans-activation or silencing of complex heteroalleles in tobacco

To study whether DNA pairing that influences gene expression can take place in somatic plant cells, a system designed to mimic transvection was established in transgenic tobacco. Pairing was evaluated by testing whether an enhancerless GUS gene on one allele could be activated in trans by an enhancer on the second allele. The required heteroalleles were obtained at four genomic locations using Cre-lox-mediated recombination. In one transgenic line, elevated GUS activity was observed with the heteroallelic combination, suggesting that trans-activation occurred. Conversely, when the unaltered allele was homozygous, GUS activity dropped to hemizygous levels in a silencing phenomenon resembling dosage compensation. Double-stranded GUS RNAs or small GUS RNAs indicative of RNA-based silencing mechanisms were not detected in plants displaying reduced GUS activity. These results suggested that a transgene locus capable of pairing, as revealed by trans-activation, could also become silenced in an RNA-independent manner, thus linking DNA pairing and gene silencing. The transgene locus was complex and comprised an inverted repeat, which possibly potentiated allelic interactions. The locus was unable to trans-activate transgenes at ectopic sites, further implicating allelic pairing in the transvection effects.