一种转录受病原物接种和机械创伤诱导的水稻WRKY基因的鉴定

WRKY基因编码一类植物特异性转录因子,该类基因目前在双子叶植物中得到了较好的研究.为了研究WRKY基因在单子叶植物中的生物学功能,从水稻(Oryza sativa L.)中分离了OsiWRKY基因的cDNA.该cDNA可编码一个含482个氨基酸残基的蛋白质,该蛋白质预测的一级结构中含有一个WRKY结构域,在该结构域中发现了一个典型的C2-H2锌指结构模块.OsiWRKY预测的一级结构中还可能含有一个细胞核定位因子;利用瞬时表达体系,发现OsiWRKY与GFP绿色荧光蛋白(GFP)的融合蛋白的确定位在水稻细胞核中.在凝胶阻滞实验中,体外表达的OsiWRKY蛋白可特异地结合W-box顺式因子.在水稻白叶枯病原细菌接种实验中,OsiWRKy基因的转录本在抗病品种(IR-26)中的诱导增加强度明显高于感病品种(Jingang 30).在模拟接种的水稻中,OsiWRKy基因的转录本也表现诱导增加,但其诱导增加的起始时间和幅度要显著低于水稻白叶枯病原细菌接种的水稻植株.这些结果表明,OsiWRKY基因可能编码了一个含有WRKY结构域的转录因子,该转录因子可能参与了水稻对病原物接种和机械创伤的反应,但OsiWRKY蛋白调节两种反应的机制可能有所不同.     

锌指结构：最普遍的核酸识别元件

锌指是最大的DNA结合蛋白家庭,是识别DNA最有效、最成功的一种结构元件。其模块性结构特点及与核酸作用的相对简单性,使其成为研究蛋白-核酸相互作用的理想材料,以及人为设计筛选新的核酸结合蛋白的最佳元件。     

Effect of various metal ions on growth of two wheat lines known to differ in aluminium tolerance

The effects of aluminium (Al), manganese (Mn), zinc (Zn), copper (Cu), boron (B), iron (Fe), gallium (Ga), scandium (Sc) and lanthanum (La) on growth of an Al-tolerant and an Al-sensitive line of wheat (Triticum aestivum L.) were measured in solution culture. The concentrations of nutrients in the basal nutrient solution were (M) 500 Ca, 100 Mg, 300 K, 600 N (150 NH₄, 450 NO₃), 600 SO₄, 2.5 P, 3 B, 2.5 Fe, 0.5 Zn, 0.5 Mn, 0.1 Cu at a pH of 4.7. The major solution nutrient concentrations were maintained at the nominal concentration with monitoring, frequent additions and weekly renewal. Differentiation in yield between the Al-tolerant and Al-sensitive line only occurred in the presence of Al indicating that, in the long term, none of the other metals tested could be used as an analog for Al. The visual symptoms in the roots of Cu toxicity (in both lines) and Al toxicity (in the sensitive line) were similar. The solution concentration (M) at which yield of the roots of the tolerant line was reduced by 50% was, in order of increasing tolerance, Cu 0.5, Sc 1.1, La 7.1, Ga 8.6, Al 15, Zn 19, Fe 84, B 490 and Mn 600.     

Active site of Zn(2+)-dependent sn-glycerol-1-phosphate dehydrogenase from Aeropyrum pernix K1

The enzyme sn-glycerol-1-phosphate dehydrogenase (Gro1PDH, EC 1.1.1.261) is key to the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate) of archaeal ether lipids. This enzyme catalyzes the reversible conversion between dihydroxyacetone phosphate and glycerol-1-phosphate. To date, no information about the active site and catalytic mechanism of this enzyme has been reported. Using the sequence and structural information for glycerol dehydrogenase, we constructed six mutants (D144N, D144A, D191N, H271A, H287A and D191N/H271A) of Gro1PDH from Aeropyrum pernix K1 and examined their characteristics to clarify the active site of this enzyme. The enzyme was found to be a zinc-dependent metalloenzyme, containing one zinc ion for every monomer protein that was essential for activity. Site-directed mutagenesis of D144 increased the activity of the enzyme. Mutants D144N and D144A exhibited low affinity for the substrates and higher activity than the wild type, but their affinity for the zinc ion was the same as that of the wild type. Mutants D191N, H271A and H287A had a low affinity for the zinc ion and a low activity compared with the wild type. The double mutation, D191N/H271A, had no enzyme activity and bound no zinc. From these results, it was clarified that residues D191, H271 and H287 participate in the catalytic activity of the enzyme by binding the zinc ion, and that D144 has an effect on substrate binding. The structure of the active site of Gro1PDH from A. pernix K1 seems to be similar to that of glycerol dehydrogenase, despite the differences in substrate specificity and biological role.     

丛枝菌根真菌菌丝体吸附重金属的潜力及特征

应用玻璃珠分室培养系统获得丛枝菌根真菌材料,研究了离体真菌菌丝体对pH缓冲体系中Zn、Cd和Mn等金属离子的吸附特征。试验结果表明,真菌菌丝体对各金属离子吸附能力差异显著,对Cd最强,Zn次之,Mn最弱。试验条件下,菌体可分别吸附相当于自身干物重1.6%的Mn、2.8%的Zn和13.3%的Cdo吸附于菌丝体的Cd2+绝大部分可以被Ca2+交换吸附。另外研究了宿主植物根系对Cd的吸附作用,证实菌根真菌侵染改变了根系的吸附特性,相对于非菌根根系,菌根的CEC较高,对Cd的吸附能力较强。试验结果为重金属污染条件下丛枝菌根强化根系的屏障作用提供了直接证据。     

针对DNA长片段靶标的锌指筛选体系

锌指蛋白能够特异性识别目标DNA序列,常被作为分子靶向因子用于定点核酸编辑以及定点转录调控等方面,具有十分广泛的应用前景.然而,目前常规采用的实验方法得到的锌指蛋白通常结合的目标DNA序列较短,结合能力不强,因而一直难以高效运用在核酸序列与调控蛋白在基因组上的原位互作等方面的研究中.为了解决这个问题,本研究构建了一个高通量筛选系统,利用N4噬菌体gp8毒蛋白和LacZ作为报告基因,以300 bp以上的DNA长片段为靶标,来筛选能够结合多位点的锌指蛋白组合,提高锌指蛋白应用的精确度以及效率.该系统针对小鼠Nrxn-1?启动子区域进行了锌指蛋白文库筛选,得到了具有序列选择特异性的混合锌指蛋白库,并对筛选结果进行了初步功能验证.研究表明,本系统具有简便快捷的特点,不仅大幅度缩短了筛选时间,而且减少了因靶标序列DNA片段较长而不得不反复设计多位点结合锌指蛋白造成的成本浪费;筛选得到的锌指蛋白库具有较高的长片段DNA靶标结合能力和一定的序列特异性,并且能够在真核细胞内特异地结合目标DNA序列.因此,本研究建立的新型锌指筛选系统不仅可以广泛应用于高通量筛选,而且在DNA-蛋白相互作用的研究中也具有重要意义.     

Quantitative and Qualitative Changes in Peroxidase of Cucurbita Pepo Cultivars Stressed with Heavy Metals

Seedlings of two cultivars of zucchini (Cucurbita pepo L.) Courgette d'Italie (CI) and Courgette d'Alger (CA) were pre-treated with various concentrations of cadmium, copper and zinc for 30 d. High accumulation of heavy metals especially in the roots was showed. Peroxidase activity was affected according to the type of metal added, concentration, and the plant cultivar used. In leaves and roots of the CI control plants peroxidase activities were 50 and 17 % higher than in the CA control plants. Treatment with Cd (5 μg g⁻¹), Cu (200 μg g⁻¹), and Zn (500 μg g⁻¹) increased peroxidase activities in CA but decreased it in CI both in leaves and roots. Heavy metals tested lead also to some qualitative changes characterized by appearance of new isoforms of peroxidase. The results show the possibility to use the activities of peroxidase as biomarkers for Cd, Cu and Zn stresses. This revised version was published online in July 2006 with corrections to the Cover Date.     

Zn对细胞保护作用机理的研究

应用扫描质子微探针和同步辐射ｘ荧光分析技术测定了细胞中元素的分布和组成,为确定Zn是细胞结构成分提供了直接的实验依据.用上述核技术结合有关生化指标,分析测定了正常和损伤细胞（脂质过氧化损伤）中Fe,Zn和丙二醛、SH基含量变化的相互关系.实验结果表明,当细胞发生脂质过氧化损伤时,Fe含量和丙二醛含量同步增高,而Zn含量和SH基量则降低.给细胞补充Zn后,提高了细胞质膜中的Zn含量,SH基量也随之增加,同时丙二醛量降低.提示Zn保护细胞完整性的作用机理之一是控制脂质过氧化作用.Zn可保护膜蛋白的SH基,减少和阻止被Fe所催化的过氧化反应.     

多聚ADP-核糖聚合酶抑制剂对高浓度锌损伤 PC12细胞的保护作用

探讨多聚ADP-核糖聚合酶(PARP)抑制剂3-氨基苯甲酰胺(3-AB)对400μmo1/L氯化锌损伤PC12细胞的保护作用及其对锌造成的细胞死亡类型的影响.应用MTT法,免疫细胞化学和Western印迹分别测定PC12细胞的存活率和PARP活性;用Hoechst 33342/PI荧光双染色、膜联蛋白V结合实验及DNA断裂分析等方法检测细胞死亡类型.结果表明在400μmol/L氯化锌的作用下,细胞存活率降至(22.7±4.6)%,PARP活性增强,坏死、凋亡和正常细胞百分比分别为(58.4±6.3)%、(18.0±5.6)%及(23.6±4.2)%;3-AB使细胞存活率提高至(76.9±4.7)%,PARP活性减弱,坏死细胞百分数降至(19.2±5.2)%,而正常和凋亡细胞百分数增加到(43.3±1.9)%和(37.5±6.5)%.实验证明,PARP参与了高浓度锌诱导的PC12细胞损伤,抑制PARP活性可提高细胞的存活率,而这种保护作用在于减少细胞的坏死而非凋亡.     

苦荞锌指蛋白基因FtLSD1的克隆及其对非生物胁迫的应答

根据苦荞(Fagopyrum tataricum)花期转录组数据,分别以苦荞DNA和cDNA为模板,克隆得到1个苦荞C2C2型锌指蛋白基因FtLSD1(GenBank登录号KP252134)的DNA序列和cDNA序列,采用实时荧光定量PCR方法,研究了FtLSD1基因在非生物胁迫下的表达模式。结果显示:苦荞FtLSD1基因DNA全长2 427bp,由6个外显子和5个内含子构成,符合GU-AG剪切原则;cDNA序列包含一个528bp开放阅读框,编码175个氨基酸,具有LSD1家族的典型结构域;UV-B照射和水杨酸处理均能使FtLSD1基因的表达量上升,且UV-B处理在6h达到最大,为0h(CK)的3.84倍;水杨酸处理于10h达到最大,为0h(CK)的3.44倍,而4℃冷胁迫下该基因表达量保持稳定。推测该基因可能参与苦荞抗UV-B和高浓度水杨酸等非生物胁迫的应答反应,为苦荞的抗逆性研究提供新的视角。