大蒜植物络合素合酶基因转化对酵母重金属抗性的提高

重金属污染是全球面临的亟待解决的生态问题。利用植物对重金属的富集作用来清除环境重金属污染即植物修复已成为重要的环境生物技术之一。这一技术的长远发展有赖于在重金属富集或耐受中起关键作用的基因的克隆和应用。植物络合素是植物体内一类重要的对重金属起螯合作用的多肽, 其合成受植物络合素合酶的催化。该文取得了如下研究结果:1)通过原子吸收测定表明,在大蒜(Allium sativum)的根部可以积累3 000 mg·kg^-1的重金属镉;2)将克隆的大蒜植物络合素合酶基因(AsPCS)置于酵母表达启动子之下,构建酵母表达载体,并将其分别转入了因CUP1和acr3基因缺失而对重金属镉和砷敏感的酵母突变体菌株后,发现来自大蒜的AsPCS基因的表达使酵母CUP1缺失菌株对镉的耐受性提高了4倍, acr3缺失菌株对砷的耐受性提高了两倍;3)表达AsPCS基因酵母的生长模式证实了AsPCS基因的表达是酵母对重金属耐受性提高的原因。这些结果暗示, 大蒜植物络合素合酶基因在大蒜对重金属的抗性及大蒜根部对镉的积累中起关键作用,可作为重要的基因元件应用到修复污染的植物基因工程中。     

植物络合素的测定方法

植物络合素是一类富含巯基,并能够与重金属离子相结合的多肽。植物络合素具有共同的结构：[γ-Glu—Cys]n Gly(n=2-11)。植物络合素是植物重金属抗性的要素之一。植物络合素的测定是一种植物重金属抗性机理的研究技术。介绍了植物络合素测定的理论,并综述了现有的植物络合素测定的方法：分光比色法、凝胶过滤法、液相色谱法和毛细管电泳法。其中,着重阐述了液相色谱法在植物络合素测定中的应用。     

植物类MT与植物络合肽

金属硫蛋白在哺乳动物的解毒方面民挥着重要作用，植物本身也存在类似的解毒机制。研究表明，植物存在基因编码的类ＭＴ，另外还有非基因编码的植物ＭＴ，又称植物络合肽，类ＭＴ在必需重金属离子（例如Ｚｎ＾２＋、Ｃｕ＾２＋）的代谢中挥着重要作用而植物络合肽在非必需重金属离子（例如Ｃｄ＾２＋）的解毒中起到关键作用。本文了类ＭＴ和植物络合肽之间的关系以及各自的生物合成途径、相关的结构与功能、在植物中的分布等。     

多年生黑麦草抗氧化酶和植物络合素对Cd2+胁迫的应答

采用水培方法研究了5 mg· L-1 Cd2+胁迫下,Cd在多年生黑麦草中的积累和Cd2+对多年生黑麦草抗氧化酶活性和植物络合素等巯基化合物浓度的影响.将具有3片展开叶的多年生黑麦草实生苗转至1/2霍格兰营养液中培养2周后,对其进行5 mg-L-1 Cd2+处理,分别在处理后的0、0.25、1、3、6d取样测定根系和叶片的Cd浓度、抗氧化酶活性和植物络合素等巯基化合物的浓度.结果表明,Cd2+处理多年生黑麦草6d后,根系中Cd浓度达到2.59 mg·g-1,叶片中Cd浓度达到0.24 mg·g-1,根中Cd向叶片的转运系数力0.093,叶中Cd的富集系数为48,多年生黑麦草属Cd高积累植物,具备在植物修复上应用的前景.Cd2+胁迫下,多年生黑麦草根叶中丙二醛(MDA)含量无显著变化,根中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性无显著变化,抗坏血酸过氧化物酶(APX)对Cd2+敏感,处理后6d活性较0d显著下降67.19％.Cd2+处理1d内,叶中SOD、APX、CAT活性显著降低.Cd2+处理后3d,叶中的抗氧化酶系统对叶中Cd浓度的升高做出了正反馈,SOD、APX、CAT的活性分别较处理后1d显著上升了14.19％、76,82％、99.26％,Cd2+处理时间延长至6d,SOD活性较处理后3d显著下降了18.58％,APX、CAT活性无显著变化.Cd2+处理后6d,多年生黑麦草根中半胱氨酸(Cys)、谷胱甘肽(GSH)、植物络合素2(PC2)、植物络合素3(PC3)、植物络合素4(PC4)、植物络合素5(PC5)和植物络合素6(PC6)浓度分别较处理0d提高了2.19、1.57、2.06、16.08、5.73、6.03和4.31倍,叶中Cys、GSH、PC2、PC3和PC4浓度分别较处理0d提高了0.69、3.21、1.64、5.73和0.27倍.根中PC3巯基比例最大,叶中GSH的巯基比例最大,二者是根、叶中巯基存在的主要形式.随着Cd2+处理时间的延长,根系和叶片中各巯基化合物的总巯基浓度显著升高,根系和叶片中植物络合素总巯基浓度与Cd浓度显著正相关.多年生黑麦草通过植物络合素等巯基化合物的快速合成降低了根叶中自由Cd2+的比例,保护了根叶中抗氧化酶的活性,间接维持了活性氧代谢的平衡.     

高等植物纤维素合酶超家族

从1996年第一个植物纤维素合酶基因的鉴定,人们对植物体内纤维素合成的研究已经走过了10年的历程。10多年中,人们取得了很大的成果,也有很多问题有待解决。该文主要介绍拟南芥和毛果杨基因组中的纤维素合酶超家族。     

环境因子对小麦体内镉的生物毒性和植物络合素合成的影响

采用水培方式,研究了不同环境因子对小麦体内Cd的生物毒性与植物络合素(PCs)合成的影响.结果表明,Cd胁迫对小麦产生明显的毒害效应,并显著诱导根合成PCs;pH、Ca和S对小麦体内Cd的吸收和生物毒性具有不同程度的影响,根中PCs的诱导量与Cd的生物毒性变化表现一致;供磷减轻了Cd胁迫的生物毒性,根中PCs的诱导量也显著降低;镁对Cd胁迫的生物毒性影响甚微,根中PCs的诱导量和Cd的吸收量均未见明显变化.本实验结果证明Cd对PCs的诱导能力与植物体内Cd的毒性之间存在一定的相关关系,可将PCs作为Cd胁迫的生物标记物.     

海藻糖合酶基因的克隆及其植物表达载体的构建

以担子菌灰树花的菌丝体中提取总ＲＮＡ,并纯化出ｍＲＮＡ,ｍＲＮＡ经反转录合成ｃＤＮＡ第一链,以ｃＤＮＡ第一链为模板经ＰＣＲ扩增海藻糖合酶（Ｔｓａｓｅ）基因,获得一长约２．２ｋｂ的片段,把该片段连接一ｐＧＥＭ－Ｔ－ｅａｓｙ ｖｅｃｔｏｒ上进行测序,其全长共２１９９ｂｐ。随后将此片段以正向插入植物表达载体ｐＢＩ１２１的ＨｉｎｄⅢ＋Ｘｂａｌ位点构建ｐＵＢ,再把海藻糖合酶基因以正向插入载体ｐＵＢ的ＢａｍＨ     

植物纤维素合酶基因研究进展

纤维素合酶催化合成的 β_1 ,4糖苷链构成植物细胞壁中含量最丰富的组份纤维素。植物体中存在着众多纤维素合酶 ,同时还具多种与之相关的纤维素合酶相似蛋白 ,它们组成了一个庞大的纤维素合酶超家族。纤维素合酶的催化机理尚不清楚 ,纤维素合酶相似蛋白的功能更有待于深入研究。本文综述了近年植物纤维素合酶及其相似蛋白编码基因的研究进展。     

植物纤维素合酶基因研究进展

纤维素合酶催化合成的β_1,4糖苷链构成植物细胞壁中含量最丰富的组份纤维素。植物体中存在着众多纤维素合酶,同时还具多种与之相关的纤维素合酶相似蛋白,它们组成了一个庞大的纤维素合酶超家族。纤维素合酶的催化机理尚不清楚,纤维素合酶相似蛋白的功能更有待于深入研究。本文综述了近年植物纤维素合酶及其相似蛋白编码基因的研究进展。     

复合胁迫下茭白体内镉、铅的亚细胞分布和植物络合素的合成

以茭白（Zizania latifoliaTurcz.）的单季茭品种‘蒋墅茭’和双季茭品种‘葑红早’为试材,进行Cd2＋、Pb2＋的单一及复合胁迫处理,测定了茭白根系和叶片中的非蛋白巯基（NPT）、谷胱甘肽（GSH）、植物络合素（PCs）的含量,同时测定了茭白植株各亚细胞组分中Cd2＋、Pb2＋的积累量,以探讨茭白对重金属镉、铅胁迫的耐性机理。结果表明：Cd2＋、Pb2＋的单一及其复合胁迫均能促进两茭白品种根系和叶片中NPT、GSH、PCs的含量及茭白各亚细胞组分中Cd2＋、Pb2＋积累量的显著增加;复合胁迫时两茭白品种的NPT、GSH、PCs含量及各亚细胞组分中Cd2＋、Pb2＋的积累量均高于单一胁迫,茭白的不同部位间,以根系中的NPT、GSH、PCs含量显著高于叶片;茭白各亚细胞组分中Cd2＋、Pb2＋的积累量表现为：细胞壁高于原生质体,而可溶性部分高于细胞器。     

植物螯合肽及其在重金属耐性中的作用

综述植物螯合肽的生物合成及其在重金属耐性中的作用．有毒重金属在土壤中的积累不仅影响作物的生长和产量形成,而且严重威胁农产品的安全性．植物对重金属的耐性和积累在种间和基因型之间存在着很大的差异,在重金属胁迫条件下植物螯合肽(PC)的合成是植物对胁迫的一种适应性反应,耐性基因型合成较多的PC谷胱苷肽是合成PC的前体,PC可与重金属螯合,并进一步转运至液泡贮存,使细胞质的重金属浓度降低,从而达到解毒效果．重金属诱导植物合成PC的遗传机理和生化途径有赖于分子生物学的深入研究,cD-敏感型拟南芥突变体Cad1-1(缺失GSH)和Cad2-1(缺失PC合成诱导酶)的分离及相关研究,佐证了PC在Cd-解毒中起关键作用．对PC在重金属污染土壤或水体的植物修复和农作物安全生产中的意义进行了讨论．     

植物螯合肽的转运与功能研究进展

植物螯合肽（phytochelatins,PCs）是由植物螯合肽合酶催化谷胱甘肽合成的一类生物小分子,结构式为（γ-Glu-Cys）n-Gly（n=2-11）,在真菌和高等植物耐受重金属胁迫机制中具有重要作用。近年来,人们在Pc介导重金属脱毒害的分子机理研究上取得了重要进展,发JLSpHMT1和SpABC2是PC在裂殖酵母中介导重金属液泡区室化的主要转运蛋白,鉴定了拟南芥液泡膜PC转运蛋AtABCC1和AtABCC2。此外,PCs也可能在超积累植物细胞内对重金属脱毒害具有重要功能。     

Phytochelatins govern zinc/copper homeostasis and cadmium detoxification in Cuscuta campestris parasitizing Daucus carota

Cuscuta sp., known with the common name of “dodder”, is an obligate parasite capable of invading stems and leaves of a wide range of host plants. Dodder stem usually coils counterclockwise around the host and, within a few days, develops haustorial structures at each point of contact. As soon as dodder haustoria reach host vascular bundles, they start tapping water, photosynthates and minerals. Metal ions such as zinc (Zn) and copper (Cu) are essential for dodder growth and metabolism, although an exceedingly high (over-homeostatic) supply of these micronutrients can result in growth inhibition and cellular toxicity. Even more so, non-essential metals such as cadmium (Cd), if transferred from the host to the parasite, need to be neutralized by timely detoxification mechanisms. In this work, we showed that Cuscuta campestris Yuncker establishes effective haustorial connections with leaf petioles and blades of Daucus carota L. (carrot), with the consequent transfer of Cd and essential metals (such as Zn and Cu) from the host vascular bundles to the parasite. Following up to this point, we detected the presence in the parasite of significant amounts of glutathione and phytochelatins, even in the absence of Cd exposure. This suggests that thiol peptides in dodder might be particularly important for Zn and Cu homeostasis as well as for Cd detoxification. Finally, we demonstrated that dodder is capable of synthesizing phytochelatins on its own, rather than massively importing them from the host, and also provided evidence for the existence of an endogenous, constitutively expressed, dodder's phytochelatin synthase.     

Phytochelatins and heavy metal tolerance

The induction and heavy metal binding properties of phytochelatins in heavy metal tolerant (Silene vulgaris) and sensitive (tomato) cell cultures, in water cultures of these plants and in Silene vulgaris grown on a medieval copper mining dump were investigated. Application of heavy metals to cell suspension cultures and whole plants of Silene vulgaris and tomato induces the formation of heavy metal–phytochelatin-complexes with Cu and Cd and the binding of Zn and Pb to lower molecular weight substances. The binding of heavy metal ions to phytochelatins seems to play only a transient role in the heavy metal detoxification, because the Cd- and Cu-complexes disappear in the roots of water cultures of Silene vulgaris between 7 and 14 days after heavy metal exposition. Free heavy metal ions were not detectable in the extracts of all investigated plants and cell cultures. Silene vulgaris plants grown under natural conditions on a mining dump synthesize low molecular weight heavy metal binding compounds only and show no complexation of heavy metal ions to phytochelatins. The induction of phytochelatins is a general answer of higher plants to heavy metal exposition, but only some of the heavy metal ions are able to form stable complexes with phytochelatins. The investigation of tolerant plants from the copper mining dump shows that phytochelatins are not responsible for the development of the heavy metal tolerant phenotypes.     

Phytochelatin synthase catalyzes key step in turnover of glutathione conjugates

Conjugation of xenobiotic compounds and endogenous metabolites to glutathione is an ubiquitous process in eukaryotes. In animals, the first and rate-limiting step of glutathione-S-conjugate metabolism is characterized by the removal of the aminoterminal glutamic acid residue of glutathione. In plants, however, glutathione-S-conjugates are generally metabolized by removal of the carboxylterminal glycine residue of the tripeptide glutathione to give rise to the S-glutamylcysteinyl-derivative. Purification of the glutathione-conjugate catabolizing activity from cell suspension cultures of the plant Silene cucubalus indicated that phytochelatin synthase catalyzes the first step of the pathway. Heterologously expressed phytochelatin synthase from Arabidopsis efficiently converted S-bima ne-glutathione to S-bimane-glutamylcysteine, the formation of which was unequivocally identified by mass spectrometry. No further products, such as S-derivatives of phytochelatins, were observed. Several different glutathione-S-conjugates served as substrates for the enzyme and were processed to the corresponding glutamylcysteinyl-adducts. Affinity-purified phytochelatin synthase preparations required divalent heavy metal ions such as Cd(2+), Zn(2+) or Cu(2+) for detectable turnover of glutathione-S-conjugates. Characterization of the enzymatic properties of phytochelatin synthase argues for both cellular functions of the gamma-glutamylcysteinyl-dipeptidyltransferase: (1) formation of heavy-metal binding peptides and (2) degradation of glutathione-S-conjugates. Mechanistically, the former role is the result of gamma-glutamylcysteinyl transpeptidation onto glutathione or derivatives thereof, while the catabolic function reflects transpeptidation of S-glutamylcysteinyl-adducts onto the acceptor molecule water. Thus, phytochelatin synthase seems to fulfil a second crucial role in glutathione metabolism.     

植物螯合肽及其在抗重金属胁迫中的作用

植物螯合肽(PCs)广泛存在于植物体中,与植物抗重金属胁迫关系密切。植物螯合肽及其复合物是一类富含半胱氨酸的低分子量化合物。现有研究证明,PCS由谷胱甘肽(GSH)为底物的酶促反应合成,其合成受相关基因的调控,从模式植物拟南芥的突变体中已分离到与PCS合成有关的几个基因。植物螯合肽首先与重金属离子结合形成低分子量(LMW)复合物,以此形态经由细胞质进入液泡后,再与一个分子的植物螯合肽结合,形成对植物组织毒性较小的高分子量(HMW)复合物,从而达到缓解重金属对植物的危害作用。就植物螯合肽及其复合物的结构、生物合成、基因调控及重金属解毒机理等进行了综述,并对今后的研究方向提出了一些看法。     

A cyanobacterial protein with similarity to phytochelatin synthases catalyzes the conversion of glutathione to gamma-glutamylcysteine and lacks phytochelatin synthase activity

Phytochelatins are glutathione-derived, non-translationally synthesized peptides essential for cadmium and arsenic detoxification in plant, fungal and nematode model systems. Recent sequencing programs have revealed the existence of phytochelatin synthase-related genes in a wide range of organisms that have not been reported yet to produce phytochelatins. Among those are several cyanobacteria. We have studied one of the encoded proteins (alr0975 from Nostoc sp. strain PCC 7120) and demonstrate here that it does not possess phytochelatin synthase activity. Instead, this protein catalyzes the conversion of glutathione to gamma-glutamylcysteine. The thiol spectrum of yeast cells expressing alr0975 shows the disappearance of glutathione and the formation of a compound that by LC-MSMS analysis was unequivocally identified as gamma-glutamylcysteine. Purified recombinant protein catalyzes the respective reaction. Unlike phytochelatin synthesis, the conversion of glutathione to gamma-glutamylcysteine is not dependent on activation by metal cations. No evidence was found for the accumulation of phytochelatins in cyanobacteria even after prolonged exposure to toxic Cd2+ concentrations. Expression of alr0975 was detected in Nostoc sp. cells with an antiserum raised against the protein. No indication for a responsiveness of expression to toxic metal exposure was found. Taken together, these data provide further evidence for possible additional functions of phytochelatin synthase-related proteins in glutathione metabolism and provide a lead as to the evolutionary history of phytochelatin synthesis.     

伴矿景天植物螯合肽合酶基因的克隆及功能分析

重金属超积累植物由于长期生长在高浓度的重金属环境中,使得经由植物螯合肽（phytochelatins, PCs）解毒途径来应对重金属毒害代价高昂。我们从Zn/Cd超积累植物伴矿景天（Sedum plumbizincicola）中克隆了植物螯合肽合酶（phytochelatin synthase, PCS）基因SepPCS。该基因在裂殖酵母和拟南芥中表达后都具有PCS活性,而且能够互补它们的PCs缺失突变体的Cd敏感表型。SepPCS在伴矿景天中的表达受到高浓度Cd处理的诱导。与其近亲非超积累生态型东南景天（S. alfredii）相比,虽然伴矿景天地上部PCs与Cd的摩尔比远低于东南景天,但是在高浓度Cd处理时PCs含量以及PCs与Cd的摩尔比急剧增加。我们推测在伴矿景天应对Cd毒害的过程中, PCs起到一定的作用,并且在高浓度Cd胁迫时地上部PCs依赖的解毒作用有所加强。     

Phytochelatin synthesis plays a similar role in shoots of the cadmium hyperaccumulator Sedum alfredii as in non‐resistant plants

Phytochelatin (PC) synthesis is considered necessary for Cd tolerance in non‐resistant plants, but roles for PCs in hyper‐accumulating species are currently unknown. In the present study, the relationship between PC synthesis and Cd accumulation was investigated in the Cd hyperaccumulator Sedum alfredii Hance. PCs were most abundant in leaves followed by stems, but hardly detected by the reversed‐phase high‐performance liquid chromatography (HPLC) in roots. Both PC synthesis and Cd accumulation were time‐dependent and a linear correlation between the two was established with about 1:15 PCs : Cd stoichiometry in leaves. PCs were found in the elution fractions, which were responsible for Cd peaks in the anion exchange chromatograph assay. About 5% of the total Cd was detected in these elution fractions as PCs were found. Most Cd was observed in the cell wall and intercellular space of leaf vascular cells. These results suggest that PCs do not detoxify Cd in roots of S. alfredii. However, like in non‐resistant plants, PCs might act as the major intracellular Cd detoxification mechanism in shoots of S. alfredii.