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。     

水稻镉积累特性的生理和分子机制研究概述

我国的稻米镉超标对国民身体健康造成严重威胁, 而选育低镉积累的水稻(Oryza sativa)品种是降低稻米镉含量行之有效的策略, 因此有必要了解水稻对镉的积累特性及其生理过程和相关功能基因。该文概述了镉在水稻根部的吸收、木质部中的装载与运输、茎节中的分配、叶片中再分配以及籽粒镉积累等过程的生理和分子机制研究进展, 以期为低镉水稻的选育和安全生产提供理论参考。     

水稻籽粒镉积累QTL定位及候选基因分析

水稻(Oryza sativa)是全世界重要的经济作物之一, 稻田镉(Cd)污染和镉积累问题严重威胁世界水稻的产量和品质以及人类健康, 如何降低水稻中镉积累已成为热点问题。以籼稻品种华占(HZ)为父本、粳稻品种热研2号(Nekken2)为母本, 连续自交多代后得到120个重组自交系群体, 对其镉积累进行检测和分析, 同时利用遗传图谱进行QTL作图。结果共检测到7个QTLs, 分别位于水稻第2、3、9和12号染色体上, 其中1个LOD值高达4.97。对这些QTL区间内与耐金属离子胁迫相关的候选基因进行定量分析, 发现LOC_Os02g50240、LOC_Os02g52780、LOC_Os09g31200、LOC_Os09g35030和LOC_Os09g37949这5个基因在双亲间的表达量差异显著, 结合亲本对不同金属离子的浓度积累数据, 推测LOC_ Os02g50240、LOC_Os09g31200及LOC_Os09g35030的高表达可能极大地提高了水稻对镉离子的吸收和胁迫耐受能力。通过QTL挖掘和分析, 发现这些基因与水稻籽粒的镉积累有关, 可能影响水稻耐镉胁迫的能力。研究结果为进一步筛选和培育耐镉胁迫的水稻品种创造了条件, 为阐明水稻镉积累的分子调控机制奠定了基础。     

小立碗藓对重金属镉胁迫的应答特征

镉是植物非必需的微量重金属元素, 镉胁迫引起植物细胞的代谢紊乱, 甚至导致细胞死亡。为了探索苔藓植物对镉胁迫的应答机制, 采用高通量测序及生物信息学技术分析了藓类模式植物——小立碗藓(Physcomitrella patens)在镉胁迫下的基因表达特征。结果表明, 在镉胁迫下, 小立碗藓细胞骨架组织、微管运动、DNA修复系统、端粒维护、配子体形成与有性生殖以及与氮代谢等相关基因的表达具有明显的镉胁迫应答特征, 暗示了这些基因可能共同参与小立碗藓对镉胁迫的调控反应。该研究结果为阐明植物对镉胁迫的应答机制提供了新的线索。     

植物超积累重金属的生理机制研究进展

土壤重金属污染已经成为一个全球性问题。重金属超积累植物在修复土壤重金属污染中具有重要的应用前景。重金属超积累植物通常具备三个基本特征,即：根系具有从土壤中吸收重金属的强大能力、能从根到地上部分高效转运重金属、在叶片中能解毒和隔离大量重金属。本文总结了重金属超积累植物吸收、转运、隔离和解毒重金属的生理机制研究进展,以期为进一步阐明植物超积累重金属的机制及其在植物修复中的应用提供参考。     

一个新的OsBRI1弱等位突变体的鉴定及其调控种子大小的功能研究

水稻(Oryza sativa)籽粒大小是影响其产量的关键农艺性状, 克隆并研究水稻籽粒大小相关基因对于提高水稻产量具有重要意义。为深入探究水稻籽粒大小的调控机制, 通过EMS诱变品种宽叶粳(KYJ), 分离了一系列水稻籽粒大小改变的突变体, 其中smg12表现为籽粒变小, 株高变矮, 一级枝梗数和二级枝梗数减少。遗传分析表明, 该小粒突变体受隐性单基因控制。细胞学分析显示, 该突变体颖壳纵向细胞长度显著变短, 表明SMG12主要影响细胞扩展。利用Mutmap方法对候选基因进行克隆, 筛选出SMG12的候选基因OsBRI1, 该基因编码油菜素内酯受体激酶。OsBRI1外显子上的第2 074个碱基发生了由C到T的置换, 产生非同义突变, 使得该位置编码的脯氨酸变为丝氨酸, 从而影响OsBRI1的功能。综上, 该研究鉴定了OsBRI1基因的1个新等位变异, 揭示了油菜素内酯途径调控水稻籽粒大小的细胞和分子基础。     

“先驱”转录因子LEC1在早期胚胎重置春化状态的机制

开花是植物由营养生长阶段向生殖生长阶段转变的重要过程, 长时间低温处理即春化对开花起到非常重要的促进作用。春化控制的拟南芥(Arabidopsis thaliana)开花中, 阻抑型转录因子FLC是重要的关节点, 春化记忆依赖于对该基因的控制。何跃辉研究组之前对拟南芥的研究揭示了转录因子VAL1或VAL2可以识别负调控开花的关键基因FLC成核区的顺式DNA元件, 协同PRC2复合体在春化过程中沉默FLC基因的表达, 并在随后的常温下继续维持FLC基因沉默直至受精结束, 使植物产生春化记忆。但在下一代中如何擦除这种记忆功能, 使FLC重新被激活, 以防止植物在过冬前或过冬时开花, 相关机制目前并不清楚。近期, 该研究组揭示了在植物胚胎发育早期一个种子特有的“先驱”转录因子参与擦除春化记忆, 重新激活FLC基因的分子机制, 并解析了胚胎中的基因激活传递到后胚胎发育(营养生长期)的表观遗传机理。该研究是开花领域的重要突破, 为作物开花调控的生产应用提供了新思路。     

不同生长调节物质对水稻生长及镉积累的影响

比较脱落酸(ABA)、乙烯利(ETH)、水杨酸(SA)和茉莉酸甲酯(MeJ A)4种植物生长调节物质(PGR)对水稻生长及籽粒镉(Cd)积累的影响差异。试验采用重金属污染土种植水稻,于分蘖期、灌浆期各进行1次PGR叶面喷施处理,分析灌浆期叶片光合指标,丙二醛(MDA)含量以及收获期各部位生物量和Cd含量。结果表明:(1)低浓度ABA(5mg/L)可维持水稻正常产量;高浓度ABA(15mg/L)则导致产量下降。ETH对水稻地上部生长和单株产量有显著抑制作用,SA和MeJ A(0.56mg/L)均可保证地上部正常生长,维持正常产量。(2)外施4种PGR均抑制灌浆期叶片气孔开放,降低蒸腾速率和光合速率,抑制效果最明显的是高浓度MeJ A(0.56mg/L)。(3)在供试浓度范围内SA、低浓度ABA(5mg/L)以及高浓度MeJ A均可降低灌浆期叶片MDA含量,减少质膜过氧化水平。(4)4种PGR均可降低水稻籽粒Cd含量,其中低浓度ABA(5mg/L)抑制籽粒Cd积累的效应最佳。相关性分析结果表明,PGR抑制籽粒积累Cd的效应与地上部向籽粒转运Cd的调控机制有关,与蒸腾速率无显著相关关系。(5)综上所述,低浓度ABA(5mg/L)处理对水稻产量无影响,且籽粒Cd含量降低程度最大。适当浓度的PGR可降低水稻籽粒Cd含量,在中低度重金属污染农田生态修复实践中具有一定的应用前景,但必须精确控制PGR的处理时间和处理浓度,避免出现抑制生长和降低产量的负效应。     

重金属污染土壤植物修复中细胞分裂素的作用与机制

植物修复因投资成本低、环境扰动少、二次污染易控制、美化环境等优点成为重金属污染土壤修复重要的治理技术。植物内源细胞分裂素调控植物生理活动,外源细胞分裂素对植物生理生态特征产生显著影响,且在植物修复中逐渐受到研究人员的关注。细胞分裂素能够调控植物根茎发育、叶片衰老、激素传递等过程,同时在重金属胁迫下也参与蒸腾、光合、抗性、解毒等系统的运转。以细胞分裂素对植物生理活动的调控作用研究为基础,阐述了细胞分裂素在植物修复中的作用机制。主要包括：增强光合作用,延缓叶片衰老,提升植物抗性能力;调控根茎叶发育,增加植物生物量,强化植物富集效果;增强转运蛋白表达,提高叶面蒸腾作用,促进重金属吸收转运;参与解毒过程,降低重金属毒性,调控重金属体内转化。最后提出了细胞分裂素在重金属污染土壤植物修复中的研究方向,这对促进细胞分裂素在植物修复中的实际应用具有重要意义。     

梯棱羊肚菌镉胁迫响应基因ATX1功能分析

梯棱羊肚菌Morchella importuna是一种可以大田覆土栽培的珍稀食用菌,而土壤重金属污染状况日益严重,对梯棱羊肚菌菌丝生长和子实体产品质量安全构成了潜在的威胁。本研究先采用镉离子胁迫处理梯棱羊肚菌菌丝体,RT-PCR检测发现候选基因ATX1的表达量显著下调。克隆梯棱羊肚菌ATX1基因,对ATX1p蛋白结构进行功能预测,发现ATX1p可能与铜离子转运及重金属胁迫相关。然后分别构建ATX1的超表达和RNAi基因沉默载体,采用农杆菌介导的转化方法,将其转入梯棱羊肚菌同核体菌株A50中,分别筛选到4个ATX1表达显著上调的超表达转化子和4个ATX1表达显著下调的RNAi基因沉默转化子,镉敏感性检测发现ATX1的RNAi基因沉默转化子表现为镉抗性增强,而ATX1超表达转化子则表现为镉抗性减弱。结果表明,梯棱羊肚菌ATX1基因表达与镉抗性呈负相关,ATX1p可能在梯棱羊肚菌镉胁迫响应过程中发挥着某种重要作用。     

不同类型土壤小麦-水稻轮作体系施用含重金属污泥的环境效应

采用贵州黄壤、石灰土和浙江水稻土,通过盆栽试验探讨了在3种土壤上施用含不同浓度重金属的污泥对小麦、水稻生长及锌(Zn)镉(Cd)吸收性的影响.结果表明： 不同土壤施用同种污泥所产生的重金属污染风险不同,在黄壤和水稻土上施用高浓度重金属污泥对作物的污染风险较高.一次施用Zn、Cd浓度分别为1789、8.47 mg·kg-1的污泥1.6%,使黄壤小麦籽粒中Zn、Cd浓度分别达109、0.08 mg·kg-1;第二次施用后种植水稻,糙米中Zn、Cd浓度达52.0、0.54 mg·kg-1.而施用污泥后石灰性土壤的两种作物其可食部分均无重金属污染风险.土壤醋酸铵提取态Zn是影响麦粒和糙米中Zn浓度的主要因素,而土壤醋酸铵提取态Cd对麦粒和糙米中Cd浓度无明显影响.施用高浓度重金属污泥使3种土壤Zn、Cd全量显著提高,且两次施用后土壤全量Zn均超过国家土壤环境质量二级标准.     

Natural alleles of a uridine 5ʹ-diphospho-glucosyltransferase gene responsible for differential endosperm development between upland rice and paddy rice

Traditional upland rice generally exhibits insufficient grains resulting from abnormal endosperm development compared to paddy rice. However, the underlying molecular mechanism of this trait is poorly understood. Here, we cloned the uridine 5ʹ-diphospho (UDP)-glucosyltransferase gene EDR1 (Endosperm Development in Rice) responsible for differential endosperm development between upland rice and paddy rice by performing quantitative trait loci analysis and map-based cloning. EDR1 was highly expressed in developing seeds during grain filling. Natural variations in EDR1 significantly reduced the UDP-glucosyltransferase activity of EDR1^YZN compared to EDR1^YD1, resulting in abnormal endosperm development in the near-isogenic line, accompanied by insufficient grains and changes in grain quality. By analyzing the distribution of the two alleles EDR1^YD1 and EDR1^YZN among diverse paddy rice and upland rice varieties, we discovered that EDR1 was conserved in upland rice, but segregated in paddy rice. Further analyses of grain chalkiness in the alleles of EDR1^YD1 and EDR1^YZN varieties indicated that rice varieties harboring EDR1^YZN and EDR1^YD1 preferentially showed high chalkiness, and low chalkiness, respectively. Taken together, these results suggest that the UDP-glucosyltransferase gene EDR1 is an important determinant controlling differential endosperm development between upland rice and paddy rice.     

湘北丘陵林-稻系统镉的迁移

为了研究中南丘陵区林-稻系统镉的迁移规律,2012年5月下旬至9月上旬(中稻稻季)在湘北汩罗市桃林林场选择两种类型的林-稻复合生态系统针对降水、地表径流、山塘水、稻田水及其中镉含量用收支平衡法进行了原位动态研究。结果表明,(1)以降水形式从区域外输入的镉是湘北地域地表系统主要的外来镉源,镉的输入主要集中在稻季的早期;(2)镉以雨水形式输入丘陵林-稻系统后,53%被林地截留,7.5%累积于山塘,11.6%存留于稻田,其余28.9%以稻田水和稻草、谷粒的形式输出林-稻系统;(3)不同林-稻系统比较,混-稻系统对外源镉的固持功能最强,松-稻系统其次,对照系统最弱,其原因在于各系统间岗地森林郁闭度存在差异,郁闭度大能增强岗地对镉的固持能力;(4)在岗地截留镉能力较弱的情况下,山塘能够减缓系统镉的迁移强度;(5)岗地输入山塘水中的大分子有机物能加强镉在稻田中累积。综上所述,林-稻系统镉的累积与雨水常年输入镉有关,系统内岗地森林郁闭度、与有机物输出相关的森林类型影响镉在系统中的迁移和分配。     

海南稻田土壤硒与重金属的含量、分布及其安全性

采集了海南省18个市(县)代表性的稻田土壤耕作层(0—20cm)样品280个,研究了硒(Se)和5种有毒重金属元素(Hg、Cd、Cr、Pb和As)的含量、分布及其相关关系,并对Se和重金属的安全性进行评价,可为合理区划清洁且富Se稻田提供理论依据。结果表明:海南稻田土壤中Hg、Cd、Cr、Pb和As平均含量均低于国家土壤环境质量一级标准值和全国土壤背景值,以绿色食品产地环境技术条件限量标准为标准,用单项污染指数法和内梅罗综合污染指数法评价海南稻田土壤重金属的污染状况,结果都是清洁的。但以海南省土壤背景值做参比值,Hakanson潜在生态危害指数达到211.54,属于强生态危害,从潜在生态危害系数来看,Hg(102.61)和Cd(98.89)达到强生态危害,分别比海南省土壤背景值增加1.56和2.3倍,今后应注意控制Hg和Cd污染源。稻田土壤Se含量从痕量到1.532mg/kg之间,平均值为0.211mg/kg,占47.5%的稻田土壤Se含量处于中等及以上水平(>0.175mg/kg)。Se含量高的稻田土壤主要集中在东北部的海口及其周边的澄迈、定安、文昌和琼海,还有东南部的万宁和保亭。由于重金属平均含量还比较低,可暂时忽略重金属污染,故可在上述Se含量高的稻田土壤上种植富Se水稻。稻田土壤Se含量与Hg、Cd和As含量呈极显著或显著正相关,因此今后应加强研究稻田土壤Se与Hg、Cd和As的有效性及其相互作用,以便生产出绿色的富Se优质大米。     

Phytochelatin and cadmium accumulation in wheat

Cadmium (Cd) is a nonessential heavy metal that can be harmful at low concentrations in organisms. Therefore, it is necessary to decrease Cd accumulation in the grains of wheats aimed for human consumption. In response to Cd, higher plants synthesize sulphur-rich peptides, phytochelatins (PCs). PC–heavy metal complexes have been reported to accumulate in the vacuole. Retention of Cd in the root cell vacuoles might influence the symplastic radial Cd transport to the xylem and further transport to the shoot, resulting in genotypic differences in grain Cd accumulation. We have studied PC accumulation in 12-day-old seedlings of two cultivars of spring bread wheat (Triticum aestivum), and two spring durum wheat cultivars (Triticum turgidum var. durum) with different degrees of Cd accumulation in the grains. Shoots and roots were analysed for dry weight, Cd and PC accumulation. There were no significant differences between the species or the varieties in the growth response to Cd, nor in the distributions of PC chain lengths or PC isoforms. At 1 μM external Cd, durum wheat had a higher total Cd uptake than bread wheat, however, the shoot-to-root Cd concentration ratio was higher in bread wheat. When comparing varieties within a species, the high grain Cd accumulators exhibited lower rates of root Cd accumulation, shoot Cd accumulation, and root PC accumulation, but higher shoot-to-root Cd concentration ratios. Intraspecific variation in grain Cd accumulation is apparently not only explained by differential Cd accumulation as such, but rather by a differential plant-internal Cd allocation pattern. However, the higher average grain Cd accumulation in the durum wheats, as compared to the bread wheats, is associated with a higher total Cd accumulation in the plant, rather than with differential plant-internal Cd allocation. The root-internal PC chain length distributions and PC–thiol-to-Cd molar ratios did not significantly differ between species or varieties, suggesting that differential grain Cd accumulation is not due to differential PC-based Cd sequestration in the roots.     

Expression in Arabidopsis and cellular localization reveal involvement of rice NRAMP,OsNRAMP1, in arsenic transport and tolerance

Irrigation of paddy fields to arsenic (As) containing groundwater leads to As accumulation in rice grains and causes serious health risk to the people worldwide. To reduce As intake via consumption of contaminated rice grain, identification of the mechanisms for As accumulation and detoxification in rice is a prerequisite. Herein, we report involvement of a member of rice NRAMP (Natural Resistance‐Associated Macrophage Protein) transporter, OsNRAMP1, in As, in addition to cadmium (Cd), accumulation through expression in yeast and Arabidopsis. Expression of OsNRAMP1 in yeast mutant (fet3fet4) rescued iron (Fe) uptake and exhibited enhanced accumulation of As and Cd. Expression of OsNRAMP1 in Arabidopsis provided tolerance with enhanced As and Cd accumulation in root and shoot. Cellular localization revealed that OsNRAMP1 resides on plasma membrane of endodermis and pericycle cells and may assist in xylem loading for root to shoot mobilization. This is the first report demonstrating role of NRAMP in xylem mediated loading and enhanced accumulation of As and Cd in plants. We propose that genetic modification of OsNRAMP1 in rice might be helpful in developing rice with low As and Cd content in grain and minimize the risk of food chain contamination to these toxic metals.     

Bioavailability of Cd in a soil–rice system in China: soil type versus genotype effects

Human exposure to toxic heavy metals via dietary intake is of increasing concern. In this regard, the bioavailability of heavy metals in the soil–crop system is considered to be a key factor controlling plant uptake and, therefore, public health risk through food chain transfer [J. Environ. Sci. Health B 34(4) (1999) 681]. In 2002, a pot experiment was conducted to elucidate the relative significance of soil types and rice genotype on the bioavailability, uptake and partitioning of Cd by two rice cultivars with distinct affinity for Cd. The results indicated that the total uptake of Cd and accumulation in grain was dependent on both soil type and genotype effects. Cd spiking enhanced high Cd uptake and partitioning in grain. Inherent differences in soil type effecting Cd bioavailability were less significant under the Cd spiking regime as compared to non-Cd spiking. In the case of Soil-P, with low Cd bioavailability as indicated by the comparatively lower MgCl₂ extractableCd, differences in metal affinity between genotypes dominated uptake. Conversely, inherent differences in soil type affecting Cd bioavailability dominated uptake in the low metal affinity cultivar treatments. Under the experimental conditions evaluated, the positive interaction between soil type and genotype results in elevated levels of Cd in rice grain with the Cd values exceeding the Chinese food guideline limit of 0.2 mg kg^–1. The results indicated that Cd bioavailability and plant uptake is dependent on soil chemical and physical properties affecting Cd mobility, rice genotype and soil pollution status. The results further suggested that caution should be paid to rice production with the new high metal affinity genotypes on soils with inherent Cd bioavailability as with acidic Red Soils of Jiangxi Provinces, China.     

Evidence for separate translocation pathways in determining cadmium accumulation in grain and aerial plant parts in rice

Background  

Cadmium (Cd) translocation and accumulation in the grain and aerial plant parts of rice (Oryza sativa L.) is an important aspect of food safety and phytoextraction in areas with contaminated soil. Because control of Cd translocation and accumulation is likely to be determined by the plants genetics, the Cd contents of grain and the aerial parts of rice may be manipulated to improve food safety and for phytoextraction ability. This study studied Cd translocation and accumulation and their genetic control in aerial parts of rice to provide a starting point for improving food safety and phytoextraction in Cd-contaminated soils.     

Mutations in rice (Oryza sativa) heavy metal ATPase 2 (OsHMA2) restrict the translocation of zinc and cadmium

Widespread soil contamination with heavy metals has fostered the need for plant breeders to develop new crops that do not accumulate heavy metals. Metal-transporting transmembrane proteins that transport heavy metals across the plant plasma membrane are key targets for developing these new crops. Oryza sativa heavy metal ATPase 3 (OsHMA3) is known to be a useful gene for limiting cadmium (Cd) accumulation in rice. OsHMA2 is a close homolog of OsHMA3, but the function of OsHMA2 is unknown. To gain insight into the function of OsHMA2, we analyzed three Tos17 insertion mutants. The translocation ratios of zinc (Zn) and Cd were clearly lower in all mutants than in the wild type, suggesting that OsHMA2 is a major transporter of Zn and Cd from roots to shoots. By comparing each allele in the OsHMA2 protein structure and measuring the Cd translocation ratio, we identified the C-terminal region as essential for Cd translocation into shoots. Two alleles were identified as good material for breeding rice that does not contain Cd in the grain but does contain some Zn, and that grows normally.