Phytoremediation of the herbicides atrazine and metolachlor by transgenic rice plants expressing human CYP1A1, CYP2B6, and CYP2C19

This study evaluated the expression of human cytochrome P450 genes CYP1A1, CYP2B6, and CYP2C19 in rice plants (Oryza sativa cv. Nipponbare) introduced using the plasmid pIKBACH. The transgenic rice plants (pIKBACH rice plants) became more tolerant toward various herbicides than nontransgenic Nipponbare rice plants. Rice plants expressing pIKBACH grown in soil showed tolerance to the herbicides atrazine, metolachlor, and norflurazon and to a mixture of the three herbicides. The degradation of atrazine and metolachlor by pIKBACH rice plants was evaluated to confirm the metabolic activity of the introduced P450s. Although both pIKBACH and nontransgenic Nipponbare rice plants could decrease the amounts of the herbicides in plant tissue and culture medium, pIKBACH rice plants removed greater amounts in greenhouse experiments. The ability of pIKBACH rice plants to remove atrazine and metolachlor from soil was confirmed in large-scale experiments. The metabolism of herbicides by pIKBACH rice plants was enhanced by the introduced P450 species. Assuming that public and commercial acceptance is forthcoming, pIKBACH rice plants may become useful tools for the breeding of herbicide-tolerant crops and for phytoremediation of environmental pollution by organic chemicals.     

Transgenic rice containing human CYP2B6 detoxifies various classes of herbicides

The human gene for CYP2B6, a cytochrome P450 monooxygenase that inactivates xenobiotic chemicals, was introduced into Oryza sativa cv. Nipponbare by Agrobacterium-mediated transformation. At germination, R(1) seeds of transgenic rice plants expressing CYP2B6 (CYP2B6 rice) showed a high tolerance to 5 microM metolachlor, a preemergence herbicide that is degraded by CYP2B6. Thin-layer chromatography after culture with (14)C-labeled metolachlor revealed that the amounts of residual metolachlor decreased in plant tissues and the medium of CYP2B6 rice faster than those of untransformed Nipponbare. CYP2B6 rice plants were able to grow in the presence of 13 out of 17 herbicides: five chloroacetamides and mefenacet, pyributicarb, amiprofos-methyl, trifluralin, pendimethalin, norflurazon, and chlorotoluron. These herbicides differ in their modes of action and chemical structures. Transgenic rice expressing a xenobiotic-degrading human CYP2B6, which has broad substrate specificity, should be good not only for developing herbicide tolerant rice but also for reducing the environmental impact of agrochemicals.     

Transgenic rice plants expressing human CYP1A1 remediate the triazine herbicides atrazine and simazine

The human cytochrome P450 CYP1A1 gene was introduced into rice plants (Oryza sativa cv. Nipponbare). One-month-old CYP1A1 plants grown in soil clearly showed a healthy growth and tolerance to 8.8 microM atrazine and 50 microM simazine, but nontransgenic plants were completely killed by the herbicides. Although transgenic and nontransgenic plants metabolized the two herbicides into the same sets of compounds, CYP1A1 plants metabolized atrazine and simazine more rapidly than did control plants. In small-scale experiments, residual amounts of atrazine and simazine in the culture medium of CYP1A1 plants were 43.4 and 12.3% of those in control medium; those of nontransgenic Nipponbare were 68.3 and 57.2%, respectively. When cultivated in soil with 2.95 microM atrazine and 3.15 microM simazine for 25 days, CYP1A1 plants eliminated 1.3 times more atrazine and 1.4 times more simazine from the soil than did control plants. Thus, CYP1A1 rice plants make it possible to remove atrazine and simazine more rapidly from the culture medium and soil than can nontransgenic Nipponbare.     

Protection against Trp-P-2 DNA adduct formation in C57bl6 mice by purpurin is accompanied by induction of cytochrome P450

Purpurin, an anthraquinone constituent from madder root, has previously been reported as antimutagenic in the Ames Salmonella bacterial mutagenicity assay and as antigenotoxic in Drosophila melanogaster, against a range of environmental carcinogens. Short-term dietary supplementation with purpurin inhibits the formation of hepatic DNA adducts in male C57bl6 mice after a single dose of the heterocyclic amine dietary carcinogen Trp-P-2 (30 mg/kg). Inhibition of adduct formation was dose-dependent. No DNA adducts were observed in animals treated only with purpurin. The decrease in adduct formation was accompanied by significant, dose-dependent inductions of hepatic cytochrome P450-dependent dealkylations of methoxy- (CYP1A2), ethoxy- (CYP1A1), and pentoxy- (CYP2B) resorufins, total cytochrome P450, and NADPH cytochrome P450 reductase. It is hypothesized that purpurin exhibits chemopreventive potential by inhibiting the cytochrome P450-dependent metabolism of heterocyclic amines to their genotoxic N-hydroxylamines.     

Phytoremediation of metolachlor by transgenic rice plants expressing human CYP2B6

We introduced the human cytochrome P450 gene CYP2B6 into rice plants (Oryza sativa L. cv. Nipponbare), and the CYP2B6-expressing rice plants became more tolerant to various herbicides than nontransgenic Nipponbare rice plants. In particular, CYP2B6 rice plants grown in soil showed tolerance to the chloroacetanilide herbicides alachlor and metolachlor. We evaluated the degradation of metolachlor by CYP2B6 rice plants to confirm the metabolic activity of the introduced CYP2B6. Although both CYP2B6 and nontransgenic Nipponbare rice plants could decrease the amount of metolachlor in plant tissue and culture medium, CYP2B6 rice plants could remove much greater amounts. In a greenhouse, the ability of CYP2B6 rice plants to remove metolachlor was confirmed in large-scale experiments, in which these plants appeared able to decrease residual quantities of metolachlor in water and soil.     

Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by human cytochrome P450-dependent monooxygenase systems

Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by monooxygenase systems dependent on 12 forms of human cytochrome P450 (CYP) was examined with the recombinant yeast microsomes containing each of the human CYP. The metabolites of PCDDs were analyzed by HPLC and GC-MS. Remarkable metabolism by the multiple CYP forms was observed toward dibenzo-p-dioxin (DD) and mono-, di-, and trichloroDDs. The metabolism contained multiple reactions such as hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, and hydroxylation with elimination of a chloride substituent. Although major CYPs in human liver such as CYP2C8, CYP2C9, and CYP3A4 showed no significant metabolism toward the PCDDs, CYP1A1 and CYP1A2 showed high catalytic activity toward DD and mono-, di-, and trichloroDDs. The kinetic parameters K(m)(app) and V(max) of the CYP1A1-dependent 8-hydroxylation activity of 2,3,7-trichloro-DD (2,3,7-triCDD) were estimated to be 0.30 microM and 51 (mol/min/mol of P450), respectively, suggesting that 2,3,7-triCDD was a good substrate for CYP1A1. However, none of the CYPs showed any detectable activity [<0.01 mol/min/mol of P450)] toward 2,3,7,8-tetraCDD. Substrate-induced absorption spectrum and inhibition studies indicated that CYP1A1 could bind 2,3,7,8-tetraCDD with considerably high affinity. It was strongly suggested that the long half-life (7.1 years) of 2,3,7,8-tetraCDD in humans was due to an extremely low activity of CYPs toward 2,3,7,8-tetraCDD in addition to its chemical stability.     

印度芥菜(Brassica juncea L.)重金属耐性机理研究进展

印度芥菜可富集/忍耐Cd、Zn 等多种重金属, 是研究植物修复技术的一种模式植物。高浓度的重金属离子会改变植物的基因表达、细胞形态、细胞结构, 最终使植物生长受抑, 甚至死亡。印度芥菜高效的抗氧化系统、损伤修复系统以及对重金属的螯合、区域化可部分解除重金属的毒性, 缓解重金属离子的毒害作用。利用基因工程技术在印度芥菜中导入重金属耐性及运输相关基因可大幅度提高其重金属富集能力, 在重金属污染修复方面具有广阔的应用前景。     

Comparison in metabolic activity of cytochrome P450 1A1 on heterocyclic amines between human and rat

In mutagenicity and antimutagenicity tests, the toxicants have been activated to the ultimate mutagenic forms usually with rat cytochrome P450 (CYP) enzymes. An understanding is important of whether these data can be available for human. In this paper are compared the activating abilities of CYP1A1 between human and rat using recombinant yeast cells that express respective CYP1A1 and yeast NADPH-CYP-oxidoreductase simultaneously. Three different types of dietary procarcinogens, heterocyclic amines, were tested by two methods: a bioassay with Salmonella mutagenicity test and a chemical determination of N-hydroxyls as the ultimate mutagenic forms. Compared with ED(50) values, saturation levels, and V(max)/K(m) values at an initial stage of the enzyme activity, human and rat CYP1A1 showed almost similar abilities for the metabolic activation on heterocyclic amines. The two enzymes also had the same preference for the tested procarcinogens and the same affinities to the specific inhibitors such as flavonoids.     

Modulation of rat hepatic and pulmonary cytochromes P450 and phase II enzyme systems by erucin, an isothiocyanate structurally related to sulforaphane

Administration of dietary doses of the isothiocyanate erucin had no effect on rat hepatic cytochrome P450 activity or protein levels, but at higher doses a rise in CYP1A/B1 protein levels was evident. In lung, treatment with erucin, as well as sulforaphane, failed to modulate cytochrome P450 activities but elevated CYP1A/B1 protein levels. In liver, erucin stimulated quinone reductase activity accompanied by a rise in protein. Glutathione S-transferase activity was unaffected, but GSTalpha and GSTmu protein levels increased. In lung, both isothiocyanates increased quinone reductase paralleled by a rise in protein levels; at the higher dose both isothiocyanates elevated moderately GSTalpha levels. Hepatic microsomes converted both isothiocyanates to metabolites that impaired cytochrome P450 activity, which was antagonized by reduced glutathione. It may be concluded that erucin may protect against carcinogens by stimulating the detoxication of quinones but is unlikely to significantly influence reactive intermediate generation through modulation of cytochrome P450 activity.     

Regulation of phenobarbital-mediated induction of CYP102 (cytochrome P450(BM-3)) in Bacillus megaterium by phytochemicals from soy and green tea

Cytochrome P450 102 (CYP102 or Cytochrome P450(BM)(-)(3)) is induced in Bacillus megaterium by barbiturates, perioxisome proliferators, estrogen, and nonsteroidal antiinflammatory drugs. We have previously demonstrated that a CYP102 construct (BMC 143) coupled with a luciferase reporter gene can be used to identify the inducers of CYP102. We now describe the effect of added phytochemicals on the induction of CYP102 by phenobarbital (PB) in B. megaterium. The isoflavones genistein, biochanin A, coumestrol, and equol, the green tea flavanoid epicatechin, and the fungal toxin zearalenone inhibit the induction of CYP102 by PB in a dose-dependent manner. However, the isoflavone daidzein, the phytoalexin glyceollin, and catechin, an epimer of epicatechin, failed to exhibit a similar inhibitory effect on PB-mediated CYP102 induction.     

Expression Pattern of Ａ Recombinant Phloem-specific Promoter from Pumpkin in Transgenic Potato Plants

以本实验室构建的重组南瓜(Cucurbita moschata)韧皮部特异启动子dENP构建了植物表达载体pBdENP。利用根癌农杆菌(Agrobacterium tumefaciens)LBA44O4介导转化马铃薯(Solanum tuberosum)品种Favorita，经过抗生素筛选，共获得转pBdENP和对照pBI121的抗卡那霉素马铃薯再生植株106株。通过PCR初步筛查，筛选出65株为转基因阳性植株。通过Southern blot对部分植株进一步分析,确证外源gus基因已经插入到转基因马铃薯植株的基因组中，插入拷贝数在1个或2个以上。对这些转基因马铃薯植株进行GUS染色结果表明, dENP和CaMV35S启动子一样均能驱动gus基因的表达，前者仅在马铃薯的韧皮部内特异表达，而CaMV35S启动子驱动的gus基因为组成型性表达。GUS酶活力测定结果进一步表明dENP和CaMV35S启动子驱动gus基因表达水平没有明显区别。以上结果证明dENP启动子驱动的外源基因在马铃薯中也具有韧皮部特异而高效表达的特征，从而可用于马铃薯抗病、抗蚜虫转基因研究。     

高羊茅细胞色素FaP450基因克隆及其在非生物胁迫下表达分析

细胞色素P450属于一种含亚铁血红素单加氧酶的超基因家族,在植物多种代谢途径与防御反应中起着重要作用。为探究细胞色素P450基因在高羊茅中的表达模式,本研究采用cDNA末端快速扩增技术从高羊茅叶片中克隆了细胞色素P450基因,命名为FaP450。FaP450全长1 737 bp,含1 437 bp的开放阅读框,共编码478个氨基酸,具有P450基因家族典型的P450结构域。系统进化树分析表明,高羊茅FaP450与禾本科植物小麦、山羊草的P450蛋白亲缘关系较近。荧光定量PCR表明,高羊茅FaP450基因受干旱、高温、氮胁迫及盐胁迫的诱导表达上调,表明该基因与非生物胁迫的抗性相关。构建FaP450超量表达载体,利用农杆菌侵染法转化拟南芥,对其进行低氮胁迫,发现低氮胁迫下FaP450基因过量表达植株的鲜重与根长显著高于野生型,表明FaP450基因的超量表达可以增强拟南芥对低氮的适应性。本研究为深入探索P450基因家族在牧草生长发育与抗逆功能的研究奠定了理论基础。     

过量硼对植物的毒害及高硼土壤植物修复研究进展

硼作为一种植物必需元素,在土壤中过量存在会对植物产生毒害,硼对植物的毒害作用以及利用植物修复高硼土壤已经日益受到关注。目前,硼对不同类型植物的毒害特点,植物的耐受机制还不十分清楚。特别是对于硼污染的植物修复,其研究还处于起步阶段。本文分别从植物的表观症状、生理生化和基因水平等层次,综述了过量硼对植物的毒害,并从高耐受性、超富集能力植物筛选,以及转基因技术应用等角度回顾了硼污染的植物修复研究进展。在此基础上,提出了当前相关研究存在的主要问题,并对未来的研究进行了展望。     

The Function of a Maize-Derived Phosphoenol pyruvate Carboxylase (PEPC) in Phosphorus-Deficient Transgenic Rice

Transgenic rice ( Oryza sativa L., a C₃ plant) lines carrying a complete phospho enol pyruvate carboxylase (PEPC) gene from maize (a C₄ plant) were tested for their performance in terms of organic acid synthesis and organic acid exudation into the rhizosphere under phosphorus (P)-deficient conditions. High PEPC activity increased the fraction of photosynthetically fixed carbon allocated to the organic acid pool, and P deficiency enhanced oxalate exudation from the roots of the transgenic plants. There was no evidence that the transformed PEPC was involved in internal P recycling in the plant. However, the root PEPC activity was positively correlated with the oxalate exudation and negatively correlated with the root P concentration, and a higher root PEPC activity led to a higher oxalate exudation. Thus, it is suggested that C₄-PEPC transgenic rice plants had acquired the ability to exude oxalate, which enhanced their capacity to adapt to low P soil conditions.     

与甜瓜性别分化相关植物激素生物合成途径分析

甜瓜性别分化是由多因素构成的一个复杂的网络系统调控,受到多种环境因素及激素水平的影响。本研究利用甜瓜(Cucumis melo L.)WI 998×TopMark 构建重组自交系群体,通过第二代高通量测序技术对F2S6群体中雌雄异花同株及全雌系植株的转录组测序,比较差异表达基因,分析与性别差异表达相关的植物激素合成途径。结果显示,雌雄异花同株获得77376 条 Unigenes,平均长度为 435 bp;全雌系转录组测序获得 80 825 条 unigenes,平均长度为 509 bp。比较雌雄异花同株与全雌系转录组,发现共有 8966个基因差异表达,4 296 个基因下调,4 670 个基因上调。对差异基因进行基因本体论(GO)功能分类,发现2 352 条 Unigenes 归入到生物学过程,4 107 条 Unigenes 归入到细胞学过程,2 507 条 Unigenes 归入到分子功能。差异表达基因共参与 121 个 Pathway,发现赤霉素(gibberellin, GA)合成途径中 GA3 - 氧合成酶(GA3-ox)(MU3674)、GA7- 氧化酶(GA7-ox)(MU36987)、GA2- 氧化酶(GA2-ox)(MU13098/MU13099)、GA20-氧化酶(GA20-ox)和 GA2- 氧化酶(GA2-ox)(MU33020)等基因差异表达;共有 38 个基因在脱落酸(abscisicacid, ABA)合成途径中差异表达,其中 19 个上调,19 个下调;油菜素内酯(brassinolide, BR)合成途径中,MU22012 (cytochrome P450),MU26893 (cytochrome P450) 基因上调 ,MU56098 (cytochrome P450,CYP724B3),MU76596(CYP724A1)下调。本研究还发现参与甜瓜性别表达,分别与赤霉素、脱落酸、油菜素内酯及玉米素等多种激素合成、信号传导等代谢途径相关。研究结果为分析甜瓜决定性别分化的可能机理,为下一步研究甜瓜性别修饰基因的克隆,提供重要依据。     

植物修复金属污染土壤上丛枝菌根真菌与植物之间的交互作用

Metal contamination in the environment is a global concern due to its high toxicity to living organisms and its worldwide distribution.The principal goal of this review is to examine the current strategies and technologies for the remediation of metalcontaminated soils by metal-accumulating plants and assess the roles of arbuscular mycorrhizal(AM)fungi in remediation of soils under hyperaccumulator or non-accumulator plants.The use of plants to remove metals from the environment or reduce the toxicity,known as phytoremediation,is an environmentally sustainable and low cost remediation technology.The mechanisms of the use of hyperaccumulator plants for phytoremediation included solubilization of the metal in the soil matrix,the plant uptake of the metal,detoxification/chelation and sequestration,and volatilization.Recently,some ecologists have found that phytoremediation with the aids of mycorrhizae can enhance efciency in the removal of toxic metals.AM fungi can facilitate the survival of their host plants growing on metal-contaminated land by enhancing their nutrient acquisition,protecting them from the metal toxicity,absorbing metals,and also enhancing phytostabilization and phytoextraction.Such information may be useful for developing phytoremediation program at metal-contaminated sites.     

Problems and prospects concerning the phytoremediation of heavy metal polluted soils: A review

The current state, problems, and prospects of phymoremediation of heavy metal polluted soils are analyzed. The main attention is paid to the phytoextraction and phytostabilization as the most widespread and alternative methods of soil phytoremediation. The efficiency of phymoremediation is related to the natural capability of plants for the accumulation and translocation of metals, their tolerance to a high content of metals, the plant biomass, and the amendments applied. The advantages and disadvantages of phytoremediation as compared to other methods of remediation of polluted soils in situ are considered. Examples of successful phytoextraction and phytomining for cleaning up of contaminated soils in Rasteburg (South Africa) and the phytostabilization of technogenic barrens nearby the copper-nickel plants in Sudbury (Ontario, Canada) and in the Kola Subarctic (Russia) are presented.