硝酸盐对硝酸还原酶活性的诱导及硝酸还原酶基因的克隆

硝酸盐在植物体内的积累过多已成为影响蔬菜品质并影响人类健康的重要因素。硝酸还原酶（NR）是硝酸盐代谢中的关键酶,提高其活性有利于硝酸盐的降解。为了解植物不同组织中NR的活性,用活体测定法检测了经50mmol/L的KNO₃诱导不同时间后的油菜、豌豆和番茄幼苗根茎叶中NR活性,同时为了明确外源诱导剂浓度与植物体内NR活性的关系,检测了经不同浓度KNO₃诱导2h后的矮脚黄、抗热605、小白菜和番茄叶片中的NRA。结果表明,不同植物组织NR活性有很大差异,叶中NR活性较高,根其次,茎最低;不同植物的NR活性随诱导时间呈不同的变化趋势,相同植物不同组织的NR活性变化趋势相似;不同植物叶片NRA为最高时KNO₃浓度不同。用30mmol/L的KNO3诱导番茄苗2h后,从番茄根和叶中提取总RNA,用RT-PCR方法获得NR cDNA,全长2736bp,编码911个氨基酸。为进一步利用该基因提高植物对硝酸盐的降解能力打下基础。     

植物硝酸还原酶的研究进展

一、硝酸还原酶的特性 硝酸还原酶(NR )是植物中显著的诱导酶之一,易被诱变得到突变体。酶结构复杂,田多个亚基组成;含有不同组分酶,其活性易受体内外因素影响,为氮素代谢的关键酶,影响农作物的总氮和蛋白氮水平,与作物的耐肥性有密切关系。该酶同时具有功能多样性的特点,在植物的其他代谢过程中,如能量代谢、铁离子同化与运转、水分胁迫、光呼吸、氯离子还原、分子O_2的分解释放等也有重要作用。NR在植物基因表达、蛋白质分子基础研究和植物代谢途径及调控研究中占有十分重要的地位。 1952年埃文斯(H.Evans)和纳松(A.Na-son)在红色链孢霉中最早发现NR,次年又在高等植物中发现,此后发现NR广泛分布于细     

磺胺比色法测定植物组织硝酸还原酶活性的改进

“植物组织硝酸还原酶（NR）活性的测定”是植物生理学矿质营养一章中必做的实验,其目的是让学生掌握植物硝酸还原酶（nitrate reductase,NR,EC．1．6．6．1）活性测定的原理和技术方法,进一步了解NR在植物氮素同化过程中的作用（白宝璋和汤学军1993;李合生等2001;郝建军等2007）。但是,在实验教学中,我们感到,按照几种植物生理学实验指导书中介绍的方法（白宝璋和汤学军1993;李合生等2001;     

硝酸还原酶的研究——Ⅲ.硝酸还原酶钝化蛋白的专一性及其对硝酸还原酶的水解作用

以同样的提取方法,分别从小麦叶片、曼陀罗愈伤组织中提取的硝酸还原酶(NR)钝化蛋白均可明显钝化小麦、水稻、玉米等叶片的NR,而从水稻、豌豆叶片中提取的NR钝化蛋白也均可明显钝化小麦叶片NR的事实显示出植物体内NR钝化蛋白存在的普遍性及不同植物种间这种钝化蛋白作用的共同性。水稻叶片及曼陀罗愈伤组织NR钝化蛋白只能钝化NR,而不能钝化与NR同为植物氮素同化关键酶的亚硝酸还原酶(NiR),小麦叶片NR钝化蛋白只能钝化NR而不能钝化与NR同为诱导酶的α-淀粉酶又表明NR钝化蛋白对NR的钝化作用具有一定的专一性。在小麦叶片NR钝化蛋白(部分Ⅰ)与NR一起保温时,同时加入作为水解酶抑制剂的大豆胰蛋白酶抑制物或丝氨酸酶抑制物PMSF,均可部分解除钝化蛋白的钝化效力,可作为此种钝化蛋白是个水解酶的进一步证明。     

番茄叶片中硝酸还原酶活性的“稳定因素”

硝酸还原酶(NR)是一种不稳定的酶,离体的NR在0℃下保藏几小时后,它的活性会显著丧失,有时甚至测不出它的活性。这一情况一直是进一步研究NR的生理生化的主要障碍。1979年,Sharrad首先发现小麦叶片中有二种因素能增进 NR活性,他认为这二种因素可能是蛋白类物质。我们(1980)证实番茄叶片中也有蛋白类物质能活化脱辅基NR酶蛋白。Purvis(1980)发现棉花种子及子叶中有一类热稳定的蛋白类物质,能稳定NR活性。现在对这类物质的认识刚刚开始,对它的性质和在植物之间的分布有待进一步研究。     

钼和6-BA对缺钼番茄叶片中硝酸还原酶活性恢复的影响

硝酸还原酶(NR)在植物的氮素营养上是一个关键酶,NO_3~-进入植物体内,首先须经过NR的催化作用才能被还原为NO_2~-,然后再经过其他种酶的作用被同化到蛋白质上。所以对NR的研究一直受到重视。 Nicholas和Nason证明NR中含有金属钼(Mo),以后又有不少工作者研究了Mo与NR的关系。肯定指出Mo构成NR的辅基,处于该酶的活性中心。但是NR又     

青霉素对草莓组培苗硝酸还原酶活性的影响

本实验利用植物组织培养的方法,在基本培养基中添加不同浓度的青霉紊对草莓进行根的诱导,以测定植株的 NR 活性。结果表明:实验剂量范围内,其 NR 活性随青霉素浓度增加呈正相关,同时发现草莓体内 NR 主要存在于地上部分。本实验设两个处理:MS 青霉素50mg/1;MS 青霉素100mg/1;对照 MS,各80瓶,在无菌条件下,分别取草莓组培丛芽接种于上述培养基中70天后进行 NR 活性的测定:取草莓组培植株0.5克,置三角瓶中,各瓶中分别加入5ml 0.1M/L 硝酸缓冲液,5ml 0.2M/L     

茶树硝酸还原酶基因克隆及表达分析

利用茶树全器官转录组文库中硝酸还原酶(NR)的EST,通过RACE技术扩增出NR基因的cDNA,并利用实时荧光定量PCR检测了NR基因在不同茶树品种中的表达。结果表明:NR基因cDNA全长2 927bp,开放阅读框2 652bp,编码一个有884个氨基酸蛋白质,GenBank登录号为JX987133。经BlastX比对,与GenBank中登录的烟草NR相似性达到74%。茶树NR蛋白属于亲水性蛋白,可能为胞质蛋白。25个茶树品种叶片中NR表达水平差异明显,最高值是最低值的22.75倍。因NR是植物氮代谢过程中的关键限速酶,推测25个茶树品种间氮吸收利用能力存在差异。     

钙对黄瓜子叶愈伤组织中硝酸还原酶(NR)活性的影响

本文采用黄瓜子叶愈伤组织,研究了不同钙培养下,对愈伤组织生长、硝酸盐吸收,NR活性以及组织中钙、镁含量的影响。结果表明,缺钙后,愈伤组织生长、硝酸盐吸收、NR活性等都比正常钙培养下的愈伤组织降低。这与用整株植物所得结果基本一致,对此结果进行了讨论。     

钙对黄瓜子叶愈伤组织中硝酸还原酶（NR）活性的影响

本文采用黄瓜叶愈伤组织,研究了不同钙培养下,对愈伤组织生长、硝酸盐吸收,NR活性以及组织中钙、镁含量的影响。结果表明,缺钙后,愈伤组织生长、硝酸盐吸收、NR活性等都比正常钙培养下的愈伤组织降低。这与用整株植物所得结果基本一致,对此结果进行了讨论。     

Preliminary Studies on the Acting Mechanism of Stabilizing Factor of Nitrare Reductase Activity (NR-SF)

Nitrate reductase (NR, E. C. 1.6.6.1), which was purified for 500-fold from crude extract of wheat leaves through the Blue-Dexdran Sepharose 4B affinity colume, could be still increased obviously by NR-SF in vitro. The results demonstrated that NR-SF increasedmainly activity of Cytochrome-C reductase (CytcR) in NR complex, but not affected activity of reduced methylviologen-nitrate reductase (MVH-NR). NR-SF was not similar to glutathione (GSH) which ieversed the inhibition of P-chloromercuribenzoate (PCMB) on activity of NR. So that the action of NR-SF was not in protecting sulfhydryl group of NR protein from oxidation alone.     

THE INFLUENCE OF LIGHT INTENSITY ON THE SUSCEPTIBILITY OF PLANTS TO CERTAIN VIRUSES

Reducing the light intensity under which plants were grown in summer to one-third increased their susceptibility to infection with tobacco necrosis, tomato bushy stunt, tobacco mosaic and tomato aucuba mosaic viruses. With the first two viruses shading increased the average number of local lesions per leaf by more than ten times and by more than five times with the second two.
Reducing the light intensity increased the virus content of sap from leaves inoculated with Rothamsted tobacco necrosis virus by as much as twenty times. As it also reduced the total solid content of sap by about one-half, purification was greatly facilitated; crystalline preparations of the virus were readily made from shaded plants but not from unshaded controls.
Reducing the light intensity also increased the virus content of systemically infected leaves; the greatest effect was with tomato bushy stunt virus with which increases of up to ten times were obtained, but with tobacco mosaic and aucuba mosaic viruses there were also significant increases.
The importance of controlled illumination in raising plants for virus work and the possible mechanisms responsible for the variations in susceptibility are discussed.     

农作物钾离子通道基因的生物信息学分析

以AtAKT1所编码的氨基酸序列为检索序列,通过对GenBank NR数据库以及EMBL、DDBJ、PDB数据库进行检索,发现水稻、小麦、大麦、玉米、烟草等作物中具有cds全长的34个疑似K~+通道基因.其中:水稻7个,玉米6个,烟草4个,马铃薯、胡萝卜、大麦各3个,葡萄、番茄各2个,蚕豆、小麦、油菜、甜瓜等各1个;有25个基因经过功能验证确定为K~+通道基因.利用比较基因组学,将剩余的9个基因确定为K~+通道基因.对34个K~+基因进化关系的分析表明,与AtAKT1的同源性较高的K~+通道基因为马铃薯的SKT1(相似性71%)、番茄的LKT1(相似性71%)、胡萝卜的DKT1(相似性69%)、烟草的NKT1(相似性62%).     

The Studies of the Stabilizing Factor of the Nitrate Reductase(E. C. 1.6.6.2) Activity from the Tomato(Lycopersicum esculentum)Leaves

A stabilizing factor of NR activity was isolated from tomato leaf homogenate by (NH4)2SO4 precipitation, boiling water treatment, Sephadex G-75 column and DEAE 52 column and dialysis against water. This factor appeared as band on polyacryl amide gel electrophoresis. This factor protected NR activity of tomato leaves from inactivation by temperature. It delayed the inactivation of N-R activity in wheat seedlings. when mixed with stabilizing factor and stored at 0 ℃ for nine days NR from wheat seedlings still kept its activity; without stabilizing factor, its activity was lost completely. Stabilizing factors both treated in boiling water in the process of isolation and isolated below 4 ℃ could stabilize and increase NR activity of tomato leaves. This factor existed in two forms, one free and one combined with some protein. It could be separated from the protein by heating during extraction. The stabilizing factor is different from FAD, haem, and proline.     

石蒜对萝卜、黄瓜、番茄和油菜幼苗的化感效应

在室内用离体生测方法研究了石蒜水浸提物对萝卜、黄瓜、番茄和油菜的化感效应.结果表明, 石蒜水浸提物对4种植物种子的萌发和幼苗生长均具有较强的抑制作用.4种受体作物中,以番茄最为敏感,最低浓度0.0125 g·ml^-1处理下,番茄种子完全受到抑制不能萌发,油菜、萝卜与黄瓜种子的萌发抑制率分别为17.73％、14.97％和2.65％.相同浓度处理时,石蒜水浸提物对萝卜、黄瓜及油菜芽的生长抑制作用高于对根的抑制作用.采用去胚乳小麦生长法和高梁河沙法测定了石蒜甲醇浸提物对光合作用和非光合作用抑制活性.结果表明,石蒜甲醇浸提物对去胚乳小麦和高梁的生长有较强的抑制作用,对高梁芽和根生长的抑制作用高于对去胚乳小麦根和芽生长的抑制,说明石蒜对植物的化感效应主要体现在抑制非光合作用活性上,但也一定程度地抑制植物的光合作用.     

某些环境条件对硝酸还原酶活性稳定因子的影响

小麦 Triticum aestivum L.苗在 NO_3~--N 完全营养液中培养比在 NH_4~ -N 完全营养液中培养,它们叶细胞内的硝酸还原酶(NR)即 NO_3-NR 比 NH_4-NR 活性增高了15倍,而它们叶片中的稳定因子(NR_(SF)),即 NO_3-NR_(SF)比 NH_4-NR_(SF)活化 NO_3-NR 的能力仅增加0.2倍,表明 NR 与 NR_(SF)不是依存关系;另外在 NO_3~--N 培养的黄化小麦叶片,及黄化缺氮、缺铝,加(?)的叶片中,所有的 NR_(SF)都十分稳定,并且保持较高活性,但这些叶片中没有测出NR 活性,因而认为,在植物叶细胞中,NR_(SF)不是调节 NR 活性的主要条件。     

AN ANALYSIS OF THE GROWTH RESPONSE OF YOUNG TOMATO PLANTS TO INFECTION BY VERTICILLIUM ALBO-ATRUM: II. THE PRODUCTION OF GROWTH SUBSTANCES

Bioassays on ether-soluble acid extracts from healthy and Verticillium -infected tomato plants, showed the presence of substances inhibiting growth of wheat coleoptiles in both healthy and infected leaves and stems, but the amounts were greater in the infected.
Assays of infected stems and leaves showed increases in growth-promoting activity expressed as indole-3-acetic acid equivalents (IAAe), up to 200% of those for healthy controls.
Similar assays of cultures of V. albo-atrum showed growth-promoting activity. No acid substance capable of inhibiting the growth of wheat tissue was detected in the culture filtrate. IAA was identified by colour test with Ehrlich's reagent on chromatograms from extracts of both infected stems and fungal culture filtrates.
The vertical distribution of IAAe was determined in healthy and infected plants at the eight-leaf stage by assaying individual leaves and four stem segments separately. In healthy plants the IAAe content was greatest in the young leaves (6–8) but no gradient was observed as between leaves 1–5. In infected leaves increases over the controls were found in leaves, 1, 3 and 6 and a decrease in leaf 8.
In healthy stems IAAe was highest in the distal segment and infected stems showed higher values at all four levels, with the relative increase greatest in the distal region.
It is suggested that the major part of the Verticillium syndrome including petiolar epinasty, tylosis, pith hyperplasia and the formation of adventitious roots is the result of an accumulation of growth substances in infected tissue.     

Effect of transgenic plants expressing high levels of a tobacco anionic peroxidase on the toxicity of Anagrapha falcifera nucleopolyhedrovirus to Helicoverpa zea (Lepidoptera: Noctuidae)

Wild type and corresponding transgenic tomato (Lycopersicon esculentum Miller) and two tobacco (Nicotiana spp.) plants that express high levels of a tobacco anionic peroxidase were used to determine what type of interactions occurred between peroxidase altered plant chemistry and the baculovirus Anagrapha falcifera nucleopolyhedrovirus (AfMNPV) for control of neonate corn earworms, Helicoverpa zea (Boddie). Transgenic plants expressed approximately five to 400 times higher peroxidase activity than corresponding tissues of wild type plants. The H. zea larvae typically fed 1.5 times less on transgenic compared with wild type leaf disks. There was only one experiment (of three with tomato leaves) where the larvae that fed on transgenic leaves were less susceptible to the virus based on nonoverlapping 95% confidence intervals for LC50 values. When the exposure dose was corrected for reduced feeding on the transgenic leaf disks, the insecticidal activity of the virus was not significantly different for larvae fed on transgenic versus wild type plants. Eight other experiments (with tomato and two species of tobacco) indicated either no significant effect or enhanced susceptibility (when corrected for feeding rates) to the virus of larvae fed on the transgenic leaves. These results indicate enhanced insect resistance in plants expressing high levels of a specific anionic peroxidase may be compatible with applications of AfMNPV. Potential reasons for this compatibility are discussed.     

Common occurrence of homologues of petunia glycine-rich protein-1 among plants

The presence of specific glycine-rich proteins (GRP) related to petunia GRP1 (ptGRP1) was examined in three species of monocots (wheat, barley and maize) and five species of dicots (rape, turnip, soybean, crabapple and tomato). Protein blot analysis showed that anti-ptGRP1 antibody cross-reacted with a single different polypeptide in all species except maize. The molecular mass of these polypeptides ranged from 14 to 55 kDa. Tissue-print immunoblots of rape petioles and stems showed that the rape ptGRP1 homologue, like ptGRP1, is primarily located in the vascular tissue, and that its expression decreases with developmental age of the tissue. In barley, the ptGRP1 homologue is found in leaf vascular bundles, and may also be present in the surrounding bundle sheaths. Unlike the dicots examined, expression of the protein did not appear to decrease significantly with developmental age.     

Properties of nitrate reductase from seedling leaves of selected barley genotypes

Crude extracts from leaves of 6-day barley seedlings of parental genotypes (cv. Aramir and primitive line R567) and selected doubled haploid (DH) lines were not found to have significant differences in the NADH:NR activity, while considerable differences between these genotypes were shown by the NAD(P)H:NR activity. The cv. Aramir and DH lines did not differ by nitrate accumulation in the leaves. However, the primitive line R567, as compared to the remaining genotypes, was characterized by an appreciably lower ability to accumulate nitrates. In partially purified leaf extracts, significant differences in total NADH:NR activity and in distal activity dependent on methyl viologen (MV:NR) were found between the parental genotypes and selected DH lines. The studied genotypes differed also in dehydrogenase NR activity, i.e. cytochrome c reductase activity in crude extracts. In the studied genotypes, the NADH:NR activity in partially purified leaf extracts did not substantially differ by Km values for nitrates. Calculated Vmax values for NADH:NR in these genotypes were similar to total NR activity in partially purified extracts. Significant differences between the parental genotypes and selected DH lines were found in the thermal NADH:NR stability in crude and partially purified leaf extracts. From the performed studies it follows that different NR stability was one of the reasons of revealed differences in total activity and in partial NR activities in the leaf extracts between the studied genotypes of spring barley. Besides, it is suggested that varied NR gene expression in the leaves of these barley genotypes could also influence NR activity.