菊酯类农药残留在武夷岩茶加工过程中的降解研究初报

以肉桂、水仙、白鸡冠、铁罗汉和水金龟5个武夷岩茶品种（名枞）为试验材料,将5种菊酯类农药（联苯菊酯、甲氰菊酯、氯菊酯、氯氰菊酯和溴氰菊酯）喷施于茶树,一周后对鲜叶进行采摘,一部分烘干固样,另一部分按武夷岩茶制作工艺加工成干茶。通过气相色谱技术,检测分析鲜叶、成茶的农药残留量。结果表明：联苯菊酯的降解率在13.02%～54.33%,甲氰菊酯的降解率在17.12%～100%,氯菊酯的降解率在17.57%～18.09%,氯氰菊酯的降解率在22.93%～87.23%,溴氰菊酯的降解率在27.33%～100%。不同菊酯类农药在不同品种的武夷岩茶加工过程中的降解率存在一定的差异,其中,水金龟的降解率最大。     

Comparison of ULV with conventional spraying on cotton in Pakistan

ULV and conventional low-volume spraying were compared using ULV and e.c. formulations of deltamethrin, cypermethrin and triazophos to control cotton pests. Both spraying techniques were effective, but Bemisia spp. were controlled better with the low-volume aqueous sprays. The ULV sprayer was much cheaper to use than the conventional knapsack sprayer and covered a five- to six-fold greater area daily.     

大肠杆菌glgC基因的定点突变及高效表达

通过PCR方法,从E.coliJM109基因组DNA中扩增到全长为1296bp的glgC基因,对其进行定点突变,获得第296位和336位氨基酸同时突变的glgC336基因。将2者亚克隆进pET-30a,成功构建了重组表达载体pET-C和pET-C336,转化大肠杆菌BL21(DE3),在1mmol/LIPTG诱导下进行表达。SDS-PAGE电泳分析显示,在约53kD处有1条明显的蛋白表达带,证明目的基因已得到高效表达,且重组蛋白表达量占菌体蛋白总量的77.3%。     

本氏烟草中异源合成抗癌药物葫芦素前体

葫芦素具有显著的抗癌活性,解析其生物合成途径对异源生产葫芦素具有重要意义。葫芦素首先由葫芦二烯醇（Cuol）在氧化鲨烯环化酶（OSCs）家族的葫芦二烯醇合酶（CBS）催化下生成2,3-氧化鲨烯,后者在细胞色素P450(CYP)等酶催化下氧化修饰产生葫芦素系列化合物。本研究在建立高效的根癌农杆菌（Agrobacterium tumefaciens）介导的瞬时表达体系的基础上,将雪胆（Hemsleya chinensis Cogn.）CBS基因（HcOSC6）与截短的燕麦（Avena strigosa）3-羟基-3-甲基-戊二酰辅酶A还原酶基因（tHMGR）共表达,通过不断优化烟草共表达体系,将本氏烟草（Nicotiana benthamiana）叶片中的Cuol产量从2.832 mg/g（干重）提高到9.48 mg/g（干重）。同时,将雪胆的HcCYP87D20共渗入烟草叶片,获得葫芦素生物合成的重要中间体11-羰基-20β-羟基-葫芦二烯醇。本研究结果将为葫芦素的高效异源合成提供理论依据。     

水稻种子脂氧合酶基因OsLOX1的原核表达、纯化及鉴定

脂氧合酶是动植物体内催化脂质降解的关键酶,也是茉莉酸合成途径的第一个关键酶。以先前克隆到的水稻种子脂氧合酶基因OsLOX1全长cDNA为模板,用含有特异酶切位点的P1、P2为引物,通过PCR方法将它构建到大肠杆菌表达载体pET30a(+)上并转化到大肠杆菌菌株BL21（DE3）中,获得相应的重组工程菌。经过20℃条件下的IPTG诱导,OsLOX1重组蛋白在BL21（DE3）菌株中得到表达,经生化特性分析,发现该重组蛋白具有LOX催化活性,其最适pH和温度分别为4.8和30℃。该重组子可进一步应用于体外生产茉莉酸和研究植物种子LOX结构与功能的关系。     

Monitoring of insecticide resistance in Spodoptera litura (Lepidoptera: Noctuidae) from four districts of Punjab,Pakistan to conventional and new chemistry insecticides

Studies were carried out to evaluate the resistance of Pakistani populations of the beet armyworm, Spodoptera litura (F) to several commonly used insecticides. Different field populations of S. litura from four districts of the Punjab were monitored from 2009 to 2011 for resistance to insecticides using a standard leaf dip bioassay method. For organophosphates and pyrethroids, resistance ratios compared with a susceptible Lab-Pk population were in the range of 8–109 fold for deltamethrin, 11–139 fold for cypermethrin, 19–143 fold for chlorpyrifos and 39–162 fold for profenofos. For new chemistry insecticides, resistance levels were 2–74 fold for spinosad, 4–216 fold for abamectin, 7–87 fold for indoxacarb, 2–77 fold for emamectin benzoate, 1.9–58 fold for lufenuron and 4–43 fold for methoxyfenozide. Pairwise correlation coefficients of LC₅₀ values showed a positive correlation with cross-resistance among deltamethrin, cypermethrin and chlorpyrifos, while resistance to profenofos showed correlations with resistances to other insecticides except chlorpyrifos. New chemistry insecticides showed no correlations between any of the tested insecticides. There were high to very high levels of resistance to organophosphates in most of the population, which suggested that the use of these should be avoided against this pest. Selective use of pyrethroids in several areas, including Bahawalpur and Lodhran, where the pest showed a low level of resistance, would appear to be acceptable, the new chemistry insecticides, lufenuron, methoxyfenozide, emamectin and indoxacarb had no, very low, low and moderate resistance levels against populations, respectively. These are considered to be safe to the environment and safer to natural enemies.     

宿根矮化病菌的竞争定量PCR检测方法建立

利用宿根矮化病菌的特异检测引物,构建竞争定量PCR检测的非同源模板,建立宿根矮化病菌的竞争定量PCR检测方法.实验中非同源模板(竞争模板)构建是采用PCR方法对大肠杆菌基因组进行低严谨度扩增,回收合适的DNA片段并将其构建到T-esay载体上,通过测序和序列比对,除两端引物序列完全相似外,其余部分没有同源性,因此可以作为定量检测的非同源模板.通过计算非同源模板的拷贝数,进行竞争定量PCR检测宿根矮化病,建立宿根矮化病菌的标准竞争曲线和直线回归方程.本研究建立的竞争定量PCR检测方法操作简单、特异性和敏感性高,适合于宿根矮化病菌的检测,并可为其它病菌竞争定量PCR检测方法的建立提供参考.     

Control of cotton bollworms with fenvalerate in India

The synthetic pyrethroid fenvalerate (Sumicidin^® 20E¹) has been tested in 64 field trials of the control of bollworms on rainfed and irrigated cotton in Maharashtra, Tamil Nadu, Punjab, Andhra Pradesh, Haryana, Rajasthan, Madhya Pradesh, Gujarat and Karnataka. Fenvalerate, at dose levels of 50–150 g a.i./ha, was tested on 15 varieties of cotton, with long, extra-long, short and medium staples. From two to nine sprays were applied at intervals of 7–30 days, depending on whether the insecticide was sprayed according to a calendar-based schedule or when needed. Fenvalerate was compared with conventional insecticides such as carbaryl, monocrotophos, endosulfan and phosalone, and also with the synthetic pyrethroids permethrin, cypermethrin and deltamethrin. The percentage of bollworm-infested plants in the fenvalerate-treated plots ranged from zero to 21·8% and in the untreated plots was up to 100%. With conventional pesticides the maximum level of infestation was 97·2%. Increases in yield of seed cotton from fenvalerate-treated plots over those from plots treated with conventional pesticides were 54%, 57%, 67%, 84%, and 86% over monocrotophos, carbaryl, quinalphos, phosalone and endosulfan, respectively, representing increases of 791–1046 kg/ha.     

Identification and Expression of Two Novel Cytochrome P450 Genes,CYP6CV1 and CYP9A38, in Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

Cnaphalocrocis medinalis Güenée can cause severe losses in rice. Cytochrome P450s play crucial roles in the metabolism of allelochemicals in herbivorous insects. Two novel P450 cDNAs, CYP6CV1 and CYP9A38, were cloned from the midgut of C. medinalis. CYP6CV1 encodes a protein of 500 amino acid residues, while CYP9A38-predicted protein has 531 amino acid residues. Both cDNA-predicted proteins contain the conserved functional domains for all P450s. Phylogenetic analyses showed that CYP6CV1 is grouped in the cluster containing CYP6B members, while CYP9A38 is in the cluster including CYP9 members. However, both clusters are contained in the same higher lineage. Homologous analysis revealed that CYP6CV1 is most similar to CYP6B8, CYP6B7, CYP6B6, CYP6B2, and CYP6B4 with the highest amino acid identity of 41%. CYP9A38 is closest to CYP9A17, CYP9A21, CYP9A20, and CYP9A19 with the highest amino acid identity of 66%. Studies of temporal expression profiles revealed that CYP9A38 showed a steady increase in mRNA level during the five instar stages, but a low-expression level in pupae, and then presented at a high-expression level again in adults. Similar expression patterns were obtained with CYP6CV1. In the fifth instar larvae, CYP6CV1 was mainly expressed in midgut and fat bodies, whereas CYP9A38 was mainly expressed in midgut. Expression studies also revealed a 3.20-fold over-expression of CYP6CV1 and 3.54-fold over-expression of CYP9A38 after larval exposure to host rice resistance. Our results suggest that both CYP6CV1 and CYP9A38 may be involved in detoxification of rice phytochemicals.     

Resistance of beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae) to endosulfan,organophosphorus and pyrethroid insecticides in Pakistan

Field populations of beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), from Pakistan were assessed for their resistance to the chlorinated hydrocarbon endosulfan, the organophosphates chlorpyrifos and quinalphos, and the pyrethroids cypermethrin, deltamethrin, bifenthrin and fenpropathrin. Using a leaf-dip bioassay, resistance to endosulfan was high during 1998–2000 but declined to very low, to low levels during 2001–2007, following a reduced use of the insecticide. Organophosphates and pyrethroids were consistently used over the past three decades, and the resistance had been increasing to these insecticide classes. Generally, the resistance to chlorpyrifos and pyrethroids remained low from 1998 to 2002–2003, but resistance increased to moderate to high levels from 2003–2004 to 2006–2007. For deltamethrin, resistance was very high during 2004–2007. Quinalphos resistance remained low during 1998–2006. Correlation analysis of LC₅₀ and LC₉₀ values showed a positive correlation between organophosphates and pyrethroids, but no correlation between endosulfan and organophosphates or pyrethroids tested herein. These results suggest that the conventional chemistries should be replaced with new chemistries for the successful management of S. exigua.     

一个水稻小热休克蛋白的异源表达及寡聚特性分析

【目的】在前期研究中,本实验室已克隆了一个水稻小热休克蛋白基因(OsSHSP17.6),并发现该基因的表达明显受到热激和病毒侵染调控,表明该蛋白可能在逆境胁迫过程中起重要作用。本研究的目的在于进一步明确OsSHSP17.6的特性。【方法】在本研究中,进一步将该基因亚克隆至原核表达载体p ET-32a并导入大肠杆菌E.coli BL21(DE3)pLysS诱导表达,通过亲和层析的方法纯化了该重组蛋白,进一步用于非变性聚丙烯酰胺凝胶电泳和Western blotting分析。【结果】异源表达的重组OsSHSP17.6能减轻IPTG对宿主菌的毒害。非变性聚丙烯酰胺凝胶电泳和Western blotting分析显示纯化的重组OsSHSP17.6蛋白在体外能形成同源二聚体和寡聚体。【结论】这些结果支持OsSHSP17.6是一个有功能的分子伴侣蛋白并表明该蛋白可能通过形成同源寡聚体的方式参与逆境胁迫反应,这为进一步明确OsSHSP17.6的功能机制奠定基础。     

Improved control of potato virus Y by mineral oil plus the pyrethroid cypermethrin applied electrostatically

Field potato plants sprayed with a mixture of a pyrethroid (cypermethrin at 40 g a.i./ha) and a paraffinic oil (Sunoco Sunspray 7E at 7 $\text{l}$/ha) were colonized by fewer aphids and were less frequently diseased by potato virus Y (PVY) than unsprayed plants or plants sprayed with either cypermethrin or the oil alone; aphids given access to leaves of PVY-infected field plants sprayed with the mixture were also less likely to transmit PVY to laboratory test plants than aphids given access to leaves of plants sprayed with either chemical alone, or to untreated leaves. When alate aphids were confined above virus-infected and healthy plants, combined oil and cypermethrin treatment also gave greater protection against virus inoculation than either chemical alone. Electrostatic charging of spray droplets increased chemical deposits on field plants, especially when the plants were small and on upper leaves. These greater deposits improved control of PVY by the oil and the cypermethrin, individually or in combination, and enhanced the aphicidal action of the cypermethrin.     

Toxicity of some pesticides to Trichoderma viride Pers.

The toxicity of several fungicides, insecticides and acaricides to the important biological control agent Trichoderma viride Pers. has been evaluated in the laboratory. The effects of the pesticides on the spores and mycelium of T. viride and on the antagonistic activity of this fungus to Fusarium graminearum are reported. The insecticides cypermethrin, deltamethrin, fenvalerate, kelevan, carbaryl, carbaryl plus gamma-HCH (lindane), dimethoate, trichlorfon, diazinon, ethion and permethrin and the fungicides wettable sulfur, quinomethionate, dinocap, copper oxychloride and folpet were non-toxic to both the spores and the mycelium of T. viride.     

外源IBA对裸花紫珠插穗营养物质含量及抗氧化酶活性的影响

以药用植物裸花紫珠(Callicarpa nudiflora)一年生半木质化枝条为材料,使用吲哚丁酸(IBA,1 500 mg/L)处理插穗,以清水为对照,每隔6 d取样1次,测定生根过程中插穗基部可溶性糖、淀粉、可溶性蛋白含量及吲哚乙酸氧化酶(IAAO)、多酚氧化酶(PPO)、过氧化物酶(POD)活性的动态变化。结果表明,扦插后0~12 d,1 500 mg/L IBA处理增加了插穗内可溶性蛋白含量,降低了淀粉含量,可溶性糖含量先降后升,IAAO、POD、PPO活性增加;扦插后12~30 d,淀粉及可溶性糖含量逐渐降低,可溶性蛋白含量先升后降,但仍都高于对照,IAAO和PPO活性逐渐下降,POD活性先降后增;扦插30 d后,可溶性糖、蛋白、淀粉含量逐步趋于稳定,IAAO活性趋于稳定,而PPO和POD活性逐渐降低。总之,较高浓度的可溶性糖、可溶性蛋白,低浓度的淀粉,较高的IAAO、POD和PPO活性有利于裸花紫珠插穗不定根形成和发育。     

多种药剂对螺旋粉虱不同虫态的毒力测定

采用Potter喷雾法,测定30种杀虫剂对螺旋粉虱成虫与15种杀虫剂对螺旋粉虱若虫的毒力。结果表明:杀扑磷、毒死蜱、辛硫磷、溴氰菊酯、马拉硫磷等对成虫、若虫均具有较高活性。对成虫杀扑磷毒力最强,其LC50值为0.9123mg/L,毒死蜱、辛硫磷、马拉硫磷、顺式氯氰菊酯、溴氰菊酯、敌敌畏及丙溴磷对成虫毒力均较高,其LC50值分别为1.571 4、1.673 1、2.248 2、2.5570、3.039 1、3.186 8、4.1895 mg/L。对若虫溴氰菊酯毒力最强,其LC50值为8.5276mg/L,杀扑磷、毒死蜱、高效氯氟氰菊酯、高效氯氰菊酯对若虫同样具有较高毒力,其LC50值分别为11.486 5、12.9503、12.973 8、13.703 6 mg/L。另通过不同龄期若虫对毒死蜱等8种杀虫剂的敏感性测定,发现1~4龄若虫对杀虫剂的敏感性随虫龄增加而降低,1龄若虫最敏感,2龄和3龄若虫的敏感性相当,4龄若虫敏感性最低。     

AA3型连续流动分析仪（CFA）同时测定橡胶叶全氮、全磷、全钾的方法研究

通过对AA3型连续流动分析仪所附试剂配制等方法的改进,研究AA3同时测定橡胶叶片氮、磷、钾的方法。结果表明,氮、磷、钾混合标准曲线相关系数均达0.999 9以上;氮、磷变异系数均小于0.5%(n=10),当钾浓度相当于标准曲线中间浓度范围时(即6～10 mg/L),变异系数为0.97%～1.13%,重现性良好;氮、磷、钾加标回收率分别为99.7%～101.1%、99.3%～100.7%、98.4%～102.8%,待测液中氮、磷、钾检出限分别为0.019、0.004、0.072 mg/L。橡胶叶片氮、磷、钾测定结果与常规法相比无显著差异。该方法用于橡胶叶片H2SO4-H2O2消煮后氮、磷、钾同时测定,快速高效,一天半可测300个样品待测液,提高工效10多倍。     

巴西橡胶树HbHDR的原核表达及HbHDR在乳管细胞中的亚细胞定位

4-羟基-3-甲基-2-(E)-丁烯基-4-磷酸还原酶(4-hydroxy-3-methyl-2-(E)-butenyl-4-diphosphate reductase,HDR)是异戊烯基焦磷酸合成途径之一甲基赤藓糖磷酸(methylerythritol phosphate,MEP)途径中的最后一个酶,催化4-羟基-3-甲基-(2E)-丁烯基-4-磷酸生成异戊烯基焦磷酸.根据笔者从橡胶树中克隆的HDR基因(命名为HbHDR,GenBank登录号:EU881977)序列,构建了HbHDR的原核表达载体,并在大肠杆菌中诱导表达获得融合表达蛋白.将融合蛋白免疫新西兰大白兔,制备HbHDR的高效价特异的多克隆抗体.Western-blot分析表明,HbHDR存在于乳管细胞的橡胶粒子和C-乳清中.     

植物生长调节剂对大豆根系氮代谢相关指标的影响

在植物生长调节剂拌种的情况下,探明了不同植物生长调节剂对大豆根系氮代谢的影响.结果表明:DTA-6和DTA-6+GA3提高了大豆的根系活力和硝酸还原酶的活性,增加了硝态氮的含量.DTA-6、GA3和DTA-6+GA3均提高了可溶性蛋白的含量,增强了蛋白水解酶的活性.GA3增加了根系内游离氨基酸积累量和输出量,降低了硝态氮的含量和硝酸还原酶的活性.综合分析表明,应用化学调控手段可以改善大豆根系内同化物代谢水平,促进根系的正常生长发育.     

栽培大豆Rubisco小亚基基因（rbcS）全长cDNA的克隆及原核表达

根据NCBI中发表的大豆Rubisco小亚基基因（rbcS）序列（AF303939-1）设计特异引物,以吉农13大豆为实验材料,采用Trizol法提取叶片总RNA,通过RT-PCR克隆大豆rbcS的cDNA。将该基因与原核表达载体pET-30a（+）连接,转化大肠杆菌E.coli BL21感受态细胞,双酶切鉴定后筛选阳性克隆,测序结果显示：rbcS全长 696bp,其中开放阅读框为537bp,编码178个氨基酸;选择含大豆rbcS cDNA阳性克隆菌液IPTG诱导,经SDS-PAGE分析,诱导表达出分子量为29.1kDa的融合蛋白,表达产物大小与预计理论值相符。诱导7h蛋白表达量最高,为64.15%。