抗苄嘧磺隆雨久花乙酰乳酸合成酶突变的研究

本研究采用cDNA末端快速扩增技术(RACE)结合RT-PCR方法克隆抗苄嘧磺隆雨久花生物型和敏感性雨久花生物型乙酰乳酸合成酶(ALS)基因cDNA序列,并对测序结果进行比对分析。结果表明:与敏感性的雨久花ALS相比,公主岭(GZL)抗性生物型中第197位脯氨酸突变为组氨酸,第556位亮氨酸突变为苯丙氨酸;柳河(LH)抗性生物型中第358位天冬酰胺突变为天冬氨酸;磐石市(PS)抗性生物型中第525位缬氨酸突变为异亮氨酸。分析表明,高度保守区Domain A的第197位氨基酸残基的突变可能是导致公主岭稻区雨久花产生抗药性的主要原因之一,而其他抗性生物型抗性产生的原因有待进一步研究。     

磺酰脲类除草剂对抗、感性雨久花乙酰乳酸合成酶活性的影响

从2004年开始,延边地区稻田大量发生抗磺酰脲类除草剂苄嘧磺隆的抗药性生态型雨久花。为解析其抗药性突变机制,采用离体法测定了抗、感性雨久花乙酰乳酸合成酶活性。结果表明,抑制抗、感性雨久花乙酰乳酸合成酶活性50%的苄嘧磺隆剂量为253.44×10-7mol/L和2.80×10-7mol/L;抑制抗、感性雨久花乙酰乳酸合成酶活性50%的吡嘧磺隆剂量为1802.15×10-7mol/L和20.85×10-7mol/L。抗药性雨久花对苄嘧磺隆和吡嘧磺隆的抗性系数(RI50/SI50)值分别为90.6和86.5,并存在交互抗药性。确认抗药性突变系由其乙酰乳酸合成酶对磺酰脲类除草剂苄嘧磺隆、吡嘧磺隆反应钝化所致。     

抗苄嘧磺隆雨久花的田间鉴定与替代药剂的筛选

对稻田雨久花进行高剂量苄嘧磺隆茎叶喷雾,以甄别其是否已对该药剂产生抗性,同时用五氟磺草胺、苯达松、2甲4氯钠对雨久花进行防除.结果表明,雨久花喷施苄嘧磺隆后生长良好,与未施药区无差异,说明雨久花对苄嘧磺隆已产生明显抗性.五氟磺草胺、苯达松、2甲4氯钠对抗性雨久花均有较好的防除效果.由于五氟磺草胺的靶标酶与苄嘧磺隆相同,都是乙酰乳酸合成酶,说明雨久花对五氟磺草胺未产生交互抗性.     

辽宁省稻田野慈姑对苄嘧磺隆的抗药性

为明确辽宁省不同稻田区野慈姑对苄嘧磺隆的抗性水平,整株测定了辽宁省大石桥(种群R1)、海城(种群R2)、苏家屯(种群R3)和开原(种群R4)共4个水稻产区野慈姑对苄嘧磺隆的抗性水平,并离体测定了各种群叶片体内乙酰乳酸合成酶(ALS)对苄嘧磺隆的敏感性。结果显示,种群R1和R2的抗药性相对较高,抗性指数分别为76.99和49.94,种群R3和R4抗性相对较低,抗性指数分别为12.48和16.91;离体测定结果表明较高水平的ALS活性可能与是否产生抗药性无关,种群R1、R2、R3、R4的抗性指数分别为81.86、67.48、10.56、24.86;抗药性程度依次为R1R2R4R3。表明4个水稻产区野慈姑对苄嘧磺隆均产生了抗药性,而其体内ALS活性降低可能是产生抗药性的原因之一。     

乙酰乳酸合成酶抑制剂的种类及其耐药性研究进展

乙酰乳酸合成酶（ALS）抑制剂在全世界已被迅速广泛认可和普遍使用。然而,由于ALS抑制剂的作用靶标单一,连续使用易诱发杂草产生抗药性等问题,ALS抑制剂类除草剂面临的杂草抗药性问题已越来越突出,并呈现出进一步恶化的趋势。本文概括了目前ALS抑制剂类除草剂的主要品种以及杂草对该类除草剂抗药性产生的主要机理,并针对因ALS基因突变导致的抗药性问题做了详细的阐述,为抗性基因应用的前景作了进一步展望。     

吉林狗尾草种群对烟嘧磺隆抗性水平和靶标抗性机理初探

为明确吉林省玉米田狗尾草Setaria viridis (L.) Beauv.对烟嘧磺隆的抗药性水平和抗性分子机理,测定了两个抗药性狗尾草种群R1、R2和一个敏感种群S对烟嘧磺隆的抗药水平,并检测了不同种群间乙酰乳酸合成酶(acetolactate synthase,ALS)基因序列差异。结果显示:R1、R2种群抗药性指数分别为21.8和23.9。扩增狗尾草ALS基因序列发现,相较于敏感种群,R1种群在376位点由GAT突变为GAA,R2种群在376位点由GAT突变为GAG,两种突变均导致天冬氨酸替换为谷氨酸(Asp-376-Glu)。表明狗尾草R1和R2种群对烟嘧磺隆产生抗性是由于靶标ALS基因突变导致,研究结果为科学防控抗药性狗尾草种群提供了理论基础。     

抗苄嘧磺隆野慈姑乙酰乳酸合成酶的突变研究

近年来,野慈姑Sagittaria trifolia L.在中国东北稻区发生和危害日趋严重,部分稻区使用苄嘧磺隆已无法有效防除该杂草。为了明确野慈姑抗药性发生的根本原因,本试验从分子水平上对野慈姑抗苄嘧磺隆的机理进行了研究。通过对抗药性(H4)和敏感性(S)野慈姑种群靶标酶乙酰乳酸合成酶(ALS)基因片段进行扩增和克隆,比较其DNA序列的差异,确定导致抗药性产生的ALS氨基酸突变位点。结果表明,与敏感性野慈姑ALS基因相比,H4种群第197位脯氨酸(Pro)突变为苏氨酸(Thr),该位点的突变可能是H4野慈姑种群对苄嘧磺隆产生抗药性的主要原因。ALS第197位Pro突变为Thr致使对苄嘧磺隆产生抗药性是第一次在野慈姑种群中报道。     

多花水苋对苄嘧磺隆的抗性水平及靶标抗性机制

为明确水稻田杂草多花水苋Ammannia multiflora对乙酰乳酸合成酶 (ALS) 抑制剂类除草剂苄嘧磺隆的抗性水平和抗性分子机制,采用整株水平测定法,测定了采自江苏省扬州市田间的多花水苋疑似抗性种群 (YZ-R) 对苄嘧磺隆的抗性指数,并分析了YZ-R种群和相对敏感种群 (YZ-S)多花水苋ALS酶对苄嘧磺隆的敏感性差异,同时比较了YZ-R和YZ-S种群ALS基因的核苷酸序列差异。结果表明:YZ-R种群多花水苋对苄嘧磺隆已表现出高水平抗性,其抗性指数 (RI) 为40.6;苄嘧磺隆对YZ-R种群ALS酶活性的抑制中浓度 (I₅₀) 为0.087 μmol/L,对YZ-S种群的I₅₀值为0.0028 μmol/L,其抗性指数为31.1。通过PCR扩增获得了多花水苋ALS基因的部分序列,该序列包含了已报道的8个氨基酸突变位点。ALS基因序列比对分析发现,YZ-R种群多花水苋植株ALS基因第197 位氨基酸由脯氨酸 (CCT) 突变为丝氨酸 (TCT)。研究表明,ALS基因发生脯氨酸 (Pro)-197-丝氨酸 (Ser) 的突变,导致多花水苋ALS酶对苄嘧磺隆的敏感性下降,是多花水苋YZ-R种群对苄嘧磺隆产生高水平抗性的主要原因。     

反枝苋对咪唑乙烟酸抗性水平及分子机制

为初步明确大豆田反枝苋对咪唑乙烟酸的抗药性水平,并从分子角度对抗药性机制进行解释,以我国四川成都和黑龙江嫩江采集的反枝苋种子为材料,通过琼脂法检测了反枝苋对咪唑乙烟酸的抗药性水平,并分别对R(嫩江抗性种群)和S(成都敏感种群)的乙酰乳酸合成酶(ALS)部分序列进行扩增和测序。皿内生测结果表明,成都种群的GI50为11.20,嫩江种群的GI50为52.26,其抗药性指数RI为4.67。分子检测结果表明,与S种群相比,R种群反枝苋ALS位于高度保守区Domain B编码574位氨基酸的基因发生突变,TGG突变为TTG,导致色氨酸被亮氨酸取代。ALS保守区域氨基酸的替换可能是嫩江反枝苋种群对咪唑乙烟酸产生抗性的重要原因之一。     

杂草对乙酰乳酸合成酶抑制剂抗药性研究进展

乙酰乳酸合成酶(ALS)抑制剂类除草剂已经成为一类广泛使用的除草剂。综述了杂草对ALS抑制剂类除草剂抗药性的产生与发展、抗性机理、抗性基因应用等方面的研究进展。其抗性产生机理主要有杂草对除草剂代谢能力增强、ALS基因突变导致对除草剂敏感性降低和ALS含量提高等。     

Effects of a lectin-like protein isolated from Acacia farnesiana seeds on phytopathogenic bacterial strains and root-knot nematode

Acacia farnesiana lectin-like protein (AFAL) showed bacterioestatic effects against Xanthomonas axonopodis pv. passiflorae (Gram-negative) and Clavibacter michiganensis michiganensis (Gram-positive), with the latter being more sensitive. This effect is probably due to the ability of AFAL to interact with the bacterial cell wall where we observed that AFAL induced macroscopic change. The maximum bacterial growth inhibition was approximately 78% when incubated with Gram-negative strains, and as high as 92% percent for the Gram-positive one. The antibacterial effect of flavonoids (rutin, quercetin and morin) was also observed using low concentrations against both bacterial strains. Prior incubation of both with AFAL at high concentrations increases the inhibitory effect of flavonoids on bacterial growth. The potential use of AFAL as a control agent against the root-knot nematode Meloidogyne incognita was investigated as well, showing anti-nematode properties involving both egg hatching and motility. In the juvenile second-stage, AFAL showed reduction in larval mobility when measured against a control group. The results suggest that AFAL is effective against M. incognita and could be used as a component of integrated pest management programs. These data also suggest that lectins probably play a role in plant defense not only against invertebrate phytopathogens, herbivores and fungi but also against bacteria.     

枯草芽胞杆菌对离体荔枝果实霜疫霉病、炭疽病的防治效果

为了进一步明确从土壤中筛选得到的拮抗细菌OR-1、OR-2、OR-3、ON-6的分类地位和生防效果,采用形态学观察、理化特性结合分子生物学方法,鉴定这4株细菌皆为枯草芽胞杆菌Bacillussubitilis.拮抗菌对荔枝霜疫霉菌、炭疽菌菌丝生长均具有明显抑制作用,ON-6菌株的发酵液对菌丝生长的抑制率最高,分别为92.34％和70.36％,其次是OR-1菌株.拮抗菌处理组褐变指数均小于对照组以及杀菌剂处理组,且差异达显著水平(P＜0.05),OR-1菌株防褐变效果最好.常温下,拮抗菌发酵液对离体鲜果病害防治效果优于对照组和杀菌剂处理组,其中ON-6菌株的防治效果最佳;4℃低温储藏处理40天后,拮抗菌发酵液处理组的防治效果高于对照和杀菌剂处理组,且差异显著(P＜0.05),以ON-6和ON-3菌株的防治效果最佳.表明拮抗菌发酵液对荔枝鲜果上的霜疫霉菌和炭疽病菌有一定的抑制作用.     

Agrobacterium-mediated transformation efficiency is altered in a novel rice bacterial blight resistance cultivar and is influenced by environmental temperature

Y73 is a progeny of asymmetric somatic hybridization from an elite japonica rice cultivar (Dalixiang) and a wild rice (Oryza meyeriana), which shows high resistance to bacterial blight. It has a similar genetic background to its recurrent parent, Dalixiang, but Y73 has a high resistance to both bacterial blight and Agrobacterium. The transformation efficiency of Y73 was 35.7%, while Dalixiang had higher transformation efficiency (71.2%) under the same co-cultivation temperature (25 °C). These results indicate that the resistance to Agrobacterium tumefaciens in Y73 was linked with its characteristic of bacterial blight resistance, and was obtained from its donor parent, O. meyeriana. Further studies also showed that the resistance to A. tumefaciens was weakened at a lower temperature (20 °C). To study its molecular mechanism, the expression levels of genes associated with Agrobacterium-mediated transformation (OsMPKs, OsVIP1s and OsPR1s) in rice were investigated by Quantitative real-time PCR (qRT-PCR) analysis. The expression levels of OsMPK genes remained unchanged at different temperatures, while all OsVIP1s and almost all OsPR1s were up-regulated and transformation efficiency of Y73 was increased notably when infected by Agrobacterium at 20 °C compared with 25 °C. This study suggests that the bacterial blight resistance genes in Y73 also have an effect on Agrobacterium-mediated transformation and that the response to temperature is associated with the regulation of MAPK/VIP1 defense signaling pathway.     

The effects of Artemisia annua L. and Achillea millefolium L. crude leaf extracts on the toxicity, development, feeding efficiency and chemical activities of small cabbage Pieris rapae L. (Lepidoptera: Pieridae)

The biological effects of two important medicinal plants, Artemisia annua L. and Achillea millefolium (L.) (viz, mortality, growth, and feeding indices as well as enzyme and non-enzymatic activities) were studied on small white Pieris rapae L a deleterious pest of cruciferous plants under controlled conditions (16:8 h L:D at 25 ± 1 °C and 65 ± 5% RH). The LC₅₀ and LC₂₅ values were 9.387% and 3.645% for A. annua L. and 4.19% and 1.69% for A. millefolium (L.), respectively. At the lowest concentration (0.625%), the deterrency was 29.826% and 44.185% for A. annua L. and A. millefolium (L.), respectively. Feeding indices were variously affected with changes in a number of parameters and an increase in larval and pupal duration. The activity level of alkaline phosphatase increased sharply while alanin and aspartate aminotransferases showed a sharp decrease. For non-enzymatic compounds, the amount of glucose and uric acid increased, but total protein and cholesterol decreased. These results indicate that these two medicinal plants might possess potential secondary metabolites that may be useful for controlling potential insect pests.     

枣镰翅小卷蛾成虫的寄主趋向和产卵选择

采用固相微萃取(SPME)和GC-MS分析研究木枣和酸枣挥发物成分,并测试枣镰翅小卷蛾触角电位(EAG)反应、寄主趋向和产卵选择,以探明枣镰翅小卷蛾的寄主选择机制。结果显示,在萌芽期,木枣和酸枣嫩叶的挥发成分均为罗勒烯、2-甲基-2-菠烯、α-法呢烯和邻苯二甲酸二丁酯4种,但相对含量稍有不同。枣镰翅小卷蛾成虫对木枣和酸枣2种寄主都有强烈的EAG反应,且同一寄主上雌蛾的EAG反应值极显著地高于雄蛾,其EAG值是雄蛾的3.3倍。枣镰翅小卷蛾对木枣表现强烈的趋向反应,而对酸枣的趋向不明显,且雌虫的趋向反应显著高于雄虫。木枣上的产卵量显著高于酸枣,且木枣枣吊上的单雌产卵量为307.9粒,极显著地高于酸枣枣吊上的产卵量(182.9粒)。研究表明,枣镰翅小卷蛾雌蛾在寄主选择中起主导作用,木枣是其嗜好寄主。     

菜豆普通细菌性疫病菌在土壤和植株残体中的越冬能力

为评估菜豆普通细菌性疫病菌地毯草黄单胞杆菌菜豆致病变种或褐色黄单胞菌褐色亚种在土壤及植物残体中的越冬能力,对采自黑龙江、内蒙古、山西、河北及新疆的18块菜豆生产田的20份土壤及14份植物残体样品进行病原菌分离和鉴定。在MT选择性培养基上有12个土壤样品和13个植株残体样品提取液产生典型的类似黄单胞菌菌落。选取29个分离物进行致病性测定,有27个分离物对菜豆品种"英国红"致病。利用地毯草黄单胞杆菌菜豆致病变种和褐色黄单胞菌褐色亚种的特异性引物X4c/X4e及褐色黄单胞菌褐色亚种特异性引物Xf1/Xf2对29个分离物进行多重PCR检测,其中17个分离物为地毯草黄单胞杆菌菜豆致病变种,10个分离物为褐色黄单胞菌褐色亚种。结果表明,菜豆普通细菌性疫病菌可以在黑龙江、内蒙古、山西、河北的一些菜豆种植区的土壤及植株残体中越冬存活。     

两株棒束孢菌的鉴定及其对茶卷叶蛾和茶小卷叶蛾的致病力

为明确分离自茶卷叶蛾和中华大刀螳僵虫的2株虫生真菌的归属及生防潜力,采用形态学特征和rDNA-ITS序列分析其分类地位,测定了其对茶卷叶蛾和茶小卷叶蛾幼虫的致病力,并初步筛选了其最适培养条件.结果表明:2株真菌均为环链棒束孢Isaria cateniannulata,分别命名为ICBS918和ICTL911.在1.0×10⁸孢子/mL浓度下,ICBS918和ICTL911对茶卷叶蛾幼虫的累计校正死亡率均达100%,LT₅₀分别为3.13 d和3.15 d;对茶小卷叶蛾幼虫的累计校正死亡率分别为100%和95%,LT₅₀分别为3.25 d和3.31 d.接菌后8 d,2菌株对茶卷叶蛾幼虫的LC₅₀分别为0.47×10⁵孢子/mL和1.01×10⁵孢子/mL;对茶小卷叶蛾幼虫的LC₅₀分别为2.20×10⁵孢子/mL和1.34×10⁵孢子/mL.2菌株23 ℃下最适生长培养基均为综合马铃薯培养基,最适产孢培养基均为蛋白胨马铃薯葡萄糖琼脂培养基;在萨氏培养基上,菌株ICBS918和ICTL911分别在24~30 ℃及24 ℃时生长速度最快,在27 ℃和21 ℃时产孢量最大.表明2株环链棒束孢对茶卷叶蛾和茶小卷叶蛾幼虫均具有较好的生防潜力,可作为生防菌进行开发和应用.     

Analysis of two acetylcholinesterase genes in Bombyx mori

Two acetylcholinesterases (AChE, EC 3.1.1.7) cDNAs were identified and cloned from silkworm, Bombyx mori. One of those, BmAChE-o cDNA, is comprised of 3197 nucleotides which encode 638 amino acids, having an amino acid sequence homology of 72% with Drosophila melanogaster Ace-orthologous AChE (AO-AChE). In some species, another AChE group based on the sequence, Drosophila Ace-paralogous AChE (AP-AChE) has been recognized in relation to organophosphate- or carbamate-resistance, but there have been few reports of AP-AChE among lepidopteran species. However, we isolated the AP-AChE from lepidopteran silkworm, and cloned full ORF as BmAChE-p, which cDNA consisted of 2465 nucleotides that encode 683 amino acids. The homologies with other AP-AChEs were over 60% when compared. Although silkworm is not a target of pesticides, the genomic information obtained in this study will contribute to insecticide-resistance study on lepidopteran pest species.     

苏云金杆菌亚致死浓度对美国白蛾及其寄生蜂生长发育的影响

为阐明苏云金杆菌Bacillus thuringiensis(Bt)与白蛾周氏啮小蜂Chouioia cunea Yang的协同作用,分别用103~108孢子/m L的6个Bt浓度处理美国白蛾Hyphantria cunea 2龄幼虫后,通过室内试验测定了Bt对其致死、亚致死效应,及亚致死浓度Bt对寄生蜂子代出蜂的影响。结果表明,浓度106孢子/m L处理组美国白蛾均未能化蛹;而106孢子/m L处理组均能化蛹并羽化,且幼虫发育历期随Bt浓度的增加而延长,平均蛹重在74.92~92.18 mg之间,均高于对照的68.66 mg,但蛹重不呈线性规律变化。当Bt对美国白蛾平均校正死亡率低于17.17%时,幼虫发育历期延迟不明显,但平均蛹重为101.11~124.34 mg,均高于对照;接蜂后能够正常出蜂,出蜂率为33.13%~51.25%,每蛹出蜂量为117.43~143.42头,均显著高于对照。表明用平均校正死亡率低于20%的Bt处理对白蛾周氏啮小蜂的寄生有利,2种生防措施协同作用存在增效潜力。     

草间钻头蛛体内沃尔巴克氏体的去除及其感染对宿主生殖和适合度的影响

采用在草间钻头蛛Hylyphantes graminicola的饮水和人工饲料中添加四环素的方法研究了蜘蛛体内沃尔巴克氏体(Wolbachia)的去除.同时,通过设置草间钻头蛛Wolbachia感染种群与未感染种群之间不同的交配方式,研究Wolbachia感染对草间钻头蛛生殖和适合度的影响.结果显示,在草间钻头蛛的饮水和人工饲料中添加四环素均能部分去除草间钻头蛛体内的Wolbachia,在供试最高浓度下,去除率分别为40％和50％.4种不同的交配组合下,草间钻头蛛雌蛛均能产卵,卵的孵化率差异不显著,Wolbachia感染对草间钻头蛛的生殖无影响.感染Wolbachia的草间钻头蛛所产第1卵袋的卵历期为6.65d,极显著高于未感染Wolbachia的卵历期(P =0.008).感染与未感染Wolbachia的草间钻头蛛种群世代存活率、性比以及适合度均无显著差异.