外源茉莉酸和茉莉酸甲酯诱导植物抗虫作用及其机理

综述了茉莉酸(jasmonic acid, JA)和茉莉酸甲酯(methyl jasmo nate, MJA)的分子结构和应用其诱导的植物抗虫作用及其机制。植物受外源茉莉酸或茉莉酸甲酯刺激后,一条反应途径是由硬脂酸途径激活防御基因,另一条途径是直接激活防御基因。防御基因激活后导致代谢途径重新配置,并可能诱导植物产生下列4种效应：（1）直接防御,即植物产生对害虫有毒的物质、抗营养和抗消化的酶类,或具驱避性和妨碍行为作用的化合物;（2）间接防御,即产生吸引天敌的挥发物;（3）不防御,即无防御反应;（4）负防御,即产生吸引害虫的挥发物。     

二氯喹啉酸对稗草生长发育及生长素含量的影响

通过外施二氯喹啉酸、生长素处理,研究二氯喹啉酸对稗草Echinochloa crusgalli生长特性、蛋白浓度、过氧化物酶及超氧化物歧化酶活性、生长素含量的影响。结果表明,800 g·hm-2二氯喹啉酸处理24 h未对S型稗草的生长产生明显抑制,而二氯喹啉酸单独处理及外施IAA的二氯喹啉酸处理均能显著增加R型稗草鲜重。在不施药、二氯喹啉酸单独处理、二氯喹啉酸与IAA共同处理的条件下,R型稗草比S型稗草的蛋白浓度分别提高23.5%、29.8%和64.0%,R型稗草表现出较高的抗逆境耐受能力。经二氯喹啉酸处理后,两种生物型稗草SOD活性上升,POD活性下降。施外源IAA能提高S型稗草POD活性,而对R型稗草SOD、POD活性影响不显著。提高生长素含量可显著促进R型稗草的生长。     

二氯喹啉酸诱导稗草EcDnaJ基因表达

DnaJ作为分子伴侣在植物抗逆中起重要作用. 但目前在二氯喹啉酸逆境下,其在抗药性稗草中表达特点却鲜有报道. 本研究采用RACE技术从抗二氯喹啉酸稗草中克隆了1个DnaJ基因, 命名为EcDnaJ1 (GenBank登录号：JX518598), 其cDNA全长为2 154 bp, 开放阅读框为1 350 bp,编码449 个氨基酸, 理论分子量为48.4 kD, 等电点为9.5. 该蛋白质氮端含有1个保守的J结构域, 中部含有4个模式为CxxCxGxG的锌指结构. Real-time PCR分别测定EcDnaJ1在二氯喹啉酸抗性和敏感的稗草生物型苗期叶、根及成株期根、茎、叶和种子中的表达, 在感、抗生物型的相对表达量分别是0.8～20.9和7.4～30.2, 其中在抗性稗草苗期叶片中相对表达量最高为30.2, 而在敏感稗草种子中最低为0.8, 抗性稗草是敏感稗草的1.4～9.2 倍. 受二氯喹啉酸诱导后, 其在感、抗稗草的相对表达量分别为35.8～72.5和84.9～261.9, 在抗性稗草苗期叶片中相对表达量最高为261.9, 而在敏感稗草的种子中相对表达量最低为35.8, 抗性稗草是敏感稗草的2.4～3.6 倍. 诱导前后, 无论是在苗期根和叶中还是成株期的根、茎、叶和种子中, EcDnaJ1表达量均是抗性稗草高于敏感稗草. 抗、感稗草生物型的EcDnaJ1在mRNA水平差异表达暗示,它可能参与了稗草对二氯喹啉酸的抗药性.     

外源茉莉酸甲酯诱导大麦对叶斑病抗性的研究

[目的]初步探讨不同浓度外源茉莉酸甲酯（MeJA）诱导大麦抗叶斑病效应差异及其分子机制,为应用MeJA防治大麦叶斑病提供理论依据。[方法]以‘蒙啤麦3号’大麦品种幼苗为材料,设置不接菌（无菌水处理叶片）、接菌（无菌水处理叶片接种麦根平脐蠕孢菌）和接菌+MeJA（0.5、1.0、1.5、2.0、2.5 mmol/L MeJA喷施叶片后接菌）3组处理,于三叶期调查叶斑病发病情况并据此筛选最适MeJA浓度,然后测定不接菌、接菌及接菌++ MeJA（最适浓度）处理不同处理时间叶片的抗氧化酶、抗病相关酶活性、丙二醛、渗透调节物质含量以及相关基因表达水平。[[结果]]：（1）叶面喷施外源MeJA提高了大麦对叶斑病的抗性,1.5 mmol/L MeJA处理叶片的病情指数较对照显著降低19.03%,诱导抗性效果最佳;（2）与单独接菌处理相比,1.5 mmol/L MeJA处理大麦叶片超氧化物歧化酶、过氧化物酶、过氧化氢酶、几丁质酶和β-,1,3-葡聚糖酶活性均显著提高,而其丙二醛、脯氨酸、可溶性糖和可溶性蛋白含量显著降低,同时其受MeJA调控转录因子及编码抗病相关酶的基因表达量显著上调。[结论]外源喷施1.5 mmol/L MeJA通过调节抗病相关酶活性和渗透调节物质含量,以及调控抗病相关酶基因及茉莉酸信号途径关键转录因子基因表达,进而提高大麦植株的叶斑病抗病性。     

茉莉酸甲酯（MeJA）诱导水稻颖花开放

水稻颖花的开放是由浆片吸水、膨大并推开内、外颖之间的钩合而造成的。王忠等［１］最早报道颖花因呼吸增强而积累的ＣＯ２是促进浆片膨大的主要因素,并建立了用外源ＣＯ２或酸溶液诱导水稻开颖的方法。可是,颖花开放之前,其呼吸强度何以能大幅度增加？浆片细胞的吸水...     

茉莉酸甲酯的生理生化及在植物抗病中的作用

茉莉酸甲酯的生理生化及在植物抗病中的作用宾金华,潘瑞炽（华南师范大学生物系,广州５１０６３１）ＭＥＴＨＹＬＪＡＳＭＯＮＡＴＥＳ：ＢＩＯＣＨＥＭＩＳＴＲＹ,ＢＩＯＰＨＹＳＩＯＬＯＧＹＡＮＤＲＯＬＥＩＮＰＬＡＮＴＲＥＳＩＳＴＡＮＣＥＢｉｎＪｉｎ－ｈｕａ;...     

外源茉莉酸甲酯诱导竹根姜对青枯菌的抗性

以竹根姜幼苗为材料,采用室内水培试验和茎基部注射接种姜青枯菌方法,探讨外源茉莉酸甲酯(MeJA)诱导竹根姜产生姜瘟病抗病性的生理生化机制。结果显示:外源MeJA处理可以显著降低竹根姜的病情指数,提高对姜瘟病的抗性,而对青枯菌的致病力没有直接的抑制作用。MeJA处理可促进竹根姜内源H2O2在前期迅速积累,同时诱导增强姜块内苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、多酚氧化酶(PPO)、过氧化氢酶(CAT)等防御酶以及病程相关蛋白几丁质酶和β-1,3-葡聚糖酶的活性,促进姜块内木质素含量上升。研究表明,MeJA能通过调控竹根姜细胞内下游信号分子H2O2的水平来提高防御相关酶的活性和病程相关蛋白的表达,促进木质素的合成,提高了竹根姜的抗病性。     

外源茉莉酸甲酯诱导番茄对烟粉虱的抗虫性

采用盆栽法研究茉莉酸甲酯(MeJA)处理后番茄叶片营养物质含量、次生代谢物含量和防御酶活性,以及MeJA处理对烟粉虱成虫的防治效果。结果表明:外源MeJA可以诱导番茄叶内营养物质含量的降低,用0.0225 mmol·L^-1的MeJA处理5 d后,番茄叶片中可溶性糖和蛋白质含量最低,诱导效应最强;外源MeJA可诱导番茄叶内次生代谢产物含量的升高,用0.0225 mmol·L^-1的MeJA处理5 d后,番茄叶中单宁和黄酮含量最高,诱导效应最强;外源MeJA能够诱导番茄叶内防御性酶(POD和PPO)活性升高,随着时间的延长(0～14 d),不同浓度MeJA处理后防御酶活性呈现出先上升后下降再上升的变化趋势,且用0.0225 mmol·L^-1的MeJA处理5 d后,番茄叶中POD和PPO活性最大,诱导效应最强;不同浓度的外源MeJA对烟粉虱的防效不同,用0.0225 mmol·L^-1MeJA处理5 d后,对烟粉虱的防治效果最好,防效可达86.96%;外源茉莉酸甲酯处理可以通过降低营养物质含量、提高次生代谢产...     

茉莉酸及其甲酯在植物诱导抗病性中的作用

茉莉酸类物质被认为是植物抗病防卫反应的内源及中间信号分子。本文介绍了茉莉酸及其甲酯在植物抗病性中的作用,从它们在体内激活的代谢途径及相关基因表达探讨有关作用机制以及有可能在农业上应用的前景。     

外源茉莉酸甲酯诱导对香菇多糖代谢及关键酶基因表达的影响

香菇多糖是重要的生物活性成分,其药用价值高,茉莉酸甲酯有助于提高食用菌多糖含量,但关于其对香菇多糖合成的影响尚不清楚。本研究以香菇新808为实验材料,对不同浓度茉莉酸甲酯（MeJA）诱导下香菇菌丝生物量和生长速率、香菇多糖代谢关键酶活性及基因相对表达量、香菇多糖含量进行比较分析。结果表明：10μmol/L的茉莉酸甲酯可显著促进香菇菌丝生长并提高菌丝体生物量,分别是对照的1.14倍、1.2倍;与香菇多糖代谢相关的UDP-葡萄糖焦磷酸化酶（UGPase）、葡萄糖磷酸异构酶（PGI）、α-葡萄糖磷酸变位酶（α-PGM）活性增加,为对照组的1.58、1.13、3.21倍;其基因相对表达量较对照组也显著上调,为对照组的5.73、1.4、1.77倍;香菇多糖合成量显著增加,为对照组1.58倍。10μmol/L的MeJA处理后,香菇多糖代谢关键酶活性及基因相对表达量与多糖含量间存在显著的正相关关系,表明添加适宜浓度的茉莉酸甲酯会影响香菇菌丝的生长及多糖合成。     

Genetic control of a cytochrome P450 metabolism-based herbicide resistance mechanism in Lolium rigidum

The dynamics of herbicide resistance evolution in plants are influenced by many factors, especially the biochemical and genetic basis of resistance. Herbicide resistance can be endowed by enhanced rates of herbicide metabolism because of the activity of cytochrome P450 enzymes, although in weedy plants the genetic control of cytochrome P450-endowed herbicide resistance is poorly understood. In this study we have examined the genetic control of P450 metabolism-based herbicide resistance in a well-characterized Lolium rigidum biotype. The phenotypic resistance segregation in herbicide resistant and susceptible parents, F1, F2 and backcross (BC) families was analyzed as plant survival following treatment with the chemically unrelated herbicides diclofop-methyl or chlorsulfuron. Dominance and nuclear gene inheritance was observed in F1 families when treated at the recommended field doses of both herbicides. The segregation values of P450 herbicide resistance phenotypic traits observed in F2 and BC families was consistent with resistance endowed by two additive genes in most cases. In obligate out-crossing species such as L. rigidum, herbicide selection can easily result in accumulation of resistance genes within individuals.     

Impact of exogenous lysolipids on sensitive and multidrug resistant K562 cells: 1H NMR studies

The ability of lysolipids to enter into a membrane bi-layer and disturb the membrane structure was used to study the behavior of K562 erythroleukemic cells, K562 wild type (K562wt) as well as the multidrug resistant cells K562adr. Both types of cells, when analyzed by proton NMR spectroscopy exhibit the high resolution signals assigned to so-called "mobile lipid" signals, which, in most cases, are located outside the lipid bi-layer as lipid droplets. In order to perform these studies, the K562wt and K562adr cells were treated for 48h with lysophosphatidylcholine oleoyl (LPC18), lysophosphatidylcholine palmitoyl (LPC16) and L-alpha-lysophosphatidyslerine (LPS). After evaluating toxicity of lysolipids, proton NMR of whole treated cells was used to analyze the mobile lipid content. Nile red staining and fluorescence microscopy were used to detect the presence of intracellular lipid droplets. Membrane lipid asymmetry perturbation was estimated by annexin V staining with use of flow cytometry. Using fluorescence spectroscopy the functioning of P-glycoprotein (P-gp) responsible for multidrug resistance was also evaluated after the treatment with lysolipids. Lysolipids were found to be more toxic for K562wt than for K562adr cells. LPS and LPC16 produced an increased of a mobile lipid NMR signal and amount of lipid droplets in K562wt cells only. LPC18, with the lowest toxicity, has shown more intense effects on NMR spectra with a large increase of lipid NMR signal without changes in lipid droplet staining. The functioning of the P-gp pump and membrane asymmetry were not modified by any of the lysolipids used.     

A mechanism of chlorotoluron resistance in Lolium rigidum

Many biotypes of Lolium rigidum Gaud, (annual ryegrass) have developed resistance to herbicides; however, few have developed resistance to phenylurea herbicides. Two biotypes with different histories of herbicide selection pressure were six to eight times less sensitive to the phenylurea herbicide, chlorotoluron, than a susceptible biotype. Resistance was not due to differences in the herbicide target site as oxygen evolution by thylakoids isolated from resistant and susceptible biotypes was similarly inhibited by diuron and chlorotoluron. There was no difference in the uptake and distribution of chlorotoluron into resistant and susceptible plants. There was a twofold greater rate of chlorotoluron detoxification in resistant plants with N-demethylation being a major detoxification reaction. Resistant plants treated with a 3-h pulse of 120 M chlorotoluron recovered net carbon fixation after 42 h, half the time taken by susceptible plants. The mixed-function oxidase inhibitor 1-aminobenzotriazole (70 M) intensified the effects of chlorotoluron in resistant plants when applied in combination with the herbicide for 7 d. 1-Aminobenzotriazole also inhibited the metabolism of chlorotoluron in both resistant and susceptible plants. The cytochrome P-₄₅₀ inhibitor, piperonyl butoxide piperonyl butoxide, interacted with chlorotoluron when applied to plants growing in soil. Chlorotoluron applied with reduced plant dry weight to a greater extent than chlorotoluron alone. It appears, therefore, that enhanced detoxification is the major mechanism of resistance to chlorotoluron in the resistant biotypes studied.Abbreviations ABT 1-aminobenzotriazole - VLR1 Victorian L. rigidum biotype 1 — herbicide susceptible - VLR69 Victorian L. rigidum biotype 69 — herbicide resistant - WLR2 Western Australian L. rigidum biotype 2 — herbicide resistant M.W.M.B, was supported by an Australian Postgraduate Research Award and a supplementary scholarship from the Grains Research and Development Corporation. We are very grateful to Dr. E. Ebert, Ciba Geigy, Basal, Switzerland for providing [¹⁴C]chlorotoluron and standards of chlorotoluron metabolites. We express our gratitude to Dr. John Huppatz of the CSIRO Division of Plant Industry for providing ABT. We also thank Ciba Geigy Australia for providing technical-grade chlorotoluron and formulated phenylurea herbicides.     

转双抗虫基因三倍体毛白杨外源基因表达及性状相关性分析

用部分改造的BtCry1Ac基因与慈菇蛋白酶抑制剂（API）基因构建的双抗虫基因表达载体,通过农杆菌介导法转化了三倍体毛白杨Populus tomentosa Carr.,获得一批转双抗虫基因株系。对转基因株系的抗虫毒蛋白表达进行了ELISA和Western Blot检测,同时用转基因株系叶片对杨扇舟蛾Clostera anachoreta Fabricius和舞毒蛾Lymantria dispar L.幼虫进行室内饲虫试验,并对各项外源基因表达指标进行了相关分析。结果表明：在检测的28个转基因株系中,对杨扇舟蛾高抗株系占总参试系号的41％,中抗系号占35.0％,低抗系号占24％,对舞毒蛾高抗株系占参试系号的70％。转基因植株可明显抑制存活幼虫的生长发育,且不同转基因株系饲养的幼虫发育存在显著差异。连续两年相关分析表明,不同转基因株系幼虫死亡率间存在极显著相关。转基因植株对舞毒蛾和杨扇舟蛾均表达出抗虫性,并存在极显著相关。ELISA检测结果表明,不同转基因株系Bt毒蛋白表达量存在差异,变化在0.0011％～0.0161％。转基因植株对害虫的杀虫效果与Bt杀虫蛋白的表达量存在显著相关,表明Bt毒蛋白在抗虫效果中占有重要地位。转基因植株对卡那霉素表现出一定的抗性,但与抗虫程度相关不明显,单纯用卡那霉素作为筛选手段并不能完全反映植株的真实抗虫效果。     

褐飞虱抗甲胺磷品系的交互抗性和抗性生化机制

用甲胺磷筛选的褐飞虱Nilaparvata lugens品系（R）,对甲胺磷的抗性达到43.74倍,对马拉硫磷、二嗪磷、异丙威、仲丁威及醚菊酯都表现出一定的交互抗性,而对氰戊菊酯和吡虫啉的交互抗性不显著。为了研究褐飞虱对甲胺磷抗性和对其它药剂交互抗性产生的机制,进行了活体增效试验和离体生化实验。用2 μg/头的增效剂预处理试虫的活体增效实验结果显示,在甲胺磷筛选品系（R）中, TPP(triphenyl phosphate, 磷酸三苯酯)对甲胺磷的增效倍数达到4.54,TPP对马拉硫磷、二嗪磷、仲丁威、异丙威都表现出一定的增效作用,增效比分别为2.76、2.07、2.17和1.64;PBO(piperonyl butoxide,胡椒基丁醚)对甲胺磷、马拉硫磷和醚菊酯有一定的增效作用;DEM(diethyl meteate, 顺丁烯二酸二乙酯)的增效作用不明显。研究离体情况下增效剂对三种解毒酶活性的影响发现,TPP对R品系酯酶活力抑制作用很强（抑制率69.04%）,PBO对多功能氧化酶（MFO）具有一定的抑制作用（抑制率29.30%）,而TPP和PBO在F品系和S品系中对酯酶和MFO的抑制作用都较小;DEM在三个品系中对谷胱甘肽-S-转移酶的抑制作用都很小。由此可见,酯酶在褐飞虱对甲胺磷的抗性中起最主要作用,在马拉硫磷、二嗪磷、异丙威和仲丁威的交互抗性中起很重要作用;MFO可能在甲胺磷抗性和醚菊酯、马拉硫磷的交互抗性中起一定作用。     

新型甲氧苄啶耐药二氢叶酸还原酶DfrB7的生化分析与宿主菌耐药机制研究

【目的】分析新型甲氧苄啶获得性耐药蛋白DfrB7的生化性质,探究其与B家族代表性的二氢叶酸还原酶(DHFR)对甲氧苄啶获得性耐药的生化基础。【方法】构建系统进化树分析B家族DHFRs与新型DfrB7的进化关系。将dfr基因分别构建到pACYC184和pET15b(+)载体并转化到相应大肠杆菌中。使用微量肉汤稀释法确定克隆菌株对甲氧苄啶的耐药性。测定DHFRs使用NADPH作为质子供体催化二氢叶酸还原的酶活反应参数。通过等温滴定量热法测定甲氧苄啶的解离常数。【结果】克隆到大肠杆菌中的新型dfrB7基因表现出对甲氧苄啶的高耐药表型。系统进化树确定了dfrB7编码B家族的DHFRs。检测并比较DfrB7和代表性DHFRs的酶活性质、抑制剂的亲和力,与染色体上DHFR相比,DfrB7与DfrB1均表现出显著降低的催化活性,通过生化实验证实B家族二氢叶酸还原酶对甲氧苄啶结合力极差。【结论】新型dfrB7基因编码的DfrB7具有B家族二氢叶酸还原酶的普遍特征。B家族二氢叶酸还原酶赋予宿主菌对甲氧苄啶的获得性耐药与该酶对甲氧苄啶的低亲和力有关。