水稻对褐飞虱抗性相关蛋白的双向电泳分析

以药用野生稻(Oryza officinalis)的转育后代B5(高抗褐飞虱(Nilaparvata lugens Stl))与感虫品种明恢63 (Oryza sativa L.)为亲本,构建了一个重组自交系群体.通过抗褐飞虱鉴定,筛选出极端抗虫株系和极端感虫株系,运用分群分析法(bulked segregant analysis,BSA)分别建成了极端抗虫集团(resistant bulk)和极端感虫集团(susceptible bulk)的蛋白质池.利用双向电泳技术,分别分析了极端抗虫集团和极端感虫集团受虫害与未受虫害的秧苗蛋白质的变化.结果发现,虫害48 h后,感虫集团的一个分子量为40 kD的蛋白质P40 (pI=6.3)的表达明显减弱甚至消失,而在抗虫集团中,P40的表达未受影响.与褐飞虱为害后抗虫株系和感虫株系不同的生理反应相联系,推测P40与水稻受褐飞虱虫害后引起的应答反应相关.     

抗感吡虫啉褐飞虱在抗虫水稻品种上的适合度比较

建立抗呲虫啉褐飞虱Nilaparvata lugens(St(a)l)种群和敏感呲虫啉褐飞虱种群在不同抗虫水稻品种(TN1、IR36)上的实验种群生命表,比较了抗感吡虫啉种群在感虫品种TN1上以及在抗虫品种IR36上饲养4代的种群趋势指数(Ⅰ)和适合度的差异.结果表明,在TN1上,抗呲虫啉和敏感吡虫啉褐飞虱种群的种群数...     

褐飞虱刺吸诱导的水稻一些防御性酶活性的变化

褐飞虱刺吸导致稻株体内丙二醛含量迅速上升,表明褐飞虱刺吸引起了水稻植株膜脂的过氧化;抗虫和感虫品种植株体内脂氧合酶(LOX)、脂氢过氧化物裂解酶(HPL)、苯丙氨酸解氨酶(PAL)的活性均显著升高;而过氧化氢酶(CAT)活性在抗虫品种稻株体内明显受抑制,H2O2含量提高,但在感虫品种植株体内CAT的活性略有提高,H2O2含量略有下降.对抗虫品种植株不同器官的测定结果表明,褐飞虱刺吸对PAL和H2O2的影响是系统性的,而对LOX的影响则仅局限于褐飞虱刺吸部位的茎杆中.     

水稻挥发性次生物质对褐飞虱寄主定向及生长的影响

从褐飞虱的抗虫与感虫水稻品种植株中提取挥发性次生物质,分析其对褐飞虱的定向选择、生长发育及成虫触角电位反应影响.结果表明,感虫品种TN1挥发性次生物质对褐飞虱生物型Ⅱ若虫的取食及生长发育未表现不良作用,对其定向选择有引诱作用;而抗虫品种IR36的挥发性物质对褐飞虱为负作用,表现为褐飞虱取食添加挥发性物质的人工饲料后,其死亡率均增加,生长发育受阻,且随着浓度的增加,不良影响加大.触角电位试验结果表明褐飞虱的电位反应总的趋势是对相同浓度的TN1挥发性物质的触角电位反应值比IR36大,且触角电位值基本都随着挥发性物质浓度的升高而增大;短翅型成虫电位值强于长翅型成虫,雄性成虫的电位值强于雌性成虫.挥发性物质气质联用检测分析结果发现抗虫与感虫水稻中挥发性次生物质的化学组成及含量相差较大,从抗感两个品种的挥发性物质中共检到44种已知化合物,部分化合物对褐飞虱有引诱作用.     

小粒野生稻导入系对稻飞虱的抗性鉴定与分析

稻飞虱是水稻生产最严重的害虫之一。野生稻拥有丰富的抗虫基因资源,导入系是鉴定和利用野生稻有利基因的有效途径。本研究通过对371份小粒野生稻导入系进行抗褐飞虱和白背飞虱接虫鉴定,分别筛选出了11份抗、72份中抗褐飞虱的材料和7份抗、45份中抗白背飞虱的材料,其中有5份材料兼抗褐飞虱和白背飞虱,这是从小粒野生稻中鉴定出抗白背飞虱材料的首次报道。通过对2份抗性导入系材料与感虫亲本杂交构建的F1和F2群体的抗虫鉴定和分析表明:K41对褐飞虱和白背飞虱的抗性受2对显性抗虫基因通过互补作用所控制;P114对褐飞虱和白背飞虱的抗性都是由1对主效的隐性基因控制。这些结果必将有利于小粒野生稻抗稻飞虱的基因定位和育种利用。     

褐飞虱对水稻苗期生长及地下部土壤活性碳氮的影响

地上部植食者对地下部土壤生态系统的影响引起了陆地生态学者的浓厚兴趣。报道了盆栽条件下褐飞虱取食不同品种水稻后对水稻苗期生长和土壤活性碳氮的影响。土壤活性碳氮水平的评价采用了土壤微生物生物量碳和氮、可溶性碳和氮及无机氮等指标,它们是反映土壤生态过程的重要变量。结果表明,褐飞虱侵害降低了苗期水稻茎叶、根系的生物量及根冠比,并与水稻品种的抗褐飞虱能力存在交互作用。褐飞虱也显著影响土壤活性碳氮水平(P0.05),并强烈依赖于水稻品种特性。一般的,褐飞虱导致感虫品种广四的土壤微生物生物量碳、可溶性碳下降,而对抗虫品种IR36的影响则相反。在褐飞虱的危害下,抗虫品种水稻对土壤微生物生物量氮、可溶性氮及硝态氮的促进程度较大。中感品种汕优63与汕优559在褐飞虱作用下对水稻茎叶、根系生物量及土壤活性碳氮的影响也不相同。汕优63的影响趋势与感虫品种广四一致,而汕优559的影响与抗虫品种IR36更接近。总之,土壤活性碳氮组分对褐飞虱危害的响应程度和趋势因水稻品种不同而不同,特别是抗虫品种在褐飞虱侵害时有利于土壤活性碳氮水平的维持,提高土壤生物活性,从而可能进一步促进土壤生态功能的发挥。     

受褐飞虱取食的水稻幼苗的抑制消减cDNA文库的构建及筛选

采用抑制消减杂交方法,以褐飞虱取食32h的水稻幼苗及未受褐飞虱取食的水稻幼苗为作为对比材料构建了消减cDNA文库,以分离水稻幼苗中褐飞虱应答基因。随机从消减cDNA文库中挑选16个白色菌落提取质粒,进行PCR扩增,发现插入片段的长度位于100～900bp之间。以在受褐飞虱取食的水稻幼苗中特异表达的基因(BpHi008A)为探针,通过斑点印迹分析发现在抑制消减后的cDNA池中,目的基因得到有效富集。利用反向总RNA斑点印迹分析和Northern杂交验证,从消减cDNA文库中筛选到了25个基因受褐飞虱取食的诱导。其中有17个克隆与编码已知功能蛋白的基因有显著的同源性,它们分别参与蛋白质的折叠与降解、蛋白质与蛋白质的相互作用及信号传递、脂类代谢、胁迫反应、物质运输和细胞生长等。总体上,参与胁迫反应和衰老的基因在褐飞虱取食后表达增强。     

褐飞虱致害性变异过程及其体内酶的变化

在室内连续用具不同抗虫基因的水稻品种TNl、IR26、Mudg。和ASD7、单管饲养褐飞虱(Nilaparvata lugens)种群,研究它对抗虫水稻品种的适应过程及其体内酶的变化规律。 结果表明：褐飞虱在抗虫品种上取食2代的若虫存活率、若虫历期和短翅成虫体重均明显比取食感虫品种TNl的低,第3代以后与取食TNl者基本相同。第2代是褐飞虱适应抗虫品种的关键期。天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)和丁-谷氨酰基转移 酶(GGT)的活性在关键的第2代最低,而超氧物歧化酶(SOD)和过氧化氢酶(CAT)的含量增加。褐飞虱在适应抗虫品种以后体内天冬氨酸氨基转移酶活性明显提高。     

抗感水稻品种受灰飞虱为害后的生理反应差异

灰飞虱Laodelphax striatellus Fallén(SPBH)是我国水稻生产上的一种重要害虫。为探明水稻抗灰飞虱的生理基础,在不同灰飞虱虫口密度(0、5、10、20头/株)及不同为害时间(3 d、6 d)下,检测了抗感水稻品种相关生理指标的变化差异。结果表明,灰飞虱为害后,抗感水稻植株中叶绿素、可溶性糖及可溶性蛋白含量均呈下降趋势,感虫品种中的下降幅度显著大于抗虫品种,且感虫品种的叶绿素、可溶性糖及可溶性蛋白含量在不同虫口密度及不同为害时间下,均存在显著差异。当灰飞虱以20头/株为害6 d后,武育粳3号、Kinmaze、DV85和Mudgo中叶绿素下降幅度分别为46.0%、46.0%、2.3%和2.1%,可溶性糖含量下降幅度分别为55.5%、46.3%、9.2%和11.7%,可溶性蛋白含量下降幅度分别为22.3%、29.5%、8.5%和6.7%。抗感水稻中过氧化物酶活性随虫量增大和为害时间延长而显著升高,过氧化氢酶活性则随虫量增加和为害时间延长而降低,但抗虫植株中保护性酶活性对虫害胁迫的响应明显早于感虫材料。游离脯氨酸和丙二醛含量均随着灰飞虱虫量增大及为害时间延长而上升,其中,游离脯氨酸在抗虫材料中的上升幅度高于感虫品种,丙二醛含量则在感虫品种的上升幅度显著高于抗虫材料。     

水稻品种对褐飞虱的田间抗性及其生态学机制

评价了7个水稻品种(浙粳22、宜香845、中组14、秀水123、二优倍九、感虫对照品种TN1和抗虫对照品种IR36)在田间成株期对褐飞虱Nilaparvata lugens(Stl)的抗性表现,并初步分析了它们的抗性机制。结果表明,在田间网室人工诱发条件下,浙粳22、宜香845、秀水123和中组14等4个品种的抗性水平与抗虫品种IR36相同,均小于3级,表现为抗虫,而二优培九则与感虫品种TN1相同,表现为感虫。在自然发生条件下的水稻分蘖期至乳熟期,宜香845和中组14上褐飞虱数量仅为TN1上的7.84%～24.44%,秀水123和浙粳22则为TN1上的30.19%～60.27%。在4个抗性品种上褐飞虱的产卵量和卵孵化率明显减少,而若虫存活率仅为TN1上的20%～30%。与IR36相同,4个抗性品种上褐飞虱的若虫存活率和种群增长倍数均显著低于TN1上,说明他们对褐飞虱的抗性主要表现为抗生性。     

Revealing different systems responses to brown planthopper infestation for pest susceptible and resistant rice plants with the combined metabonomic and gene-expression analysis

Brown planthopper (BPH) is a notorious pest of rice plants attacking leaf sheaths and seriously affecting global rice production. However, how rice plants respond against BPH remains to be fully understood. To understand systems metabolic responses of rice plants to BPH infestation, we analyzed BPH-induced metabolic changes in leaf sheaths of both BPH-susceptible and resistant rice varieties using NMR-based metabonomics and measured expression changes of 10 relevant genes using quantitative real-time PCR. Our results showed that rice metabonome was dominated by more than 30 metabolites including sugars, organic acids, amino acids, and choline metabolites. BPH infestation caused profound metabolic changes for both BPH-susceptible and resistant rice plants involving transamination, GABA shunt, TCA cycle, gluconeogenesis/glycolysis, pentose phosphate pathway, and secondary metabolisms. BPH infestation caused more drastic overall metabolic changes for BPH-susceptible variety and more marked up-regulations for key genes regulating GABA shunt and biosynthesis of secondary metabolites for BPH-resistant variety. Such observations indicated that activation of GABA shunt and shikimate-mediated secondary metabolisms was vital for rice plants to resist BPH infestation. These findings filled the gap of our understandings in the mechanistic aspects of BPH resistance for rice plants and demonstrated the combined metabonomic and qRT-PCR analysis as an effective approach for understanding plant-herbivore interactions.     

Feeding‐induced changes in defence enzymes and PR proteins and their implications in host resistance to Nilaparvata lugens

Six rice genotypes showing susceptible and resistant reactions to brown planthopper (BPH), Nilaparvata lugens were studied for feeding‐induced changes in defence enzymes and pathogenesis‐related (PR) proteins. The high resistant genotypes PTB 33, ADT 45 and ASD 7 and moderately resistant genotypes CO 43 and KAU 1661 recorded the greater expression of defence enzymes peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, total phenol and β‐1,3 glucanase in response to N. lugens feeding at 1 day after infestation (DAI) compared with susceptible genotype TN1. The greater activity of chitinase was observed in resistant cultivars at 3 DAI and the activity was sustained for more than 1 week compared with susceptible TN1. In conclusion, the current study revealed that these defence enzymes and PR proteins might attribute to the resistance mechanisms in rice plants against BPH infestation.     

Differential expression of vitellogenin mRNA and protein in response to rice resistance genes in two strains of <Emphasis Type="Italic">Nilaparvata</Emphasis><Emphasis Type="Italic">lugens</Emphasis> (Hemiptera: Delphacidae) with different levels of virulence

Controlling the brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is a difficult task in rice (Oryza sativa L.) production. We focused on vitellogenins (Vg), which are the major yolk protein precursors of vitellins and play an important role in the reproduction of oviparous species, including insects. We studied the accumulation of Vg mRNA and protein in a virulent BPH strain, Nagasaki-03, and a nonvirulent strain, Hatano-66, after rearing them on four rice lines. The rice lines used were two single resistance gene introgression lines, Norin-PL3 (Bph1 carrier) and Norin-PL4 (bph2 carrier), a pyramided line in which both genes were combined, and a susceptible japonica recurrent parent Tsukushibare. RT-PCR and quantitative RT-PCR analyses showed that the Vg mRNA level decreased greatly in Hatano-66 on the resistant lines. In contrast, the level of reduction on the resistant lines was much less in Nagasaki-03. Immunoblot analysis showed that Nagasaki-03 retained comparable levels of 175 kDa Vg protein on both the susceptible and resistant lines, whereas in Hatano-66, no Vg protein was detected on the resistant lines. Our results showed that BPH resistance genes caused differential reduction in the accumulation of Vg mRNA and protein, leading to the retardation of BPH reproduction on the resistant host rice plants.     

Phenotypic mechanisms of host resistance against greenbug (Homoptera: Aphididae) revealed by near isogenic lines of wheat

Interactions between biotype E greenbug, Schizaphis graminum (Rondani), and wheat, Triticum aestivum L., were investigated using resistant and susceptible near isogenic lines of the greenbug resistance gene Gb3. In an antixenosis test, the greenbugs preferred susceptible plants to resistant ones when free choice of hosts was allowed. Aphid feeding resulted in quick and severe damage to susceptible plants, which seemed to follow a general pattern spatially and was affected by the position where the greenbugs were initially placed. Symptom of damage in resistant plants resembled senescence. Within-plant distribution of aphids after infestation was clearly different between the two genotypes. Significantly more greenbugs fed on the first (oldest) leaf than on the stem in resistant plants, but this preference was reversed in the susceptible one. After reaching its peak, aphid population on the susceptible plants dropped quickly. All susceptible plants were dead in 10-14 d after infestation due to greenbug feeding. Aphid population dynamics on resistant plants exhibited a multipeak curve. After the first peak, the greenbug population declined slowly. More than 70% of resistant plants were killed 47 d after infestation. Performance of both biotype E and I greenbugs on several Gb3-related wheat germplasm lines were also examined. It seems that the preference-on-stem that was characteristic of biotype E greenbugs on the susceptible plants was aphid biotype- and host genotype-dependent. Results from this study suggested that antixenosis, antibiosis, and tolerance in the resistant plants of wheat might all contribute to resistance against greenbug feeding.