RNAi介导的HvCDA1基因沉默影响茄二十八星瓢虫存活和发育

【目的】茄二十八星瓢虫Henosepilachna vigintioctopunctata是茄科(Solanaceae)植物上的重要害虫。昆虫体内几丁质脱乙酰酶1(chitin deacetylase 1, CDA1)催化N-乙酰氨基-D-葡萄糖胺脱去乙酰基,促使几丁质转化为壳聚糖,控制昆虫体内几丁质纤维有序堆积,并维持角质层结构的完整性。抑制虫体中CDA1基因的表达会抑制壳聚糖的合成,影响昆虫表皮结构的形成,使昆虫不能正常发育而亡。【方法】利用RT-qPCR方法测定HvCDA1基因在茄二十八星瓢虫不同发育阶段(卵、1-4龄幼虫和预蛹)和4龄幼虫不同组织(表皮、脂肪体、中肠和马氏管)中的表达模式。通过饲喂茄二十八星瓢虫1龄幼虫不同浓度dsHvCDA1溶液浸泡处理1 min的茄子叶片后及直接饲喂4龄幼虫不同浓度dsHvCDA1溶液,探究沉默茄二十八星瓢虫HvCDA1基因对其幼虫存活和发育以及HvCDA1基因表达量的影响。【结果】发育表达谱结果表明,HvCDA1在茄二十八星瓢虫的各发育阶段均有表达,但在1龄末和2龄末幼虫中的表达量最高。组织表达谱结果显示,在茄二十八星瓢虫4龄幼虫的表皮中H...     

马铃薯瓢虫和茄二十八星瓢虫体表细微结构的比较研究

本研究利用扫描电镜对马铃薯瓢虫Henosepilachnavigintioctomaculata (Motschulsky)和茄二十八星瓢虫H .vigintioctopunctata (Fabricius)体表的细微结构进行了比较研究。结果表明 ,马铃薯瓢虫和茄二十八星瓢虫在细微结构上存在明显差异。第一 ,2种瓢虫鞘翅表面凹陷内 ,毛的着生位置不同 ,马铃薯瓢虫的毛着生于凹陷中心 ,茄二十八星瓢虫的毛着生于凹陷边缘 ;第二 ,鞘翅表面凹陷的深浅不同 ,马铃薯瓢虫的凹陷较深于茄二十八星瓢虫。这为 2种瓢虫的形态学研究提供了细微结构方面的依据     

茄子品种对茄二十八星瓢虫及其寄主蜂生长发育的影响

茄子不同品种对茄二十八星瓢虫及瓢虫标腹姬小蜂的影响结果表明,上海紫茄、呼杂34^#、托塔姆3^#、洋红茄4个品种中,取食上海紫茄的瓢虫发育历期明显短于取食其它3个品种的瓢虫。其幼虫－蛹历期比在托塔姆3^#上取食的瓢虫发育历期快5.69d。瓢虫柄腹姬小蜂在取食上海紫茄的瓢虫幼虫内发育出蜂数多、性比高,但寄生率稍低。而在托塔姆3^#上取食的幼虫内发育出蜂数少、羽化率低、蜂体小、但寄生率稍高。该蜂喜在3,4龄幼虫内寄生,在其上寄生蜂出蜂多、性比高、蜂的个体大。     

瓢虫柄腹姬小蜂对茄二十八星瓢虫功能反应的研究

研究了瓢虫柄腹姬小蜂功能反应。结果表明,在供试温度下,瓢虫柄腹姬小蜂对茄二十八星瓢虫的功能反应均符合Holling圆盘方程Ⅱ型,以25℃下的搜寻效率最高,其模型为Nα=1．94N／(1 0．39N),在茄枝叶、龙葵枝叶、酸酱枝叶和西红柿枝叶4种叶片上,以茄枝叶、龙葵枝叶上的搜寻效率最高,分别为1．85和1．94。瓢虫柄腹姬小蜂对不同密度的2、3、4龄幼虫的功能反应,以4龄的搜寻效率最高,其次为3龄,最低为2龄,实验中未发现瓢虫柄腹姬小蜂寄生1龄的幼虫。不同处理方法、不同药剂影响瓢虫柄腹姬小蜂对不同寄主密度的功能反应,其中阿维菌素组的搜寻效率与对照组没有显著性差异,苦皮藤索组的搜寻效率稍有降低,而敌敌畏、氯氰菊酯处理组的搜寻效率显著下降,处理时间相对延长。     

茄二十八星瓢虫为害对不同基因型番茄叶内三种抗性酶活性的影响

【目的】研究茉莉酸合成相关基因在番茄抗茄二十八星瓢虫Henosepilachna vigintioctopunctata Fabricius中的作用,探讨茉莉酸信号因子在植物防御过程中起到的作用。【方法】利用3种基因型番茄茉莉酸合成缺失突变体spr2、茉莉酸合成过量表达体35s、野生型番茄Wt为材料,通过经2龄茄二十八星瓢虫分别取食6、24、48 h后,利用分光光度计测定3种基因型番茄叶片内苯丙氨酸解氨酶(Phenylalanineammonialyase,PAL)、胰蛋白酶抑制剂(Protease inhibitors,PI)和脂氧合酶(Lipoxygenase,LOX)酶活性的动态变化。【结果】35s型番茄叶片内PI活性显著高于另两种番茄叶片,而spr2型番茄叶片PI活性最低。瓢虫为害后,3种基因型番茄PI酶活性明显提高,在35s和Wt型番茄中,PI和LOX酶活性在胁迫24 h和6 h达到最高值,而spr2型番茄没有显著变化。受到瓢虫侵染的系统叶中,PI和LOX酶活性也受到了诱导,系统叶变化趋势同危害叶相同,但诱导的PI和LOX酶活性明显低于危害叶。在危害处理过程中,均呈现上升-下降的趋势,在系统叶中,则PAL活性没有显著变化。番茄叶片受害后,PI和LOX酶活性上升辐度大于PAL,说明PI和LOX对取食胁迫响应比PAL更敏感。【结论】35s茉莉酸合成过量表达体的番茄对茄二十八星瓢虫有一定的抗虫作用,因此,茉莉酸在植物抗性中起了一定作用。     

蠋蝽对茄二十八星瓢虫的捕食功能反应

【目的】明确蠋蝽对茄二十八星瓢虫的捕食能力。【方法】在实验室条件下测定蠋蝽5龄若虫对茄二十八星瓢虫2和4龄幼虫的捕食功能反应及搜寻效应。【结果】蠋蝽5龄若虫对茄二十八星瓢虫2和4龄幼虫的捕食行为均符合Ⅱ型功能反应;随着茄二十八星瓢虫密度的增加,蠋蝽的搜寻效应逐渐降低,蠋蝽5龄若虫对茄二十八星瓢虫2龄幼虫的搜寻效应明显大于4龄幼虫;蠋蝽5龄若虫对茄二十八星瓢虫2和4龄幼虫的实际最大捕食量分别为15和10头。【结论】蠋蝽5龄若虫对茄二十八星瓢虫幼虫具有较好的捕食作用。     

马铃薯瓢虫与茄二十八星瓢虫SCAR标记的建立及应用

为了探索快速鉴定马铃薯瓢虫Henosepilachna vigintioctomaculata(Motschulsky)和茄二十八星瓢虫Henosepilachna vigintioctopunctata(Fabricius)的分子生物学方法,本研究在随机扩增多态性DNA(random amplified polymorphic DNA,RAPD)的基础上,分别设计了可以鉴别两个物种的序列特征扩增区域(sequence characterized amplified regions,SCAR)标记。从随机合成的60条引物中筛选出来2条特异性引物(分别为OPI-6和OPJ-15),引物OPI-6在马铃薯瓢虫中扩增出约750 bp的特异性条带,引物OPJ-15在茄二十八星中扩增出约750 bp的特异性条带,根据测序结果设计了两对SCAR引物对筛选结果进行验证,发现根据OPI-6的测序结果所设计的SCAR引物(OPI-6 test)仅能在马铃薯瓢虫中扩增出645 bp的条带,而根据OPJ-15的测序结果所设计的SCAR引物(OPJ-15 test)仅能在茄二十八星瓢虫中扩增出436 bp的条带。这两对SCAR引物能够准确、稳定且快速地区分马铃薯瓢虫与茄二十八星瓢虫,对这两种害虫的精准防控具有重要意义。     

茄二十八星瓢虫在茄子叶片上垂直方向的附着力

本文采用电子天平称重法对茄二十八星瓢虫的2龄幼虫、3龄幼虫、4龄幼虫、雌成虫和雄成虫在茄子叶片上垂直方向的附着力进行测定,为进一步研发和利用气流控制技术防治农业害虫提供技术参数。研究结果表明,2龄幼虫、3龄幼虫、4龄幼虫、雌成虫和雄成虫垂直方向的最大附着力分别为7.3±0.3 mN,8.6±0.6 mN,12.0±0.3 mN,15.8±1.3 mN和13.0±1.0 mN。茄二十八星瓢虫垂直方向的附着力从大到小的顺序均为:雌成虫雄成虫;4龄幼虫3龄幼虫2龄幼虫。幼虫的附着力/虫体体重系数由大到小的顺序为:2龄幼虫3龄幼虫4龄幼虫,所有处理的附着力与虫体体重的相关性均不显著。附着力/虫体重量的系数范围为47.8-90.8,身体重量占附着力1.1%-2.1%。结果进一步表明,在设计旋风式气流吸虫机的频率等参数时,要考虑茄二十八星瓢虫在茄子叶片上的附着力远大于其体重的情况。     

茄二十八星瓢虫成虫天花板爬行行为研究

【目的】探明茄二十八星瓢虫Henosepilachna vigintioctopunctata(Fabricius)成虫的天花板爬行行为,为气流控制技术防治农业害虫提供技术参数。【方法】本研究通过采用录像观察法对茄二十八星瓢虫雌雄成虫的天花板爬行行为进行观测,明确其成虫的运动行为参数,并分析了其步速分别与步距、步频和负荷因数的关系。【结果】茄二十八星瓢虫雌雄成虫天花板爬行时的速度为3.77~19.32 mm/s,步距为2.63~4.51 mm,步频范围为1.32~4.74 Hz。步距和步频均与速度之间存在极显著线性关系,速度的增加同时依靠步距和步频的增加,但步距的变化相对步频的变化小;前足、中足和后足的负荷因数均随着速度的提高而降低,且负荷因数均与速度存在显著相关性;一个步态周期内,足的摆动时间均远小于支撑时间。【结论】茄二十八星瓢虫成虫天花板爬行时速度的增加主要是依靠步频的提高来实现。     

茄子品种对茄二十八星瓢虫及其寄生蜂生长发育的影响

茄子不同品种对茄二十八星瓢虫及瓢虫柄腹姬小蜂的影响结果表明 ,上海紫茄、呼杂 3 4# 、托塔姆 3 # 、洋红茄 4个品种中 ,取食上海紫茄的瓢虫发育历期明显短于取食其它 3个品种的瓢虫。其幼虫-蛹历期比在托塔姆 3 # 上取食的瓢虫发育历期快 5 69d。瓢虫柄腹姬小蜂在取食上海紫茄的瓢虫幼虫内发育出蜂数多、性比高 ,但寄生率稍低。而在托塔姆 3 # 上取食的幼虫内发育出蜂数少、羽化率低、蜂体小、但寄生率稍高。该蜂喜在 3 ,4龄幼虫内寄生 ,在其上寄生蜂出蜂多、性比高、蜂的个体大     

Oral delivery of dsHvlwr is a feasible method for managing the pest Henosepilachna vigintioctopunctata (Coleoptera: Coccinellidae)

RNA interference(RNAi)techniques have emerged as powerful tools that facilitate development of novel management strategies for insect pests,such as Henosepilachna vigintioctopunctata(Coleoptera:Coccinellidae),which is a major pest of solanaceous plants in Asia.In this study,the potential of oral delivery of in vvYro-synthesized and bacterially expressed double-stranded H.vigintioctopunctata lesswright(Iwr)gene(dsHvlwr)to manage of H.vigintioctopunctata was investigated.Our results showed that the gene Hvlwr had a 480-bp open reading frame and encoded a 160-amino acid protein.Hvlwr expression levels were greater in the fat body than other tissue types.Hvlwr silencing led to greater H.vigintioctopunctata mortality rates and appeared to be time-and partially dose-dependent,likely as a result of the number of hemocytes increasing with dsRNA concentration,but decreasing with time.Bacterially expressed dsHvlwr that was applied to leaf discs caused 88%,66%,and 36%mortality in 1st instars,3rd instars,and adults after 10,10,and 14 d,respectively;when applied to living plants,there was greater mortality in 1 st and 3rd instars,but there was no effect on adults.Furthermore,dsHvlwr led to improved plant protection against H.vigintioctopunctata.Our study shows an effective dietary RNAi response in H.vigintioctopunctata and that Hvlwr is a promising RNAi target gene for control of this pest species.     

A gene silencing of V‐ATPase subunit A interferes with survival and development of the tomato leafminer,Tuta absoluta

RNA interference (RNAi) technology is not only considered as a tool to analyze gene function, but it is also potentially considered as a strategy to develop novel biopesticide. In the current study, a double‐stranded RNA specific to v‐ATPase subunit A of the tomato leafminer, Tuta absoluta (Meyrick; Lepidoptera: Gelechiidae), was orally administered. A gradual decrease in the expression of the gene was observed from Day 1 to 3 and resulted in significant larval mortality. These results suggest that v‐ATPases A can be considered as a promising target gene by RNAi technology to be used in the management of the tomato leafminer.     

RNAi knockdown of CAPAr gene affects survival and fecundity of Bemisia tabaci (Gennadius)

Bemisia tabaci (Gennadius) Asia II 1 cause significant economic losses to cotton crop, directly by feeding on sap and indirectly through virus transmission. Although the current control methods are the combination of different tactics but still the chemical control is heavily relied. B. tabaci has gained resistance to majority of the insecticides which has made it difficult to manage this pest using synthetic chemicals. RNAi based gene silencing has been evolved as potential biochemical tool for specific gene silencing of target insect-pest and has been successfully demonstrated for Hemipteran insects. Here, we report the successful silencing of the CAPAr gene in B. tabaci which encodes for the G-protein coupled receptor (a transmembrane membrane peptide). Silencing of CAPAr gene resulted into reduced survival and fecundity of B. tabaci. Oral delivery of dsRNA mediated artificial diet corresponding to CAPAr gene resulted into significantly high adult mortality of 30.74% in treatment dsCAPAr 1.0 µg/µl after 48 hr of dsRNA feeding. A significant reduction in whitefly female fecundity was recorded for all the dsCAPAr concentrations with lowest value of 53.51 eggs per female in dsCAPAr 1.0 µg/µl. The relative gene expression of CAPAr gene further confirms the down regulation of CAPAr gene in knock down adult whiteflies. Our investigation proves the importance of CAPAr for B. tabaci survival and may be exploited as potential tool which can be strategic in integrated management programme for B. tabaci.     

dsRNA介导植物基因沉默及其应用

植物双链RNA(double stranded RNA,dsRNA)能有效干扰同源基因的表达,近年来已成为功能基因组学研究上的新方法。本文综述了植物dsRNA介导的转基因沉默现象及其特点、分子作用机制、主要介导方法,以及近年来在植物功能基因组学研究上的应用情况。     

油菜BnCr4基因RNAi载体构建及植株转化

该实验构建了含甘蓝型油菜黄化相关基因BnCr4特异片段反向重复结构的RNA干扰(RNAi)载体pFGC5941-Cr4,通过根癌农杆菌介导转化油菜,获得47株抗Basta的抗性再生油菜植株,其中10株经PCR鉴定为阳性转基因植株.随机选取3株经鉴定的转基因阳性油菜植株进行半定量RT-PCR分析,结果显示,相对于非转基因的野生型油菜,3株转基因植株中BnCr4基因的表达量分别降低了78.5%、8.5%、11.8%,表明该干扰载体转入油菜能特异引起植株BnCr4基因表达量下降.