梨小食心虫普通气味结合蛋白2（GmolGOBP2）cDNA的克隆与原核表达

【目的】克隆梨小食心虫(Grapholita molesta)普通气味结合蛋白2(GmolGOBP2)的全长cDNA序列并进行原核表达,为研究该蛋白在梨小食心虫化学感受系统中的作用奠定基础。【方法】利用RT-PCR和RACE技术克隆GmolGOBP2的全长cDNA序列,并使用原核表达载体pET-32a在大肠杆菌BL21(DE3)中进行表达,用SDSPAGE和Western blot检测其表达情况。【结果】GmolGOBP2的cDNA全长序列为637bp(GenBank登录号:JN857940),开放性阅读框长度为483bp,编码161个氨基酸残基,成熟蛋白分子质量为15.98ku,等电点为4.85。GmolGOBP2预测蛋白的N末端具20个氨基酸残基组成的信号肽序列,并且氨基酸序列中具有6个保守半胱氨酸的典型气味结合蛋白家族标志。将GmolGOBP2编码序列重组到表达载体pET-32a中,转入大肠杆菌BL21(DE3)进行原核表达,SDS-PAGE和Western blot检测结果显示,梨小食心虫普通气味结合蛋白基因在大肠杆菌中成功地表达出分子质量约为32ku的融合蛋白,与预测的融合蛋白分子质量大小一致。【结论】克隆并原核表达了GmolGOBP2的cDNA序列,可用于研究该蛋白分子结构及其在化学感受系统中的功能。     

梨小食心虫滞育与非滞育幼虫抑制性消减文库的构建与分析

【目的】分离和鉴定梨小食心虫（Grapholita molesta（Busck））滞育相关基因。【方法】分别以梨小食心虫滞育和非滞育幼虫为材料,通过抑制性消减杂交技术（SSH）,构建梨小食心虫滞育与非滞育正、反向差减cDNA文库,从中筛选梨小食心虫滞育相关基因,并对其进行测序和分析。【结果】在获得的128个滞育特异和132个非滞育特异的EST中,有滞育差异表达EST 42条（17条功能未知）、非滞育差异表达EST 46条（22条功能未知）。对差异表达EST同源检索后推测,其功能大部分与滞育或非滞育特性相关。在滞育个体中,代谢酶和滞育关联蛋白基因表达量较高;在非滞育个体中,贮存蛋白和信号传递基因表达量较高。【结论】这些EST基本反映了梨小食心虫在滞育与非滞育期间的基因表达谱,为今后深入研究梨小食心虫滞育的分子机理奠定了基础。     

两种农药不同亚致死浓度对梨小食心虫生长发育和繁殖的影响

在室内将梨小食心虫即将孵化的卵置于浸药幼果上,测定高效氯氟氰菊酯和氯虫苯甲酰胺不同亚致死浓度对梨小食心虫不同龄期幼虫发育时间、幼虫和蛹的发育历期、化蛹率和成虫羽化率、成虫产卵量的影响。结果表明,两种药剂不同亚致死浓度均使害虫不同龄期发育时间、幼虫和蛹发育历期显著延长,且随药剂浓度增加而增加;对化蛹率影响较小;两种药剂较高浓度会显著影响羽化率。两种药剂不同亚致死浓度对蛹重和产卵量影响显著:高效氯氟氰菊酯3个浓度均会显著减轻雌、雄蛹重量,且对雌蛹重量抑制率要高于雄蛹,并使成虫产卵量减少;氯虫苯甲酰胺LC50剂量也会显著减轻雌、雄蛹重量,使成虫产卵量显著减少,当药剂浓度降至LC20和LC30时,氯虫苯甲酰胺对雌、雄蛹重量和成虫产卵量影响不显著,但抑制率出现负值。     

Sex-related differences in the tolerance of Oriental fruit moth (Grapholita molesta) to organophosphate insecticides

In vivo toxicity assays have shown that organophosphate insecticides are less toxic to male than to female Oriental fruit moths Grapholita molesta. While male moths have higher levels of acetylcholinesterase and general esterase activities, female moth acetylcholinesterase enzymes are less sensitive to aromatic and aliphatic organophosphates than male enzymes. Elevated esterase and acetylcholinesterase activities in male moths explain their greater tolerance to aromatic and aliphatic organophosphates. Male and female acetylcholinesterase enzymes are equally tolerant to heteroaromatic organophosphates, the most widely used of this class of insecticides in G molesta control. This observation, in contrast to the greater sensitivity of male acetylcholinesterases to aromatic and aliphatic organophosphates, shows the potential for the evolution of insensitive target sites in male moths, which would increase male G molesta tolerance to these insecticides. Significant sex-linked differences in insecticide tolerance have not been reported previously in lepidopterans. The practical implications of the observed differences in tolerance in male and female G molesta question the practice of using pheromone traps to monitor populations of these moths in orchards.     

Management of insecticide resistance in Oriental fruit moth (Grapholita molesta; Lepidoptera: Tortricidae) populations from Ontario

The development of resistance in the Oriental fruit moth, Grapholita molesta (Busck) to organophosphorus (OP) insecticides (azinphos-methyl and phosmet) is a serious threat to the tender fruit industry in Ontario (50% crop losses in 1994). Resistance to carbamate insecticides and increased survival of field-collected moths at diagnostic concentrations of pyrethroids were widespread. As a result, four different treatment regimes were tested to manage resistance in G molesta, and the changes in resistance frequencies under each treatment regime were monitored from 1996 to 1999. The data indicated that the levels of resistance were significantly influenced by the various treatment regimes. The seasonal pattern of resistance was similar for all treatment regimes, in that resistance peaked in mid-season and declined in the late season. Levels of resistance in G molesta to OPs decreased from 55% to 14% and that to pyrethroids declined from 30% to 10% from 1996 to 1999 under a treatment regime consisting of endosulfan-organophosphate-pyrethroid rotation. Similarly, under a treatment regime implemented in commercial orchards (organophosphate-pyrethroid rotation), resistance to OP insecticides declined from 50% to 12% and resistance to pyrethroids evolved to around 16%. The overall data indicated that resistance was unstable; a strategy based on rotation of insecticides by class for each generation of G molesta was successful in managing resistance to both OP and pyrethroid insecticides. The rotational strategy has been widely adopted by growers and is applied to ca 85% of the acreage.     

梨小食心虫气味受体GmolOR10基因克隆及表达谱分析

为研究梨小食心虫Grapholita molesta Busck嗅觉通讯分子机制,本研究应用RT-PCR克隆获得了梨小食心虫气味受体基因GmolOR10的完整开放阅读框,并对其序列结构进行了相关生物信息学分析,最后采用实时荧光定量PCR对GmolOR10在梨小食心虫成虫不同组织(触角、头、胸、腹、足、翅)与不同发育阶段的表达模式进行了定量分析。结果表明,GmolOR10编码区长1 194 bp,编码397个氨基酸,等电点和相对分子量分别为8.57和46.14 kD,有7个跨膜结构域。多序列比对与系统发育树结果显示GmolOR10与卷蛾科苹果蠹蛾Cydia pomonella CpomOR18和山毛榉卷叶蛾Cydia fagiglandana CfagOR18的氨基酸序列一致性较高,分别为84.38%和83.38%。GmolOR10在雄成虫触角中表达量较高,极显著高于雌虫触角(P<0.01),在雌、雄成虫头部也有较高的表达量,在成虫其他组织表达量很低。不同发育阶段的表达谱显示,GmolOR10在梨小食心虫的不同发育阶段均有表达,在成虫期的表达量最高(在雌雄成虫中的表达量均呈现出随着...     

福州地区桃园梨小食心虫发生动态研究

为了解福州地区桃园梨小食心虫的发生规律,找到合适的防治时期和方法,对福州地区桃园梨小食心虫进行了连续4年的发生动态监测以及危害情况调查,结果表明：虽然每一年的发生动态都有差异,但总体表现出了一定规律,每年3月初桃树开花展叶时期,梨小食心虫呈现暴发状态,数量出现高峰,另外一个高峰位于6月中旬至7月新梢生长的时期,4、5、8、9月数量较少,10月至第二年2月蛰伏越冬,根据折梢率和蛀果率的调查也得出结论,认为在福州地区梨小食心虫的危害主要在新梢上,对果实危害较小,而防治梨小食心虫的最佳时间段为每年的3月、6月和7月。     

梨小食心虫成虫行为节律研究

【目的】研究梨小食心虫成虫行为节律,为开展梨小食心虫化学生态学研究及其综合治理奠定基础。【方法】在人工气候箱(温度(24±0.5)℃、相对湿度(70±10)%、光周期为15h光期和9h暗期)条件下,系统观测了梨小食心虫成虫羽化、交配及产卵行为节律。【结果】梨小食心虫成虫的羽化具有明显的昼夜节律,主要发生在光期05:00-10:00这个时段,此时成虫的羽化数量占总羽化数量的90%以上,其中以05:00-06:00羽化率最高,与其他时段差异显著;成虫的求偶交配行为多发生在羽化后第3天,呈"一"字形交配,单次交配持续时间为11～35min,平均为22.07min,成虫交配活动主要发生在17:00-21:00,交配率超过90%;梨小食心虫雌成虫的产卵节律与其交配节律颇具相似性,其产卵活动也主要发生在17:00-21:00,该时段所产卵量达到总产卵量的87.43%,显著高于其他时段。【结论】梨小食心虫成虫的羽化、交配及产卵行为具明显的生物节律,其中羽化行为主要发生在上午05:00-10:00,交配和产卵行为主要发生在傍晚17:00-21:00。     

梨小食心虫滞育过程中几种代谢酶活力的相关变化

梨小食心虫Grapholita molesta是一种重要的蛀果害虫,以老熟幼虫滞育越冬。为明确梨小食心虫滞育幼虫对低温的生理适应性,用3,5-二硝基水杨酸比色法、双试剂法及代谢酶活力测试盒分别测定了梨小食心虫幼虫体内海藻糖酶（trehalase,TRE）、山梨醇脱氢酶（sorbitol dehydrogen-ase,SDH）及己糖激酶（hexokinase,HK）、丙酮酸激酶（pyruvate kinase,PK）和碱性磷酸酶（alkalinephosphatase,ALP）的活力变化。在低温条件下,滞育幼虫体内各酶活力明显不同,TRE活力在滞育初期显著提高,而后随滞育时间的延长而逐渐降低;SDH活力总体变化较为平缓,变化量较小;HK、PK和ALP活力在滞育前期均显著降低,且随后一直保持在较低水平。滞育期间,5种代谢酶的活力均与滞育发育及温度有关;通过相关代谢酶的调节,梨小食心虫的代谢速率维持在较低水平,提高了虫体的耐寒能力,从而顺利完成滞育。