Feeding and oviposition preference studies of Helopeltis theivora Waterhouse, on leaves and stems of tea, Camellia sinensis L.（O）Kuntze, revealed that the insect mostly preferred the second leaf for feeding in compa...Feeding and oviposition preference studies of Helopeltis theivora Waterhouse, on leaves and stems of tea, Camellia sinensis L.（O）Kuntze, revealed that the insect mostly preferred the second leaf for feeding in comparison to the first, and the third leaf and stems; while the fifth instar nymphs were the most voracious feeders among the life stages, producing themost and largest feeding lesions. The insect preferred to oviposit mainly on stems and very few eggs are found on leaves.展开更多
A procedure is described to isolate intact RNA from tissues not previously undertaken-highly infested and wilted apical buds and leaves of tea [Camellia sinensis （L.） O. Ktze.]. The protocol uses a final concentrati...A procedure is described to isolate intact RNA from tissues not previously undertaken-highly infested and wilted apical buds and leaves of tea [Camellia sinensis （L.） O. Ktze.]. The protocol uses a final concentration of 450 mM β mercaptoethanol （βME） and 10% Polyvinylpyrrolidone （PVP） to circumvent problems associated with large amounts of polyphenols, polysaccharides, pigments and other secondary metabolites not easily removed by conventional procedures. Furthermore, the proposed protocol is applicable to normal tissues and other plant tissues with similar stresses, containing compounds that interfere with RNA extractions. Total RNA could be used for downstream applications such as mRNA isolation, reverse transcription, quantitative Polymerase Chain Reaction （PCR）, cDNA library construction and Rapid amplification of cDNA ends （RACE）.展开更多
为明确腰果角盲蝽Helopeltis theivora气味结合蛋白3(odorant binding protein 3,OBP3)功能及其嗅觉感受机制,利用PCR技术结合c DNA末端快速扩增(rapid amplification of c DNA ends,RACE)技术克隆其cDNA全长序列,利用多个生物信息学软...为明确腰果角盲蝽Helopeltis theivora气味结合蛋白3(odorant binding protein 3,OBP3)功能及其嗅觉感受机制,利用PCR技术结合c DNA末端快速扩增(rapid amplification of c DNA ends,RACE)技术克隆其cDNA全长序列,利用多个生物信息学软件对其进行序列分析,并通过实时荧光定量PCR(real-time quantitative PCR,qPCR)技术检测其在腰果角盲蝽成虫不同组织中的表达量。结果显示,腰果角盲蝽HtheOBP3基因(GenBank登录号为QHI06949)开放阅读框为474 bp,编码157个氨基酸残基,预测蛋白分子量约为17.15 kD,等电点为5.14,无信号肽和跨膜结构,蛋白氨基酸序列中具有6个保守半胱氨酸残基和性信息素结合蛋白-普通气味结合蛋白(pheromone binding protein-general odorant binding protein,PBP-GOBP)家族的保守结构域。HtheOBP3蛋白具有6个α-螺旋和3对二硫键,其中5个α-螺旋形成1个结合口袋。腰果角盲蝽HtheOBP3与其他20种半翅目昆虫OBP的6个保守半胱氨酸位点完全一致,腰果角盲蝽HtheOBP3的氨基酸序列与薇甘菊颈盲蝽Pachypeltis micranthus PmicOBP4的氨基酸序列一致性最高,为55.56%。在37种不同昆虫的OBP蛋白中,腰果角盲蝽HtheOBP3与6种半翅目昆虫OBP聚为一个分支,其中与薇甘菊颈盲蝽PmicOBP4亲缘关系最近。腰果角盲蝽成虫各组织中HtheOBP3均有表达,其中在触角中表达量最高,其次为足。表明腰果角盲蝽HtheOBP3是典型的气味结合蛋白,其可能兼具嗅觉和非嗅觉感受等生理功能。展开更多
文摘Feeding and oviposition preference studies of Helopeltis theivora Waterhouse, on leaves and stems of tea, Camellia sinensis L.（O）Kuntze, revealed that the insect mostly preferred the second leaf for feeding in comparison to the first, and the third leaf and stems; while the fifth instar nymphs were the most voracious feeders among the life stages, producing themost and largest feeding lesions. The insect preferred to oviposit mainly on stems and very few eggs are found on leaves.
文摘A procedure is described to isolate intact RNA from tissues not previously undertaken-highly infested and wilted apical buds and leaves of tea [Camellia sinensis （L.） O. Ktze.]. The protocol uses a final concentration of 450 mM β mercaptoethanol （βME） and 10% Polyvinylpyrrolidone （PVP） to circumvent problems associated with large amounts of polyphenols, polysaccharides, pigments and other secondary metabolites not easily removed by conventional procedures. Furthermore, the proposed protocol is applicable to normal tissues and other plant tissues with similar stresses, containing compounds that interfere with RNA extractions. Total RNA could be used for downstream applications such as mRNA isolation, reverse transcription, quantitative Polymerase Chain Reaction （PCR）, cDNA library construction and Rapid amplification of cDNA ends （RACE）.
文摘为筛选特定条件下实时荧光定量PCR检测体系中能稳定表达的茶刺盲蝽Helopeltis theivora内参基因,从其转录组中筛选并克隆11个候选基因Actin、β-tubulin1、RPL13A、EF1α、RPS3A、GAPDH、18S RNA、G6PDH、EIF4A、TBP和UBQ,测定它们在不同温度及药剂胁迫下mRNA的表达水平,并通过geNorm、NormFinder、BestKeeper、Delta Ct和RefFinder软件(算法)分析各基因的稳定性。结果表明,11个候选基因引物均具有良好的特异性和扩增效率(90.10%~96.87%)。温度胁迫下,EIF4A有最小的平均变异度(mean variability,M)、稳定值(stability value,SV)和几何平均值,分别为0.200、0.096和2.060;RPS3A的平均标准偏差(average of standard deviation,STDEV)最小,为0.605;二者是表达最稳定的基因。药剂胁迫下,RPL13A的M值和STDEV最小,分别为0.123和0.660;RPS3A的SV、标准偏差(standard deviation,SD)和几何平均值均最小,分别为0.063、0.336和1.189;二者是表达最稳定的基因;Actin为最不稳定基因。全样品评价中,RPL13A(M值为0.282、几何平均值为1.565)和RPS3A(SV为0.099、STDEV为0.614)的评价值同比最小,是表达最稳定的基因;最不稳定的基因为Actin、β-tubulin1和TBP。geNorm分析结果还显示所有处理条件下最适内参基因数目均为2个。表明EIF4A和RPS3A可作为茶刺盲蝽与温度相关的抗性基因表达研究的内参基因,RPS3A和RPL13A可作为该虫抗药基因表达研究的内参基因。
