【作者】 张长禹；

【导师】 雷朝亮；

【作者基本信息】 华中农业大学 ， 农业昆虫与害虫防治， 2010， 博士

【摘要】 棉铃虫Helicoverpa armigera (Hubner)隶属于鳞翅目夜蛾科,广泛分布于亚洲、欧洲、非洲及澳洲等地。棉铃虫对紫外光(Ultraviolet, UV)具有很强的趋性,是一种典型的趋光性昆虫。因此,本研究以棉铃虫为试验材料,研究了棉铃虫适应UV胁迫的生理生态对策,以及UV胁迫下棉铃虫热激蛋白的表达情况。其主要结果如下：1.UV照射对棉铃虫造成的氧化损伤分析本研究通过测定丙二醛(MDA)和蛋白羰基含量,分析了UV照射对棉铃虫造成的氧化损伤。当UV照射棉铃虫成虫30、60和90 min时,其体内蛋白质羰基含量显著升高,MDA含量没有发生显著变化。表明UV照射引起的氧化损伤体现在棉铃虫成虫体内蛋白质羰基含量的大幅度增加,进一步证实UV照射对棉铃虫是一种胁迫,可能会导致蛋白质结构功能的破坏,或加快蛋白的氧化进程。2.UV照射对棉铃虫成虫寿命、生殖力及子代发育的影响本研究测定了UV照射对棉铃虫成虫寿命、生殖力和子代发育的影响。每天对棉铃虫成虫进行不同时间的UV照射(O、1、5、9h/day),直到雌雄蛾全部死亡。研究发现,棉铃虫雌雄蛾的寿命均随着UV照射时间的延长而缩短,且在照射时间为5和9h/day的UV处理组达到显著水平。当UV照射棉铃虫成虫1和5 h/day时,每雌总产卵量与对照相比有所增加,且在5 h/day达到显著水平,相似地,UV处理组雌虫的平均产卵量显著高于对照组的雌虫。UV照射对棉铃虫子代发育影响的结果显示,当棉铃虫接受UV照射后,其子代的卵孵化率、化蛹率和羽化率没有受到显著影响,但是,长时间的UV照射(5和9h/day)会导致子代累计存活率的降低,及子代幼虫的发育历期的显著延长。我们推测,UV照射后,棉铃虫成虫的寿命和生殖之间存在一种补偿效应,而这种生殖补偿正是棉铃虫适应UV胁迫的生态对策。3.UV照射对棉铃虫体内保幼激素的影响为了研究UV照射对棉铃虫成虫体内保幼激素的影响,本实验采用了高效液相色谱法,测定了不同日龄(1、2、3、4、5日龄)和不同UV照射时间(0、1、5、9h/day)下保幼激素JHI、JHII和JHIII的滴度。棉铃虫体内JHI、JHII和JHIII的滴度随着日龄的变化发生了显著的改变；棉铃虫体内JHI、JHII和JHIII的滴度随着UV照射时间的延长也发生了显著的变化,与对照组相比,5 h/day与9 h/day的UV处理组JHI、JHII和JHⅢ的滴度均显著下降；其中,9 h/day的UV处理组JHII滴度最低；此外,日龄与UV照射的交互作用显著。本章的研究结果说明了UV照射能够影响到棉铃虫体内的保幼激素代谢水平,特别是长时间的UV照射能够显著降低棉铃虫成虫体内的保幼激素含量,可能意味着UV照射对棉铃虫造成的环境胁迫,迫使其改变了体内的激素分泌水平。4.棉铃虫hsp90、hsp70和hsc70在热激和UV胁迫下的表达本研究采用Northern-blot技术分析了棉铃虫hsp90、hsp70及hsc70在热激和UV胁迫下的表达情况,发现随着热激时间的延长,hsp90和hsp70的表达量都明显上升,hsc70的表达未发生明显变化；当热激处理时间60 min时,hsp90和hsp70的表达量达到最大值,随后表达量逐渐下降。UV照射对棉铃虫体内的Hsps也有影响,随着UV照射时间的延长,hsp90的表达量有上升的趋势,当UV照射时间在120 min时,棉铃虫体内的hsp90的表达量达到最高峰,在150 min和180 min时表达量下降。hsc70的表达则不受UV照射的影响,此外,在UV照射的处理组中均未能检测到hsp70的表达。我们推测hsps的表达升高可能是为了适应环境胁迫因子。5.棉铃虫hsp90与hsp70的克隆与序列分析本研究利用分子生物学技术得到了棉铃虫hsp90和hsp70两种重要基因的碱基序列全长。通过软件分析,我们推导出hsp90编码区有2154bp,推定的蛋白质序列由717氨基酸残基组成,分子量82.51kDa,等电点为4.92；hsp70编码区有1905bp,推定的蛋白质序列由634个氨基酸残基组成,分子量69.98 kDa,等电点为5.70。基于氨基酸序列全长,我们对Hsp90和Hsp70蛋白进行了多重序列对比,并构建了系统发育树,结果表明Hsp90和Hsp70具有很高的保守性,棉铃虫的Hsp90和Hsp70的氨基酸序列与谷实夜蛾H.zea的同源性最高,同属于鳞翅目夜蛾科。这也证实了本实验所得序列确为棉铃虫相应基因序列,并为深入了解昆虫在抗胁迫环境下的分子适应机制奠定了基础。更多还原

【Abstract】 The cotton bollworm, Helicoverpa armigera (Hubner) (Lepidoptera:Noctuidae) is one of the most serious insect pest in the world. The moths of this nocturnal insect display a conspicuous positive phototactic behaviour to light stimuli, and are especially sensitive to Ultraviolet (UV) light. In the present study, we study the effects of UV light irradiation on adult longevity and reproduction, molecular cloning and characterization of hsp90 and hsp70 and their expression patterns during thermal stress and UV light irradiation in H. armigera. Main results were summarized as follows:1. Oxidative damage analysis in H. armigera adults after exposure to UV lightThe effects of UV light irradiation on malondialdehyde (MDA) and protein carbonyl contents were investigated in H. armigera adults. The adults were exposed to UV light for various time periods (0,30,60 and 90 min). We found that exposure to UV light for 30, 60 and 90 min resulted in increased protein carbonyl content. However, there was no significant difference in MDA content in H. armigera adults following UV light exposure. We have confirmed that UV light may disturb the functional activity of protein and intensify the activity of protein oxidation process. This indicates that UV light is threatening to H. armigera adults.2. Effects of UV light irradiation on longevity and reproduction of H. armigera, and development of its F1 generationThe effects of UV light irradiation on adult longevity and reproduction of H. armigera were investigated, as well as the development of the F1 generation. Paired adults were exposed to UV light for various time periods (0,1,5 and 9 h/day), until the end of adult life. The results showed that adult longevity decreased with increasing exposure time for both sexes, and a significant decrease was observed after exposure for 5 and 9 h/day. Fecundity increased when adults were exposed for 1 and 5 h/day, and a significant difference was observed in the 5 h/day group. Oviposition rate of females in all treatments were significantly higher than control. Exposure to UV light for longer periods (5 and 9 h/day) caused a decline in cumulative survival of F1 immature stages, but no significant differences were found in egg hatching, pupation and eclosion. The developmental periods of F1 larvae were significantly prolonged after exposure to UV light for 5 and 9 h/day. UV light irradiation had no significant effects on F1 pupae period. All these physiological effects under UV irradiation may be strategies for insect adaptations.3. Effects of UV light irradiation on juvenile hormone titres of H. armigeraThe effect of UV light irradiation and age on juvenile hormone titres from H. armigera were measured with HPLC. The results indicated that JHI, JHII and JHIII titres varied signficantly across age and UV light irradiation. Female H. armigera showed the expected response to age and temperaturs and UV light irradiation. JHI, JHII and JHIII titres in the 5 and 9 h/day group decreased significantly. JHI, JHII and JHIII titres are lowest in the 9 h/day group. This indicates that UV light is threatening to H. armigera adults.4. The expresson patterns of hsp90 and hsp70 during thermal stress and UV light irradiation in H. armigeraThree heat shock protein transcripts, hsp90, hsp70, hsc70, isolated from H. armigera, were evaluated for their responsiveness to thermal stress and UV light irradiation. The expression patterns after heat shock indicated that hsp90 and hsp70 were up-regulated, and hsc70 was indeed a constitutively expressed member of hsp70 family. The expression patterns after UV light irradiation indicated that hsp90 was also up-regulated. Our results also indicated that hsps are responsive to thermal stress and UV light irradiation in H. armigera.5. Molecular cloning and characterization of hsp90 and hsp70 cognate cDNAs in H. armigeraThe sequence data of hsp90 and hsp70 indicates a 2476-bp full-length hsp90 cDNA with a 2154-bp ORF starting at nucleotide 163 and terminating at nucleotide 2316, which encodes 717 amino acid residues with a putative molecular mass of 82.51 kDa and a calculated pI of 4.92, a 2246-bp full-length hsp70 cDNA with a 1905-bp ORF starting at nucleotide 159 and stopping at 2063, which encodes 634 amino acid residues with a putative molecular mass of 69.98 kDa and a calculated pI of 5.70. Multiple sequence alignments indicate that the deduced amino acid sequences of H. armigera Hsp90 and Hsp70 show very high homology to their corresponding sequences in other species.Two phylogenetic analysis of Hsp90 and Hsp70 sequences were consistent with well known classification of insects.更多还原