【作者】 杨丽雯；

【导师】 李飞；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2011， 硕士

【摘要】 乙酰胆碱酯酶(AChE, EC3.1.1.7)是神经突触传导中的关键酶,其经典功能是将神经突触传导中的化学信号传递物质乙酰胆碱(Ach)水解为乙酰和胆碱,防止由于过兴奋而导致神经传导的去极化阻断。目前,对于许多研究也发现乙酰胆碱酯酶具有“非经典”功能,如胚胎致死、行动不协调、细胞粘连以及影响突触发生等。乙酰胆碱酯酶是有机磷和氨基甲酸酯类杀虫剂的作用靶标之一。目前,在双翅目、同翅目以及鳞翅目等几十种昆虫中发现存在两个乙酰胆碱酯酶基因。其中,ace1编码的乙酰胆碱酯酶被认为是有机磷和氨基甲酸酯类杀虫剂的主要作用靶标。然而,在双翅目的部分昆虫如果蝇、绿蝇、家蝇中,只存在ace2基因。在小菜蛾和二化螟等鳞翅目害虫中,ace1的表达量显著高于ace2。而家蚕作为重要的鳞翅目经济昆虫,与其他鳞翅目害虫有所不同,其ace2的mRNA丰量比ace1高。为了研究农业害虫和经济昆虫中两个乙酰胆碱酯酶在表达及功能上的差异,较利用RNA干扰技术分别沉默了家蚕和二化螟中的两个乙酰胆碱酯酶基因,通过分析被干扰后昆虫生理生化特性和行为学的差异,对两种昆虫的不同乙酰胆碱酯酶基因的功能进行了比较分析。主要结果如下：1)通过显微注射法将荧光标记的siRNA注射到试虫体内,结果表明siRNA可以通过血脑屏障,可以实现神经系统基因的干扰。2)分别干扰了5龄家蚕和二化螟的乙酰胆碱酯酶基因表达,研究干扰前后不同组织的表达量丰度,进一步证实家蚕中ace2的mRNA表达量比ace1高,二化螟中ace1的mRNA表达量比ace2高。RNA干扰后,家蚕和二化螟的头部和肌肉中的乙酰胆碱酯酶基因的表达丰度均有明显下降,而在中肠组织中却有所上升。3)重点研究了三龄家蚕干扰后乙酰胆碱酯酶被的mRNA丰度、酶活力和行为学变化关系。结果表明,RNAi处理96小时后,乙酰胆碱酯酶活力降低了约25%,而ace2干扰组的酶活力下降幅度大于ace1干扰组,证明Bm-AChE2是家蚕中主要的乙酰胆碱酯酶。RNAi处理120小时后,试虫取食活动明显受阻。RNAi处理14天后,处理组的体长体重与对照组相比有显著差异。RNAi处理21天后,处理组与对照组的结茧有显著差异,说明RNAi效力能维持较长时间。处理组试虫生长发育迟缓,茧出现畸形,表明AChE在昆虫生长发育中起重要作用。更多还原

【Abstract】 Acetylcholinesterase (AChE, EC3.1.1.7) is the key enzyme in insect nervous system.Its classical function is to hydrolyze the Ach, the transmitter of chemical information, by which nervous synapse transmits, into acetic acid and choline, preventing nervous transmission from depolarization disconnecting caused by over excitation. AChE is the target of organophosphorus and carbamates pesticides. But it has recently been found that AChE has many non-classical functions including embryo lethality, action incoordination, cell adhesion, synapse genesis.It has been found that multiple AChE genes exist in Diptera,Homopterous and Lepidop terous insects.Multiple AChE genes appeared before the differentiation of arthropods. However, there is only one AChE gene, ace2, in Drosophila, posssibly due to a second lose event in the evolutionary process. It is generally believed that AChEl is the main target of pesticides in many insects. In this study, the expression of two AChE genes in stripped rice borer and silkworm was interfered by RNAi and their functions were investigated.The major results are as follows:1) FAM labeled siRNA was injected into the bodies of silkworm and rice stem borers. The results showed that FAM-labeled siRNAs can pass through brain-blood barrier (BBB). The expression of AChE genes were knockdown sucessfully.2) We knockdown Bm-Ace and Cs-Ace respectively. The mRNA abundance in different tissues before or after RNAi treatment was estimated using qRT-PCR. The results showed that the expression of Bm-ace2was significantly higher than that of Bm-acel. However, the mRNA abundance of Cs-ace1was significantly higher than that of Cs-ace2. The Ace genes in the treated insects had reduced most in the head and muscle.3) The results showed that the mRNA abundance of two Ace genes in the third instar of silkworm had reduced at72h after siRNA injection, The AChE activities were reduced by25%at96h after siRNA injection.Interfering of Bm-ace2but not Bm-acel led to high reduction of AChE activity, indicating that Bm-AChE2is the major enzyme in silkworm.The feeding capacity of both si-ace1and si-ace2treated insects was significantly affected at5-days after injection. The weights and lengths were significantly reduced at14day after injection. The pupa malformation of si-ace treated insects were observed at21d after injection, proving that AChE plays an important role in the growth and development in insects.更多还原