崇明拟异小杆线虫gsa-1基因的功能鉴定  

Functional identification of gsa-1 in Heterorhabditidoides chongmingensis

作　　者：马丽君 黄金 胡天翼 黄小娜 张克云[1] MA Lijun;HUANG Jin;HU Tianyi;HUANG Xiaona;ZHANG Keyun(College of Life Sciences,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学生命科学学院

出　　处：《南京农业大学学报》2020年第1期89-97,共9页Journal of Nanjing Agricultural University

基　　金：国家重点研发计划项目(2018YFC1200201);江苏省自然科学基金项目(BK20131320);国家大学生创新训练项目(12019038)

摘　　要：[目的]昆虫病原线虫(entomopathogenic nematode,EPN)的嗅觉趋化性及适应性是影响其在实际应用中的重要因素。本文目的在于鉴定崇明拟异小杆线虫Heterorhabditidoides chongmingensis DZ0503CMFT(DZ)品系中gsa-1基因的生物学功能,探讨该基因对DZ线虫的嗅觉趋化性及适应性的影响。[方法]根据前期构建的DZ线虫与其自身共生菌菌株Serratia nematodephila DZ0503SBS1 T(S1)及其非自身共生菌S.nematodiphila DR186(186)的数字基因表达谱(digital gene expression profile,DGE)文库中的差异基因,选取化感神经元中广泛表达的G蛋白Gα亚基的基因gsa-1作为研究对象。利用饲喂法将gsa-1的dsRNA导入线虫体内进行RNAi,从而降低线虫体内gsa-1基因的表达量。利用荧光定量PCR检测RNA干扰效果,并统计线虫的趋化性、嗅觉适应性、体长、寿命、发育历期及雌虫比例等变化。[结果]DZ/S1单菌侵染期线虫与DZ/186相比其趋化性及适应性均有显著差异。DZ/S1组的线虫对苯甲醛及活体大蜡螟幼虫的趋化性指数分别是DZ/186组的2.99倍和8.62倍。DZ/S1组的线虫对苯甲醛的嗅觉适应性较DZ/186组更强。gsa-1基因在DZ/S1单菌侵染期线虫中的表达量显著下调,相比DZ/186组下降了34.30%。gsa-1 RNAi后,其表达量相比对照组被抑制了54.19%。gsa-1 RNAi组线虫对苯甲醛的趋化性及对活体大蜡螟幼虫的趋化性明显升高,其趋化性指数分别是对照组的4.22倍和2.14倍。gsa-1 RNAi组与对照组之间嗅觉适应性、体长、寿命、发育历期及雌虫比例没有显著差异。[结论]通过RNAi方法初步验证了DZ线虫gsa-1基因的生物学功能,该基因负调控崇明拟异小杆线虫的嗅觉趋化性。[Objectives]Olfactory chemotaxis and adaption are two important factors that influence the practical application of entomopathogenic nematode(EPN).This study aims to identify the biological functions of gsa-1 gene in Heterorhabditidoides chongmingensis DZ0503CMFT(DZ)and to clarify the roles of the gene in the olfactory chemotaxis and adaption of DZ nematodes.[Methods]According to the digital gene expression profile(DGE)of DZ nematodes associated with its native symbiotic bacterial strains Serratia nematodephila DZ0503SBS1 T(S1)and non-native bacterial strain S.nematodiphila DR186(186),gsa-1 one of the differential expression genes,which regulates G protein Gαsubunit widely expressed in amphid neurons,was selected.The dsRNA of gsa-1 was transferred into DZ nematodes by traditional feeding method to knockdown the expression level of gsa-1 by RNAi.The effect of RNAi was detected by real-time PCR.The changes of the chemotaxis,olfactory adaption,body length,lifespan,spans of four physiological stages and female ratio in nematodes were recorded and statistically analyzed.[Results]DZ/S1 infected juveniles(IJ)showed significant differences in chemotaxis and olfactory adaptation compared with DZ/186 IJs.The olfactory chemotaxis of DZ/S1 IJs to benzaldehyde and alive Galleria mellonella larvae significantly increased,with a chemotaxis index 2.99 and 8.62 times higher than those of the DZ/186 IJ,respectively.The olfactory adaption of DZ/S1 group to benzaldehyde was stronger than that of DZ/186 group.The expression level of gsa-1 gene was significantly down-regulated in the DZ/S1 IJ,and its expression level was down-regulated by 34.30%compared with DZ/186 group.After gsa-1 RNAi,the expression level of gsa-1 of DZ was significantly repressed of 54.19%.After gsa-1 RNAi,the chemotaxis of DZ nematode to benzaldehyde and live Galleria mellonella larvae significantly increased,with the chemotaxis index of 4.22 and 2.14 times of the control,respectively.However,there were no significant difference between the gsa-1 RNAi group and

关 键 词：昆虫病原线虫 崇明拟异小杆线虫 gsa-1 RNAI 嗅觉 

分 类 号：Q965.8[生物学—昆虫学]