›› 2018, Vol. 61 ›› Issue (8): 932-940.doi: 10.16380/j.kcxb.2018.08.006

• 研究论文 • 上一篇    下一篇

崇明拟异小杆线虫daf-16基因的功能鉴定

詹成修, 熊阳杰, 张涛, 张克云*   

  1. (南京农业大学生命科学学院, 南京 210095)
  • 出版日期:2018-08-20 发布日期:2018-08-20

Functional identification of daf-16 in Heterorhabditidoides chongmingensis (Rhabditida: Rhabditidae)

ZHAN Cheng-Xiu, XIONG Yang-Jie, ZHANG Tao, ZHANG Ke-Yun*   

  1.  (College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China)
  • Online:2018-08-20 Published:2018-08-20

摘要: 【目的】昆虫病原线虫(entomopathogenic nematodes, EPNs)的寿命和抗逆性是影响其商品化的重要因素。本研究旨在鉴定崇明拟异小杆线虫Heterorhabditidoides chongmingensis的模式品系DZ0503CMFT(DZ)的DAF-16基因的生物学功能,探讨该基因在DZ线虫的寿命和抗逆性中的作用。【方法】根据DZ线虫和其自身共生菌Serratia nematodiphila DZ0503SBS1T(S1)及非自身共生菌S. nematodiphila DR186(186)共培养时生长发育的差异,结合已经构建的DZ线虫的数字基因表达谱(digital gene expression profile, DGE)文库,选取调控生长发育的胰岛素/类胰岛素信号通路(insulin/insulin-like growth factor signaling pathway, IIS)的主要转录输出信号DAF-16作为研究对象。通过同源性分析选取daf-16基因中特异性片段作为干扰片段,利用L4440质粒载体构建干扰片段的dsRNA表达载体,并转化到大肠杆菌Escherichia coli HT115(DE3)中。用饲喂法将daf-16 dsRNA导入DZ线虫体内沉默基因;利用实时定量PCR检测RNA干扰效果,统计daf-16基因表达下调后的DZ线虫寿命、4个生理阶段的时长、抗逆性(热应激和氧化应激)以及表型(体长)和繁殖性状(48 h内产卵量、卵孵化率和雌虫率)等的变化。【结果】 DZ线虫的daf-16 (GenBank登录号: MH101641) cDNA全长2 164 bp,其中开放阅读框(open reading frame, ORF)长1 353 bp,编码450个氨基酸。RNAi后DZ线虫体内的daf-16基因表达量被显著敲低,相对于对照组空载L4440降低了49.05%。RNAi组线虫平均寿命为14.96 d,比空载L4440组(17.60 d)降低了15.00%。DZ线虫daf-16 RNAi组在繁殖阶段和快速身体运动阶段的时长比空载L4440组的短,而快速咽吸阶段和咽抽吸阶段的时长比空载L4440组的长。处理7 d后DZ线虫daf-16 RNAi组在热应激(37℃)和氧化应激(10 mmol/L百草枯水溶液)处理时存活率显著低于DZ线虫的空载L4440组。在产卵量、孵化率、雌虫率和体长上daf-16 RNAi组与对照组之间没有显著差异。【结论】通过RNAi方法初步验证了DZ线虫的daf-16基因的生物学功能,该基因调控了崇明拟异小杆线虫的寿命和抗逆性。

关键词: 昆虫病原线虫, 崇明拟异小杆线虫, daf-16, RNAi, 寿命, 抗逆性

Abstract: 【Aim】 Lifespan and stress resistance are two important factors that influence the commercial production of entomopathogenic nematodes (EPNs). This study aims to identify the function of DAF-16 gene in DZ0503CMFT (DZ), the type strain of Heterorhabditidoides chongmingensis, and to clarify the roles of the gene in the lifespan and stress resistance of DZ nematodes. 【Methods】 DAF-16, a major transcription factor of IIS pathway, was selected from digital gene expression profile (DGE) according to differential development of two monoxenic DZ nematodes associated with different symbiotic bacterial strains of Serratia, i.e., a non-native bacterial strain DR186 (186) and a native strain S1 of S. nematodiphila. The specific fragment of daf-16 was selected as the interference fragment by homology analysis, L4440 plasmid was used to construct the dsRNA expression vector of the interference fragment, and then the dsRNA expression vector of the interference fragment was transformed into Escherichia coli HT115 (DE3). daf-16 dsRNA was introduced into DZ nematode by feeding with the constructed engineering bacterium, and the effect of RNAi was detected by real-time quantitative PCR. The changes of the lifespan, spans of four physiological stages, stress resistance (heat stress and oxidative stress) and phenotype (body length) and reproduction characters (the number of eggs laid in 48 h, egg hatching rate and proportion of females) of DZ nematodes after daf-16 RNAi were observed and recorded. 【Results】 daf-16 (GenBank accession no.: MH101641) has a full-length cDNA of 2 164 bp with an open reading frame (ORF) of 1 353 bp in length, encoding 450 amino acids. After daf-16 RNAi in DZ nematodes, the expression level of daf-16 was significantly repressed with an average reduction of 49.05% as compared with that of the control group (L4440 group) in qPCR assay. The average lifespan of DZ nematodes of the daf-16 RNAi group (14.96 d) was reduced as compared with that of the L4440 group (17.60 d), with a decrease of 15.00%. Spans of the reproductive stage and fast body movement stage of DZ nematodes of the daf-16 RNAi group were shorter than those of the L4440 group, whereas the spans of the fast pharyngeal pumping stage and pharyngeal pumping stage of DZ nematodes of the daf-16 RNAi group were longer than those of the L4440 group. The survival rate of DZ nematodes of the daf-16 RNAi group was significantly lower than that of the L4440 group when the nematodes were exposed to heat stress (37℃) and oxidative stress (10 mmol/L paraquat solution) for 7 d. There was no significant difference in the number of eggs laid in 48 h, egg hatching rate, proportion of females and body length between the daf-16 RNAi group and the control group. 【Conclusion】 The biological functions of daf-16 in DZ nematode were identified by RNAi, which regulates the lifespan and stress resistance of DZ nematode.

Key words: Entomopathogenic nematode, Heterorhabditidoides chongmingensis, daf-16, RNAi, lifespan, stress resistance