›› 2018, Vol. 61 ›› Issue (8): 932-940.

• 研究论文 •

### 崇明拟异小杆线虫daf-16基因的功能鉴定

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

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.