Chinese Journal OF Rice Science >
Gene Cloning, Polyclonal Antibody Preparation and Expression Localization of Two dynamin-1-like Genes from Nilaparvata lugens (Hemiptera: Delphacidae)
Received date: 2016-12-12
Revised date: 2017-03-22
Online published: 2017-07-10
【Objective】To study the biological function of two dynamin-1-like genes, NlDNM1L-1 and NlDNM1L-2 in Nilaparvata lugens,【Method】 two dynamin-1-like genes were cloned from N. lugens by polymerase chain reaction (PCR), and their bioinformatics analysis was conducted. Using fluorescence quantitative real-time PCR technology, the relative expression levels of NlDNM1L-1 and NlDNM1L-2 in different tissues and at different developmental stages of N. lugens were detected. The expression vectors of the two genes were constructed and recombinant proteins were expressed in Escherichia coli Rosetta. The expressed recombinant proteins were purified by Ni-NTA agarose gel affinity system and then injected into New Zealand white rabbit to generate polyclonal antibodies. The titer of the antibodies was monitored by ELISA, and the immune specificity was determined by Western blotting hybridization. Using above antibodies, the localization of target proteins in ovary was detected by immunofluorescence. 【Result】The two dynamin-1-like genes were cloned, and their GenBank accession numbers were KY082900 and KY082901, respectively. The phylogenetic tree showed that NlDNM1L-1 and NlDNM1L-2 were conserved in Hemipterans. The results of protein structure prediction and space-time expression pattern analysis indicated that NlDNM1L-1 and NlDNM1L-2 might play different roles in N. lugens. The ELISA and Western blotting results showed that the polyclonal antibodies had high titer and good specificity. Immunofluorescence showed that NlDNM1L-1 and NlDNM1L-2 were widely distributed in the follicle cells of N. lugens ovary, indicating that they may be related to the development and maturity of N. lugens ovary. NlDNM1L-1 and NlDNM1L-2 may also be associated with the invasion of the yeast-like symbionts to N. lugens ovary. 【Conclusion】 The DNA sequences, bioinformatics and expression pattern of NlDNM1L-1 and NlDNM1L-2 were clarified. And the polyclonal antibodies of NlDNM1L-1 and NlDNM1L-2 were obtained and their localizations in the ovary have preliminarily been understood. These results lay a foundation for further biological function investigation of NlDNM1L-1 and NlDNM1L-2 in N. lugens.
Chenxing ZHAO, Yewei YU, Yipeng XU, Xiaoping YU . Gene Cloning, Polyclonal Antibody Preparation and Expression Localization of Two dynamin-1-like Genes from Nilaparvata lugens (Hemiptera: Delphacidae)[J]. Chinese Journal OF Rice Science, 2017 , 31(4) : 345 -354 . DOI: 10.16819/j.1001-7216.2017.6165 345
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