Construction of cytomegalovirus promoter-driven gene expression system in Laodelphax striatellus

The small brown planthopper(SBPH,Laodelphax striatellus)is a significant rice pest,responsible for transmitting rice stripe virus(RSV)in a persistent and propagative manner.RSV is one of the most detrimental rice viruses,causing rice stripe disease,which results in considerable loss of rice grain yield.While RNA interference and gene knockout techniques have enabled gene downregulation in SBPH,no system currently exists for the overexpression of endogenous or exogenous genes.Consequently,the development of a protein expression system for SBPH is imperative to serve as a technical foundation for pest control and gene function investigations.This study aimed to construct an expression vector using the promoter of the constitutive-expressed tubulin gene of SBPH,and promoter of human cytomegalovirus(CMV).Fluorescence experiments demonstrated that both tubulin and CMV promoter could drive green fluorescent protein(GFP)expression in SBPH,and could also facilitate the expression of a nucleocapsid protein(NP)-GFP fusion protein containing viral NP with comparable efficiency.Through expression vector optimization,we have identified that the 3 tandem CMV promoters display a significantly higher promoter activity compared with both the 2 tandem CMV promoters and the single CMV promoter.In addition,the incorporation of Star polycation nanoparticles significantly enhanced the expression efficiency in SBPH.These results provide a promising technical platform for investigating gene functions in SBPH.

Establishment of Human Interferon-Alpha Transgenic Mice Model

[ Objective] To establish human interferon-alpha （hlFN-alpha） transgenic mice, which can be used to study antiviral activity of IFN-alpha in vivo. [Method] The expression vector was constructed by inserting the human IFN-alpha gene into a vector harboring human cytomegalovirus （CMV） promoter. The transgenic mice were created by the method of microinjection. The genotype of transgenic lines was identified by PCR and southern blotting. [ Result] Four lines of hlFN-alpha transgenic mice were established. Lymphocytes were collected from PCR-positive ,=1 generation mice and identified by RT-PCR, and mice of three lines were positive. ELISA and microplate dye-binding assay showed the expression of human IFN-alpha in the serum of mice which were positive in RT-PCR identification. [ Conclusion] The transgenic mice carrying CMV-controlled hlFN-alpha gene has been established successfully, which is essential for studying antiviral activity of IFN-alpha and genetic engineering breeding of disease-re- sistant animals.