关键词: 灰飞虱, 水稻条纹病毒, 差异表达基因, DDRT-PCR, 正交试验

Abstract:

Rice stripe virus (RSV) is mainly transmitted by insect vector Laodelphax striatellus in circulative-propagative and transovarial manners. The interaction between RSV and L. striatellus is largely unknown. To investigate the effects of RSV on gene expression in L. striatellus, viruliferous and virus-free L. striatellus populations were detected to reveal the differentially expressed genes with five random primers and three anchor primers by differential display RT-PCR (DDRTPCR). Furthermore, positive-cross test was performed to find optimal conditions of DDRT-PCR by analyzing six critical parameters including cDNA template, random primer, anchor primer, dNTPs, Mg²⁺ and Taq. The results showed that the optimal conditions of DDRT-PCR (25 μL) included cDNA template 3.0 μg, random primer 2.0 μmol/L, anchor primer 2.5 μmol/L, dNTPs 200 μmol/L, Mg2+ 2.0 μmol/L, and Taq 2.0 U. Thirty-five differentially expressed cDNA fragments were isolated by DDRT-PCR. Six of them were verified by RNA dot blot hybridization, and four positive cDNA fragments were obtained. Three positive cDNA fragments were from viruliferous L. striatellus population and shared high homology with 5-hydroxytryptamine receptor 1D gene, gyrase B gene and 60S ribosomal protein L40 gene, respectively. The positive cDNA fragment from virusfree L. striatellus population had no similarity with sequences in NCBI nucleotide databases. The optimal DDRT-PCR system and the differentially expressed cDNA fragments obtained might provide a basis for further study on interaction between RSV and L. striatellus.

Key words: Laodelphax striatellus, rice stripe virus (RSV), differentially expressed gene, DDRT-PCR, positive-cross test