Expression of Rice Gall Dwarf Virus Outer Coat Protein Gene (S8) in Insect Cells

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.

Efficiency for Gene Silencing Induction in Nicotiana Species by a Viral Satellite DNA Vector

Virus-induced gene silencing （VIGS） is a useful technique for rapid plant gene function analysis. We recently reported a new VIGS vector modified from Tomato yellow leaf curl China virus （TYLCCNV） DNAβ （DNAm β）. In this study we compared in detail DNAmβ-induced gene silencing in four Nicotiana species including N. benthamiana, N. glutinosa, N. tabacum and N. paniculata. We found that DNAmβ-induced gene silencing in the four species was distinct in developing dynamics, tissue specificity, efficiency, and constancy in the plant life span. It was most efficient in N. benthamiana, where development of VIGS was most rapid, without tissue specificity and nearly 100% efficient. DNAmβ-induced gene silencing in N. glutinosa was also efficient despite being slightly less than in N. benthamiana. It initially occurred in veins, later was scattered to mesophyll, finally led to complete silencing in whole leaves. In both species, VIGS constantly expressed until the plants died. However, DNAmβ-mediated VIGS in the other two Nicotiana species, N. tabacum and N. paniculata, was significantly less efficient. It was strictly limited within the veins of the silenced leaves, and constantly occurred only over 3-4 weeks. The upper leaves that emerged later stopped showing the silencing phenotype. DNAmβ-induced gene silencing in N. benthamiana and N. glutinosa was not significantly influenced by the growth stage when the plants were agro-inoculated, and was not sensitive to high growth temperature up to 32℃. Our results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in some Nicotiana species.

Identification and characterization of a cytochrome P450 C YP6CX1 putatively associated with insecticide resistance in Bemisia tabaci

The novel full length of cytochrome P450 gene has been isolated in insecticideresistant （named CYP6CXlvl） and -susceptible （named CYP6CXlv2） Bemisia tabaci, which was identified as B biotype, in Shangjie, Fujian, China （Sj）. CYP6CX1 （1 940 bp contained a 1 557 bp open reading frame） included conserved domains common to CYP6 members, such as heme-binding motif PFGEGPRFCIA, putative ＂meander＂-binding sequence ETLR and PERF in helix-K, oxygen-binding motif AGLDPV and conserved sequence PEKFNP near the carboxyl end. There were four different replacements of amino acid residues between R and S B. tabaci （Thr300 Ala, Thr354Pro, Arg486His and Ile503Thr）, among which the substitution Ile503Thr was located in the substrate recognition sites region. The mRNA transcription level of CYP6CXlvl was 2.38-fold as high as that of CYP6CXlv2. The results indicated that the CFP6CX1 from the B biotype B. tabaci in Sj was one of the CYP6 members, and enhanced CYP6CX1 expression and substitute of amino acid residues might be involved in the resistance mechanisms in field B. tabaci.