Electroporation is well known today as a powerful transfection technique and is useful for the study of gene expression. The advantage of the electroporation method is that large quantity of silkworm (Bombyx mori) eggs can be transformed in a very short time. However, how to use it for introducing foreign gene into silkworm eggs needs systematical investigation. In our silkworm transgenesis program, we needed an efficient technique to evaluate the functionality of transgenes before their injection into eggs. The goal of this experiment was to find an alternative efficient method of generating transgenic silkworm eggs using a commercially available electroporation device. The Gene Pulser Xcell (Bio-Rad Laboratories, USA) were used. In contrast to other electroporation devices, which are based on a single pulse with exponential decay or square wave technology. We investigated pigmentation-rate and hatching-rate of the silkworm eggs of electroporation. We used foreign gene LacZ, EGFP, Ds-red induced vector system with selection marker for transgenic silkworm. The LacZ gene in 3rd instar larva DNA can be detected by β-galactosidase stain. During these technical studies we found that optimizing parameters such as electrical voltage, number of pulses and their frequency, and conductivity of the buffer was important. These results confirmed that electroporation is available technique for transfecting B. mori egg.

In this study, we constructed viral vector for soybean by using Soybean yellow common mosaic virus (SYCMV) infecting both Glycine max and Glycine soja. SYCMV-derived viral vector was tested to use as Virus-induced gene silencing (VIGS) vector for functional analysis of soybean genes and as protein expression vector for foreign protein expression. In vitro transcript with 5’ cap analog m7GpppG from a full-length infectious vector of SYCMV driven by T7 promoter was inoculated to soybean to test infectivity of the clone (pSYCMVT7-full). 5’-capped transcript was able to infect soybean plants. The symptoms observed in soybean plants infected by either the vector or the sap from SYCMV-infected leaves were indistinguishable, suggesting that the vector had an equal biological activity shown by virus itself. To further utilize the vector, an additional DNA-based vector was constructed. The full-length cDNA was inserted into a binary vector flanked by CaMV 35S promoter and the nopaline synthase terminator (pSYCMV35S-full). To test if the vector infects soybean and subsequently induces gene silencing, we prepared two constructs containing fragments of Phytoene desaturase (PDS) gene (pSYCMV35S-PDS1) and small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS) gene (pSYCMV35S-rbcS2) from soybean plant. Plants infiltrated with the constructs through Agrobacterium-mediated method showed distinct symptoms such as photobleaching in plants infiltrated with pSYCMV-PDS1 and pale green or yellowing in plants infiltrated with pSYCMV-rbcS2. In addition, down-regulations of mRNA levels of two genes were confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). To test if the vector can be used for foreign protein expression in soybean plants, we prepared a construct encoding amino acids 135-160 of VP1 FMDV serotype O1 Campos (O1C) (pSYCMV35S-FMDV). Plants infiltrated with the construct through Agrobacterium-mediated method showed that soybean plant infiltrated with pSYCMV35S-FMDV only was detected by Western blotting using O1C antibody. These results support that SYCMV-derived viral vector can be used as VIGS vector or protein expression vector in soybean plants.