Pelargonium zonate spot virus (PZSV)는 group IV (+) ssRNA viruses, Bromoviridae에 속하는 식물 병원체로, 일반적으로 토마토, 국화, 아티초크 및 제라늄에 감염된다. 본 연구는 검역 현장에서 PZSV를 신속하고 특이적으로 진단 할 수 있는 PCR module을 개발하는 것을 목적으로 하였다. PZSV를 검출하기 위한 RT-PCR 프라이머 선발 결과, 각각 513 및 320 bp를 증폭하는2개 조합을 선발하으며, 더욱 높은 검출감도로 검출할 수 있을 뿐아니라 RT-PCR을 검증할 수 있는 nested PCR 프라이머 조합을 개발하였다. 또한, 제한효소 Xho I 부위를 삽입한 유전자변형-양성대조구 플라스미드를 설계하여, PCR module에서 대조구로부터 오염을 검증할 수 있도록 개발하였다. 본 연구에서 개발한 PCR module은 토마토, 국화, 아티초크 및 제라늄 등에서 PZSV를 간편, 신속 및 특이적으로 검출하여, 지속적으로 식물검역에 활용할 수 있을 것으로 기대된다.

종자의 수입 시, 검역관련 종자전염바이러스는 가장 문제가 되는 식물병이다. 본 연구에서 PCR 검역체계가 보고되지 않은 3종의 종자전염바이러스, Cherry rasp leaf virus (CRLV), Spinach latent virus (SpLV) 및 White clover mosaic virus (WClMV)를 검출하기 위하여 reverse transcription polymerase chain reaction (RT-PCR)과 nested polymerase chain reaction (nested PCR) 방법을 도입하였다. 각각의 바이러스별로 2 세트의 RT-PCR primer가 선발되었으며, 증폭산물에서 더욱 높은 감도로 검출 할 수 있는 nested PCR primer set를 개발하였다. 본 연구에서 사용한 RT-PCR과 nested PCR 방법은 종자로부터 CRLV, SpLV 및 WClMV를 검역하는 고효율적 진단시스템으로 제공될 것이다.

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.