Dong-Il Shin

Transient β-glucuronidase expression in lily (Lilium longflorum L.) pollen via wounding-assisted Agrobacterium-mediated transformation

A pollen-based transient expression system has been developed. Lily pollen grains, wounded by vigorous shaking in the presence of aluminum oxide particles, were transformed by infiltration with Agrobacterium tumefaciens LBA4404 cells harboring the β-glucuronidase (GUS) gene construct, pBI121. In histochemical and fluorometric GUS analysis, the wounding processes allowed efficient transformation and, in cDNA blot hybridization, GUS mRNA synthesis was clearly detected. Lily pollen with appropriate wounds, therefore, can be used conveniently for the rapid production of recombinant proteins.

Oral vaccination of mice with Tremella fuciformis yeast-like conidium cells expressing HBsAg

Tremella fuciformis yeast-like conidium (YLC) cells were transformed by co-cultivation with Agrobacterium cells harboring the hepatitis B surface antigen (HBsAg) gene construct under the control of the CaMV35S promoter. Integration of HBsAg DNA into the YLC genome was confirmed by PCR and dot-blot hybridization. Immunoblotting verified expression of the recombinant protein. Oral administration of YLC cells expressing HBsAg in mice significantly increased anti-HBsAg antibody titer levels using a double prime-boost strategy that combined parenteral and oral HBsAg boosters.

Mechanical Wounding of Yeast-Like Conidium Cells of Tremella fuciformis Makes Them Susceptible to Agrobacterium -Mediated Transformation

We developed an efficient protocol that makes possible the practical use of Agrobacterium to transform the yeast-like conidia of Tremella fuciformis. Mechanical wounding of fungal cells prior to co-cultivation with Agrobacterium cells greatly improved transformation efficiency.

Acceleration of the Mycelial Growth of Trametes veriscolor by Spent Coffee Ground

구름버섯은 온대지역에서 흔히 발견되는 목재 부후균으로서 중요한 약용버섯의 일종이다. 본 연구에서는 커피박, 과립커피 및 과립 디카페인 커피의 구름버섯 균사체 생장에 미치는 영향을 조사하였다. 그 결과, 커피박은 10% 농도에서까지 그리고 과립커피의 경우 1% 미만에서 균사체 생육을 촉진시키는 것으로 확인되었다. 1% 커피박의 경우 무처리에 비해 10배 정도의 건조중량 증가가 측정됨으로써 구름균사체의 생육촉진제로서의 커피 효능을 확인할 수 있었다. 또한 커피는 균사체의 페놀화합물 및 항산화 효능 증대를 위한 유용 물질로 판단되었다. 【Trametes versicolor, a common inhabitant of dead hardwoods in temperate climates, belongs to one of the important medicinal mushrooms. In this study, spent coffee ground(SCG), instant coffee powder(ICP) and instant decaffeinated coffee powder(IDCP) were examined for their effect on the mycelial growth of T. versicolor. Adding SCG was proven to be significantly beneficial at the concentration as high as 10%. ICP and IDCP, both containing concentrated polyphenols, were also beneficial at low concentration less than 1%. 1% SCG culture resulted in ten-fold increased yield of dry cell mass compared to the control culture. Adding coffee substances was recommended as a useful tool for accelerating the growth and strengthening the physiological activity of the mycelium.】

Genetic Transformation of the Mycelia of Tremella fuciformis and Changes of Cytotoxicity

Tremella fuciformis, as one of higher basidiomycetes, can asexually reproduce yeast-like conidium (YLC) cells by budding. We have developed an efficient method to introduce pCambia1300 plasmid containing hph gene into YLC cells using Agrobacterium. This was successful only when YLC cells were wounded by NaOH treatment before co-cultivation. In average, 40~50 transformants were produced out of YLC cells investigated. The T-DNA transfer was confirmed by PCR. Methanolic extracts from transformants demonstrated different levels of toxicity against SKOV-3 cervical cancer cells.

Agrobacterium-Mediated Transformation of Flammulina velutipes with NaOH Treatment

Agrobacterium harboring vector pCHBs with hygromycin phosphotransferase(hph) and hepatitis B virus surface antigen(HBsAg)gene was transformed into the mycelial culture of Flammulina velutipes. In particular, mild NaOH solution was treated to the mycelia before Agrobacterium infection step. This was purposed to generate putative surface wounds in the mycelial cell walls. The results showed that hygromycin-resistant(hyg r ) mycelia could be obtained only from NaOH-treated mycelia but not from intact mycelia. The integration of hyg r gene in fungal genome was confirmed by PCR. In addition, a single transgene integration and heterologous protein expression in F. velutipes could be verified by Southern blot hybridization and western blot analysis, respectively. This study demonstrated an efficient tool for the Agrobacterium-mediated transformation of F. velutipes mycelia.