미치광이풀 부정근으로부터 탈분화 및 재분화에 미치는 세포소기관의 형태 및 tropane alkaloids의 함량 변화를 TEM과 HPLC를 사용하여 조사하였다. Scopolamine and hyoscyamine 및 세포 소기관의 형태변화는 세포분화 및 탈분화 단계마다 차이를 보였다. 부정근은 배양 20일후에는 캘러스가 형성되었고, 배양 60일부터 줄기원기가 발생하였으며, 이후 완전한 줄기로 발달하였다. 부정근 정단부 조직을 TEM 분석 결과 핵, 액포, 전분체, 미토콘드리아, 골지체, 세포벽 등 다양한 세포소기관들이 형성되었으나, 배양 20일후부터는 소기관들이 적게 관찰되었다. 그러나 분화가 시작되는 배양 40일 후 callus 세포에는 다수의 전분체 등 다양한 세포소기관들이 다시 관찰되었고, 배양 60일 이후에는 다시 전분립의 수가 감소하였다. 배양 80일째 세포는 전분립이 거의 관찰되지 않았으나, 미토콘드리아, 골지체 등 다양한 세포소기관들이 다시 관찰되었다. 한편, scopolamine과 hyoscyamine 함량은 배양 60일부터 다소 증가하다가 이후 90일까지 급격히 증가하였고, 이후에는 급격한 감소를 보였다. 본 연구 결과 tropane alkaloid 생합성은 탈분화 및 분화 과정에 영향을 받으며, 세포 내 전분립 생성은 기관분화와 더불어 물질생합성과 매우 밀접한 관련성이 있을 것으로 추정된다.

Scopolamine and hyoscyamine are important anticholinergic compounds. To increase the productivity, we have selected various elicitors and developed culture system using a bubble column bioreactor (BCB). As the same manner of elicitation in flask cultures, the elicitors were introduced into BCB cultures and the productivity was investigated. Except the bacterial elicitor of Staphyllococcus aureus, the elicitors inhibited hyoscyamine production. In scopolamine production, the elicitors revealed different responses from the results obtained in flask cultures. The elicitors of KCl and Candida albicans less increased the production than flask cultures. However, methyl jasmonate and S. aureus showed stronger positive effects on tropane alkaloid production. In particular, S. aureus was the most effective elicitor on scopolamine production and the elicitor resulted in the highly increased production, approximately 10 times higher than the control culture.

Scopolia parviflora Nakai, a rare and endangered species, is the sole plant producing tropane alkaloids (TA) among the Korean native species. In order to enhance TA productivity the SP72 root line was selected by screening 100 of root line, and the optimal culture media for root growth and TA production were investigated with the SP72 roots. Based on the several media, SH and 2B5 medium were determined as growth medium and White and NN medium as production medium. Among the four combinations of two-stage culture, 2BN (2B5 as growth medium plus NN as production medium) showed more enhanced root growth and TA production as compared with production media of White and NN medium and growth media of SH and 2B5 medium, respectively. However, bubble column bioreactor (BCB) cultures applying two-stage culture did not reveal the effective results despite of the each successful operation of two-stage culture in conical flasks and BCB cultures.

Total RNAs were isolated from cultured roots of Scopolia parviflora, poly(A)+ RNA was obtained through the mRNA purification, cDNA library of Hnh6h was constructed. Recombinant baculoviruses in Spodoptera frugiperda (Sf) cells were constructed by use of the transfer vector pBacPAK, which has the AcNPV sequence under the polyhedrin promoter. The expression vector carrying Hnh6h gene was transferred to S. parviflora and obtained transgenic hairy root lines. Our results confirmed the over expression of the H6H protein was used by anti-pBacPAK about cDNAs of S. parviflora. This study will served for production of tropane alkaloids by metabolic engineering.

Hyoscyamine and scopolamine are two most common tropane alkaloids found in the Solanaceae. The pEB expression vector carrying Nspmt gene was transformed to Agrobacterium rhizogenes. The growth of transgenic hairy roots were approximately to 80% of the wild type root. Transgenic hairy roots are less developed on only their branch roots than those of the wild type root. The extracts from Nspmt transgenic hairy root lines, 3 and 5 contained between 3.52 and 4.23 mg/g dry weight as hyoscyamine and did between 5.23 to 6.40 mg/g dry weight as scopolamine. These results showed that the overexpression of the pmt gene enhanced tropane alkaloids production of S. parviflora transformed roots and this improvement affected both hyoscyamine and scopolamine production.

미치광이풀의 모상근 뱌양에 의해 의학적으로 중요한 물질인 scopolamine과 hyoscyamine 생산을 증진시키기 위해 3종의 곰팡이와 1종의 효모 유래의 생물학적 elicitor를 supernatant와 homogenate의 형태로 처리하였다. Elicitor 중 Candida albicans는 supernatant와 homogenate 모두 scopolamine 생산성을 증가시켰다. Fusarium solani의 homogenate와 C. albicans의 supernatant 처리로 hyoscyamine 함량이 증가되었다. 그러나 나머지 fungus strain도 대조구에 비해 tropane alkaloids의 생산을 다소 증가시켰다. 본 연구에서는 C. albicans가 tropane alkaloids 생산에 적합한 elicitor임으로 구명되었다. 본 연구 결과는 미치광이풀 모상근 대량배양에 의한 tropane alklaoids 대량생산에 기여할 것이다.