Candida pseudotropicalis의 원형질체 융합: 원형질체 형성 및 재생과 융합 조건

Protoplast fusion of Candia Pseudotropicalis: The conditions for protoplast formation, regeneration and fusion

Candida pseudotropicalis CBS 607에서의 야생형과 영양요구성 돌연변이 균주에 대한 원형질체 형성 및 재생과 상보적 영양요구성 균주간의 융합에 관해서 연구하였다. 야생형과 his tidine 혹은 adenine요구성 균주에서 원형질체 형성율은 96-100%이였으나 methionine과 tryptophan요구성 균주는 각각 52%와 72%였다. Myoinositol (0.5 mg/ml)을 첨가한 배지에서 전배양한 methionine과 tryptophan조 구성 균주는 원형질체 형성시 bovine serum albumin (BSA, 4 mg/ml)을 첨가하면 96-98%의 원형질체 형성율이 얻어졌다. 원형질체 재생 빈도는 18-20% 였으나. BSA와 myoinositol을 첨가하면 adenine요구성 균주(약 20%)을 제외한 나머지 영양요구성 균주에서는 재생율이 20% 이상 증가했다. 원형질체 융합에 대한 PEG와 $CaCl_2$,최적농도는 20%와 100mM이었고 최적 pH와 반 응시간은 각각 6.0과 30분이 있다. 상보적 영양요구성 균수의 융합빈도는 $1.5{\times}10^{-3}\;to\;8.8{\times}10^{-3}$ 였으며 원행점체의 재생 증진으로 인해 융합율이 현저히 증가했다. Histidine요구성 균주와 tryptophan 요구성 균주을 융합시켰을 때 몇개의 융합세포를 얻었는데 측정된 DNA양과 액상의 차이로 보아 aneuploid나 diploid상태임을 알 수 있었다.

Protoplast formation and regeneration from wild-type and auxotrophic mutants of Candida pseudotropicalis CBS 607 as well as fusion between complementary mutants were carried out. Frequencies of protoplast formation from wild-type and histidine or adenine requiring mutants ranged from 96 to 100% whereas those from methionine or tryptophan auxotrophs were 52 and 72%, respectively. When bovine serum albumin(4mg/ml, BSA) was added to protoplasting buffer for cells of methionine or tryptophan auxotrophs grown in a medium supplemented with myoinositol(0.5mg/ml), 96-99 % of cells were converted to protoplasts. Protoplasts were regenerated at the frequencies ranging from 18 to 20%. However, the addition of BSA to protoplasting buffer and the supplement of myoinositol to a medium of cell growth doubled the regeneration rate except adenine auxotroph in which such an improvement was not observed. It was found that optimal concentrations of polyethylene glycol and $CaCl_2$ are 20% and 100mM while optimal pH and exposure time are 6.0 and 30min. The fusion frequencies between complementary mutants ranged from $1.5{\times}10^{-3}\;to\;8.8{\times}10^{-3}$ and were enhanced by the improvement in the rate of protoplast regeneration. When histidine auxotroph was fused with tryptophan mutant, several fusion products were obtained which were found to be in the state of aneuploid or diploid, judging from DNA content and the presence of a large nucleus in the products.