| In this paper, the studies were carried out to study sporulation medium, hydrolysis conditions, protoplast preparation from Pecilomyces militaris, the anamorph of Cordyceps militaris. In addition, electrophoretic karyotype of Paecilomyces militaris was analyzed by use of contour-clamped homogeneous electric field (CHEF electrophoresis with the chromosomal size standards from Hansenula wingei and Schizosaccharomyces pombe.With investigating the influence of various sporulation medium, the results showed that the optimum sporulation medium was SMAY medium except for the stain RCEF0848. Cazapk medium was the best sporulation medium for the stain RCEF0848.There are various influencing factors on enzyme digestion, such as the variety of enzyme, temperature of enzymolysis and time of enzymolysis. Conclusively, the action effect of enzyme mixture was superior to that of single type. The combination which mixed with the same concentration of snail enzyme, cellulose and solubilization wall enzyme was the best enzyme solution; the protoplast yield was almost same when the enzymolysis temperature was in the range of 30-35℃. As the ambient temperature turned higher or lower than this range, the protoplast yield decreased slowly under these temperatures. Considering the quality of protoplast, the optimum enzymolysis condition was temperature 31℃, enzymolysis time 2.48h and shaking by 120r/min that could be obtained with higher concentration for the future experiment.A series of phenomenon could be observed on the process of releasing. During the process, the volume of protoplast changed that it was smaller at the earlier stage than that at the later stage. The releasing of protoplasts could be classified into three forms- apex, side and releasing in situ. The releasing in situ was mainly emerged at the later stage. In addition, the nature fusion could be found when two protoplasts were so closed.After screening of electrophoresis conditions, two phases were needed for the optimal of electrophoresis separation. The electrophoresis time, temperature, electrophoresis buffer, agarose concentration, voltage, pulse time and angle would be considered which could have impact on the result of electrophoresis. The first phase: electrophoresis time 48h, temperature 14℃, electrophoresis buffer 1×TAE, agarose concentration 0.8%, voltage 2v/cm, pulse time 20-30min, angle 106°; in correspondence with the first phase, the second phase was 16h, 14℃, 1×TAE, 0.8%, 3v/cm, 500s, 106°.Using molecular weight markers, the analysis of chromosomes and molecular karyotype of Paecilomyces militaris was carried out. The largest chromosomes of stain RCEF0096 was 5.76Mb while the smallest one was 2.04Mb, and the total genome sizes were estimated to be 31.0Mb; the largest and the smallest one of stain RCEF4544 respectively was 5.69Mb and 2.04Mb. It was estimated that at least the total size of the karyotype was about 28.2Mb; as the size of stain RCEF0848 was between 2.0 and 5.7Mb, at least the total size of the nuclear type was 28.0Mb. So the molecular karyotype of Paecilomyces militaris was at least 28.0-31.0Mb.The result of electrophoretic karyotype showed that there was polymorphism among the stains of Paecilomyces militaris, which related to the living environment, the diversity of host, degradation in culture easily and so on. However, the size of the largest and the smallest was almost the same among the stains, and the chromosome numbers were also the same. It suggested that the differences between them were not so remarkable which equated with the report of the references that the differences among species of electrophoretic karyotype were more than that among stains and less than that among genera.
|
PDF Full Text Request