| In this paper, the conditions of sporulation, the best hydrolysis and protoplast preparation techniques from 5 strains of Paecilomyces tenuipes and conditions of pulsed field gel electrophoresis from six different origins of Paecilomyces tenuipes were investigated, and the main results were as follows.After cultivated in different media, different temperatures for two weeks, there were significant differences in sporulation and mycelial growth of different strains, so was the best light conidiation. The results of the experiment showed that the best condition of conidia production for pt165 was that the strains were cultivated in Richard's medium, 27 oC and in the dark; the best condition for pt173 of conidiation and the hyphal volume was to cultivate it in PDA and illuminate for 2d in 29 oC; the one of pt208 and pt223 both was cultivated in Richard's medium and light culture in 23 oC for 6d; the best condition of conidiation about pt45 was in PDA, 27oC under the light of a longer period of time .The method of acquiring mycelium was different, and the enzymatic effect of mycelium was different. When cultivated for a period of time after the liquid seed was accessed to fresh medium, the phases of mycelial growth were inconsistent, resulting in different protoplast volume in same hydrolysis time, so that it was difficult to carry out following experiments. The spore suspension of consistent growth was added in the shake-bottle, the condition of cryptogamic germination was the same, and it was easy to control the hydrolysis and quantity in experiments.The time of spore germination from different strains had great impact on the enzyme. When the spore concentration of pt165 was 107 spores/ml, the time of mycelial germination was shorter than the one of 108 spores/ml, the effect of enzyme was better than the one of higher concentration, and other strains were the same.More or less, the components of different strains in cell wall were different, so was the best system of enzymysis and the best concentration. The effect of enzymolysis for pt165 was best in the combination of 2% snail enzyme with 2% cellulase for 4h, reaching to 15.04×106 / ml or more; It was also very effective for pt173 and pt45 in this combination of enzymatic concentration, the volume of protoplasts respectively reached to 15.6×106/ ml, 25.88×106/ ml for 3.5h, 4h; The effect of enzymysis for pt208 was best in the combination of 2% snail enzyme with 2% solubilization wall enzyme for 3.5h, reaching to 15.12×106/ ml; The effect of pt223 was also very effective in this combination of enzymatic concentration for 3.5h, reaching to 9.23×106/ ml. The best time of obtaining volume of protoplast was also not the same in different enzyme system for strains. It was found that the effect of cellulase was slower, the best time of enzymplysis was longer under the existence of cellulase in the same enzymatic condition, but the volume of protoplasts ultimately exceeded the one of other enzyme systems, so we chose a different enzyme system in accordance with the purpose of different experiments, this experiment was to choose shorter enzymatic digestion system and the concentration in which the time of enzymatic digestion was shortest and the effect of enzymolysis was best.The different temperatures of enzymolysis had significant impact on the effects of enzyme. In this experiment, the results of enzymolysis in several temperature gradient filter were: the enzymatic effects of pt165, pt208 and pt223 were best in 37 oC, the effect of enzymolysis was lower when the temperature increased or decreased; the effect of enzymolysis for pt45 and pt173 both was best in 29 oC.In the process of selection of electrophoresis conditions, the total time for electrophoresis, pulse time, voltage and angle, and electrophoresis buffer had impact on the result of electrophoresis. Different strains had different length polymorphism, so was the molecular weight of chromosome bands, there were great changes about the conditions of electrophoresis for different strains, through a series of screening, the results were as follows: the total electrophoresis time of the first phase was 48h, the temperature was 14 oC, electrophoresis solution for 1×TAE, the concentration of agrose for 0.8%, voltage for 2v/cm, pulse time for 20 ~ 30min, the conversion angle for 106 o; the total time of the second phase was 16h, the temperature for 14 oC, the electrophoresis solution for 1×TAE, the concentration of agrose for 0.8 %, the voltage for 3v/cm, the pulse time for 500s, the conversion angle for 106 o.After analyzing the bands in selection of condition , It was obvious that there was polymorphism for chromosome lengths and numbers, the detailed results were that there were five or more than five bands of chromosome for pt173 and pt223; there were six or more than six bands for pt45, at least seven different sizes of chromosome bands for pt97 and pt208. take mb for units, the sizes respectively were: 5.56, 3.77, 3.11, 2.87, 2.44 and 0.31 Mb for pt165, it was estimated that at least the total size of the karyotype was about 18.06 Mb; 5.78, 4.70, 3.94, 3.28, 2.88, 2.45 and 0.43 Mb for pt97, in which the second band from top to down was abnormal bright, it was estimated that this band was composition of more than one band, so the karyotype size of pt97 was at least 25.91Mb; 5.75, 3.05, 2.90, 2.80 and 2.29 Mb for pt223, it was estimated that the size of karyotype was 16.79 Mb; 5.37, 4.49, 3.50, 2.99, 2.72, 2.46 and 0.44 Mb for Pt208, the nuclear-type size was about 21.97 Mb; 5.89, 3.75, 3.06, 2.91, 2.29 for pt173, and the nuclear-type size was at least about 17.9 Mb; 6.04, 3.96, 3.15, 2.98, 2.75 and 0.49 Mb for pt45, but it was observed from the top to down that the brightness and width of the first, second, fourth band were larger, it was estimated that two bands or more than two bands togather, at least the size of the nuclear type was 25.76Mb.
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