【作者】 韩建荣；

【导师】 袁静明；

【作者基本信息】 山西大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 从山西汾阳混交林土壤中分离到一株能在菌核内积累类胡萝卜素的青霉PT95菌株。分别观察了PT95菌株在查氏酵母膏琼脂（CYA）、麦芽汁琼脂（MEA）、25%甘油硝酸盐琼脂（G25N）和查氏琼脂（CA）上的菌落特征和个体形态特征。结果表明:PT95菌株在CYA培养基上25℃培养7d后能形成由大量的菌核和少量分生孢子构成的菌落,菌落表面呈放射状的沟纹和皱缩;菌核区颜色为粉红色,后变为深红色,产孢区颜色为青灰色;菌核球形或不规则形,直径约300μm;帚状枝为严格单轮生,分生孢子梗生自基质或气生菌丝;菌丝分枝较少;瓶梗3～8个轮生,安瓿形,排列较紧密,8μm～10μm×1.5μm～2.5μm;分生孢子椭圆形,长轴2.5μm～3.0μm。在CYA培养基上37℃不生长,5℃偶尔生长。经检索,PT95菌株属于汤姆青霉系。 比较了CTAB、SDS-CTAB和氯化苄这3种DNA提取方法提取青霉DNA的效果。实验结果表明:CTAB法和SDS-CTAB法只能适合于部分青霉菌株的DNA提取。另外一些菌株则提取不出谱带清晰的DNA。而氯化苄法适合于供试的所有青霉菌株的DNA提取,所有菌株的DNA凝胶电泳图谱带清晰,效果很好。同时研究比较了液氮研磨和提取液的pH值对氯化苄法提取DNA效果的影响。 运用RAPD技术对PT95菌株、4株PT95的突变株和6株标准青霉菌株进行了比较。从70个10bp引物中筛选出13个有效引物,用于所有供试菌株基因组DNA样品的扩增。这13个引物对11株供试菌株均能扩增出清晰的谱带。对RAPD结果进行聚类分析,构建了各供试菌株基因型的亲缘关系图。从聚类图上可以看出,PT95菌株与同属汤姆青霉系的3种标准菌聚为一类,而产黄青霉、娄地青霉和嗜松青霉聚为另一类,这与形态分类的结果相一致。但PT95菌株在电泳图谱上与汤姆青霉系的3种已知标准菌均有一定的差异,根据聚类图可以清楚地将PT95菌株与其他菌株分开,这说明PT95菌株不同于任一汤姆青霉系的已知菌,很可能是一新种,有待进一步研究。 初步研究了氧胁迫和外源β-胡萝卜素对青霉PT95菌株菌核分化和类胡萝卜素产率的影响。在本实验中,光照和培养基中加Fe³⁺作为高氧胁迫的培养条件,黑暗作为低氧胁迫的培养条件。结果表明,在培养基更多还原

【Abstract】 A strain of Penicillium sp. PT95 was isolated from soil collected close to Fenyang, Shanxi Province. The strain can form abundant sclerotia in which carotenoid is accumulated. Colonies and microscopic characters of PT95 strain in 4 kinds of media, i.e. Czapek yeast extract agar (CYA), Malt extract agar (MEA), 25%Glycerol nitrate agar (G25N) and Czapek agar (CA), were observed respectively. Colonies on CYA for 7d at 25 ℃ typically 32 mm ~ 40 mm diam, radially sulcate or winkled, usually enveloping abundant sclerotia and overlaid by scattered penicilli. Sclerotia, pink, becoming scarlet in age, globose or irregular, about 300um diam. Conidiogenesis sparse, caesious; pinicilli monoverticillati strictly, conidiophores borne from surface or subsurface hyphae; phialides ampulliform, 8 μm -10 μm× 1.5 μm~2.5 μm; conidia ellipsoidal, 2. 5 μm ~3. 0 μm long. Usually no germination at 5℃; occasionally germination observed. No growth at 37℃. Based the characters above, PT95 strain was identified to P. thomii series.Three kinds of methods (i.e. benzyl chloride, CTAB and SDS-CTAB) were tried to extract DNA from eleven strains of Penicillium, Results showed that the benzyl chloride method was able to be used to extract the DNA of Penicillium strains, and bands occurred from gel electrophoresis were very clear. However, both CTAB and SDS-CTAB method was only suitable to extract DNA in some strains of Penicillium, and moreover, DNA bands of some strains were clear, but others were not obvious. The effect of pH and grinding mycelia in liquid nitrogen on DNA amounts were also discussed.Eleven strains of Penicillium, i.e. PT95 strain and its 4 mutants, and 6 standard strains which were purchased from Institute of Microbiology, Academia Sinica, have been analyzed for their genotype by RAPD. Among 70 random primers, 13 primers were amplified available for all strains. The genetic relationship had been evaluated by similarity cofficient obtained from these profiles. The cluster showed that all strains could be divided into two parts. The first part consisted of PT95 and all strains of Pinicillium thomii series, and the second part was P. chrysogenum、 P. roqueforti and P. pinophilum. The genotypic typing of species seems to be related to morphological classification. But PT95 strain differed from all standard strains of P. thomii series in gel electrophoresis. According to cluster, PT95 strain could be obviously distinguished from other strains. The result showed that PT95 strain was not any species known of P. thomii series and probablywas a new species.The influence of oxidative stress and exogenous P -carotene on sclerotial differentiation and carotenoid yield of Penicillium sp. PT95 was studied. In this experiment, high oxidative stress was applied by inclusion of FeCl3 in the growth medium and by light exposure. Low oxidative stress was applied by omitting iron from the growth medium and incubation in the dark. Middle oxidative stress was applied by light minus iron or by dark plus iron. The results showed that in the medium supplemented with exogenous 3 -carotene, the time of both exudates and sclerotial initials of PT95 strain were delayed. The higher the concentration of exogenous 3 -carotene was, the longer the time of delay. But the time of sclerotial maturation was not changed. On the other hand, the exogenous 3 -carotene also caused a concentration-dependent reduction of both lipid peroxidation and content of carotenoid in sclerotia. The growth condition at high oxidative stress favored the sclerotial differentiation and pigment accumulation of PT95. The interaction of light and iron achieved a better degree of oxidative stress than any either factor alone. The sclerotial biomass and carotenoid yield of PT95 at high oxidative stress was increased 53% and 255% respectively as compared with that at low oxidative stress (dark). These data prompted us to consider that in order to attain higher sclerotial biomass and pigment yield, the strain PT95 should be grown under high oxidative stress and in the absence of antioxidants.Lipid peroxidation and ascorbic acid content were measured at different developmental stages of Penicillium sp. PT95 in Joham’s medium. It was found that PT95 strain produced ascorbic acid at levels dependent on its developmental stages. During early differentiation, there was a gradual increase in ascorbic acid level. The proportion of reduced ascorbic acid in whole ascorbic acid increased during early differentiation. The lipid peroxidation level was low in the undifferentiated mycelia, but reached its maximum at the sclerotial initials stage, the latter was 1.92 fold higher than the former. After middle differentiation stage, lipid peroxidation and ascorbic acid level decreased to those of undifferentiated stage. The influence of exogenous ascorbic acid on sclerotial differentiation and lipid peroxidation of PT95 was also studied. The result showed that in the Joham’s medium supplemented with exogenous ascorbic acid, the time of both exudates and sclerotial initials were delayed. The higher the concentration of exogenousascorbic acid was, the longer the time of delay. But the time of sclerotial maturation was not changed. On the other hand, the exogenous ascorbic acid also caused a concentration-dependent reduction of lipid peroxidation.This study examined the respective effect of various inorganic salts, carbon and nitrogen sources on sclerotial biomass and carotenoid yield in surface cultures of PT95 strain. Among inorganic salts tested, K2HPO4 was more essential to the sclerotial formation and carotenogenesis of strain PT95 than KC1, MgSO4 or FeSC>4. It was also shown that the combination of K2HPO4, KC1 and MgSC>4 could produce the best positive cooperation and gave the highest sclerotial biomass (782 mg/plate) and pigment yield (328 jig/plate). All of five carbon sources, i..e. glucose, sucrose, lactose, maltose and soluble starch, could be utilized by the strain PT95, and maltose was the best. Among 8 nitrogen sources, yeast extract favored the sclerotial formation, and peptone favored the pigment accumulation; amine salts and urea were unfavorable to form sclerotia. The medium containing 0.24 g/L ~ 0.48 g/L sodium nitrate-nitrogen was effective to both the sclerotial formation and the carotenoid production of strain PT95 when available maltose-carbon concentrations were at 5.26 g/L~ 21.05 g/L. The optimal C/N ratio was found to be 25:1.Six kinds of heat-released soluble cell-wall fragments (elicitors) were prepared from Neurospora crassa, Monascus purpureus, Sporolomyces roseus, Rhodotorula rubra, Nocardia sp. N89 and Actinoplanes sp. A05, respectively. When Penicillium sp. PT95 was grown on corn meal solid medium containing appropriate mass fraction (MF) of elicitors, both its sclerotial biomass and carotenoid content in sclerotia were enhanced significantly (P<0.01). Every one of elicitors except that from M. purpureus could also increase significantly the P -carotene fraction of total pigment (/><0.01). Among the elicitors tested, the elicitor from M. purpureus gave the highest carotenoid yield. When the MF of M. purpureus elicitor in medium was 100 p.g/g, carotenoid yield per 100 g corn meal reached 14446 |ag, which was 1.76 times higher than that of control. Experimental result also showed that the elicitor from M. purpureus could inhibit effectively the occurrence of sectoring during solid-state fermentation of strain PT95.Various grains and inocula were evaluated for carotenoid production by solid-state fermentation using Penicillium sp. PT95. The millet medium was more effective to both the sclerotia growth and carotenoid production of strain PT95 than other grain media. An更多还原