指状青霉（Penicillium digitatum）再生体系的建立以及CYP51基因功能初探
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摘要
通过对制备材料、酶液浓度、酶解时间、酶解温度、渗透压稳定剂的种类和浓度等因素的实验研究，得到了一套制备指状青霉(Penicillium digitatum)原生质体的有效方法，并在双层培养基上初步实现了原生质体的再生，再生率可达24.9％。此外，还对原生质体的释放和再生方式进行了跟踪观察。原生质体释放有顶端、侧端和原位释放3种方式。原生质体的再生有2种方式，一种是原生质体一端突出形成酵母出芽状细胞；另一种是在原生质体一端直接长出菌丝。
本研究构建了指状青霉对抑霉唑敏感和抗性的两个CYP51基因的pCB1004表达载体，并对基因的插入方向和拷贝数进行了分析，证明了两个载体连接的基因均为正向连接和单拷贝插入。采用PEG法和电击法两种方法进行指状青霉原生质体的转化，然后将得到的转化产物转移到查氏再生培养基中培养，遗憾的是没有得到理想的转化子。
采用高效液相色谱技术(HPLC)分析了对抑霉唑敏感和抗性的两种指状青霉菌株(分别为pd23和pd07)中游离麦角甾醇的含量并比较了两者之间的差异。结果表明，在抗性菌株pd07不加抑霉唑的情况下，pd07和pd23中游离麦角甾醇的含量采用Duncan's新复极差测验在5％水平没有明显差异；但当pd07中加入0.1μg／ml抑霉唑时，三次重复测定结果显示抗性菌株pd07中麦角甾醇的含量明显地要比敏感菌株pd23中的含量高，而且两者在5％水平上差异显著。推测抗性菌株中麦角甾醇含量的积累是需要抑霉唑等药物诱导的。与敏感菌株相比，抗性菌株CYP51基因启动子上游多了4个126bp的串联重复序列，推测该重复序列可能是导致CYP51基因过量表达的内在原因。
The effect of some factors, including the appropriate materials for isolating protoplasts, the concentrations of enzyme, period of digestion and temperatures, and osmotic pressure stabilizers on the isolation and regeneration of protoplasts in Penicillium digitatum were studied. The results demonstrated that the purified protoplasts could regenerate through double layers of Czapek medium containing 0.7mol/L NaCl. The regeneration rate could reach 24.9%. Additionally, the mode of protoplast releasing and regeneration were investigated. It indicated that the primary protoplasts of P. digitatum were released mainly from the hyphalapex, and occasionally from the subapical or original sites. The regeneration of protoplasts could be classfied into two forms: the yeast-like cell developed from the protoplast and the mycelium grew from the protoplast directly.
The CYP51 genes cloned from both isolates of imazalil-resistant and imazalil-sensitive of P. digitatum were cloned into the pCB 1004 expression vectors, respectively. The direction and number of inserted copy were confirmed by restriction enzyme digestion. These vectors were tried to transform into the P. digitatum imazalil-sensitive isolate by the methods of PEG-mediated or electroporation transformation. After that, the transformed protoplasts were regenerated on Czapek medium containing hygromycin at 300#g/ml. Unfortunately, the ideal transformants could not be obtained.
In order to analyze the differences of the content of ergosterol in the imazalil-sensitive and imazalil-resistant isolates of P. digitatum, the technology of HPLC was used. The results showed that the contents of ergosterol between imazalil-sensitive and imazalil-resistant isolates were not significantly defferent (P=0.05, DMRT) when they grew in the medium without imazalil. Interestingly, when imazalil (0.1#g/ml) was added to the medium, the content of the ergosterol in imazalil-resistant isolate (pd07) was significantly higher than that in imazalil-sensitive isolate (pd23) (P=0.05, DMRT). These results suggested that higher accumulation of ergosterol might be induced by imazalil in the isolate of pd07, which contained
4-126bp tandemly repeated in CYP51 gene more than that in the pd23. It also suggested that these tandemly repeated sequence might be an element that could regulate the expression of CYP51 gene.
本研究构建了指状青霉对抑霉唑敏感和抗性的两个CYP51基因的pCB1004表达载体，并对基因的插入方向和拷贝数进行了分析，证明了两个载体连接的基因均为正向连接和单拷贝插入。采用PEG法和电击法两种方法进行指状青霉原生质体的转化，然后将得到的转化产物转移到查氏再生培养基中培养，遗憾的是没有得到理想的转化子。
采用高效液相色谱技术(HPLC)分析了对抑霉唑敏感和抗性的两种指状青霉菌株(分别为pd23和pd07)中游离麦角甾醇的含量并比较了两者之间的差异。结果表明，在抗性菌株pd07不加抑霉唑的情况下，pd07和pd23中游离麦角甾醇的含量采用Duncan's新复极差测验在5％水平没有明显差异；但当pd07中加入0.1μg／ml抑霉唑时，三次重复测定结果显示抗性菌株pd07中麦角甾醇的含量明显地要比敏感菌株pd23中的含量高，而且两者在5％水平上差异显著。推测抗性菌株中麦角甾醇含量的积累是需要抑霉唑等药物诱导的。与敏感菌株相比，抗性菌株CYP51基因启动子上游多了4个126bp的串联重复序列，推测该重复序列可能是导致CYP51基因过量表达的内在原因。
The effect of some factors, including the appropriate materials for isolating protoplasts, the concentrations of enzyme, period of digestion and temperatures, and osmotic pressure stabilizers on the isolation and regeneration of protoplasts in Penicillium digitatum were studied. The results demonstrated that the purified protoplasts could regenerate through double layers of Czapek medium containing 0.7mol/L NaCl. The regeneration rate could reach 24.9%. Additionally, the mode of protoplast releasing and regeneration were investigated. It indicated that the primary protoplasts of P. digitatum were released mainly from the hyphalapex, and occasionally from the subapical or original sites. The regeneration of protoplasts could be classfied into two forms: the yeast-like cell developed from the protoplast and the mycelium grew from the protoplast directly.
The CYP51 genes cloned from both isolates of imazalil-resistant and imazalil-sensitive of P. digitatum were cloned into the pCB 1004 expression vectors, respectively. The direction and number of inserted copy were confirmed by restriction enzyme digestion. These vectors were tried to transform into the P. digitatum imazalil-sensitive isolate by the methods of PEG-mediated or electroporation transformation. After that, the transformed protoplasts were regenerated on Czapek medium containing hygromycin at 300#g/ml. Unfortunately, the ideal transformants could not be obtained.
In order to analyze the differences of the content of ergosterol in the imazalil-sensitive and imazalil-resistant isolates of P. digitatum, the technology of HPLC was used. The results showed that the contents of ergosterol between imazalil-sensitive and imazalil-resistant isolates were not significantly defferent (P=0.05, DMRT) when they grew in the medium without imazalil. Interestingly, when imazalil (0.1#g/ml) was added to the medium, the content of the ergosterol in imazalil-resistant isolate (pd07) was significantly higher than that in imazalil-sensitive isolate (pd23) (P=0.05, DMRT). These results suggested that higher accumulation of ergosterol might be induced by imazalil in the isolate of pd07, which contained
4-126bp tandemly repeated in CYP51 gene more than that in the pd23. It also suggested that these tandemly repeated sequence might be an element that could regulate the expression of CYP51 gene.
引文
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