模拟空间环境诱变温莪术内生真菌及代谢产物生物活性研究
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摘要
目的：
模拟空间微重力、高磁场环境对温莪术内生真菌EZG0807进行诱变,筛选出抗生活性更高的正突变株。研究比较突变株与出发菌株EZG0807的菌体形态、细胞生长及其代谢产物的抗生活性。
方法：
采用稀释涂布平板法对诱变得到的菌株进行分离纯化；通过滤纸片扩散法和MTT法分别研究诱变株代谢产物对大肠杆菌、普通变形杆菌、枯草芽孢杆菌、金黄色葡萄球菌的抗菌活性和抗乳腺癌细胞MCF-7的活性,筛选出正突变株；将得到的正突变株与出发菌株EZG0807通过肉眼和显微观察法比较两者的菌落、菌丝及孢子形态；十字交叉法测量菌落直径和干重法测定生长曲线比较两者的生长速度；通过滤纸片扩散法抑菌实验和MTT抗肿瘤实验比较其抑菌及抗肿瘤细胞活性。
结果：
1.温莪术内生真菌EZG0807经模拟空间环境诱变后共分离得到129株菌,其中24 h,0 g条件下得到15株菌；24 h,1 g 14株；24 h,2 g 16株；48 h,0 g 17株；48 h,1 g 6株；48 h,2 g 22株；72 h,0 g 13株；72 h,1 g 14株；72 h,2 g 12株；
2.滤纸片扩散法初筛结果显示,129株菌中17株菌的代谢产物对金黄色葡萄球菌、普通变形杆菌、大肠杆菌、枯草芽孢杆菌有较好的抑制作用；
3.MTT法复筛结果表明：初筛的17株菌中诱变菌722-6的代谢产物对MCF-7细胞的抑制率比出发菌株EZG0807高58.08%；
4.菌落形态上,正突变株722-6的菌丝较出发菌株EZG0807纤长,膨松,且菌丝呈直立状,基内颜色偏暗；两株菌的菌丝及孢子形态相似,第4天时出发菌株EZG0807在光学显微镜下观察到孢子囊,而正突变株菌722-6没有；
5.通过比较菌落直径大小和生长曲线发现：正突变株722-6在PDA培养基上的菌落生长速度比出发菌株EZG0807慢,在查氏液体培养基中的生长速度也较慢,但进入稳定期后,两株菌的菌体干重基本相同；
6.在生物活性方面,正突变株722-6的代谢产物对普通变形杆菌的抑制作用与出发菌株EZG0807相当,对其它三种供试菌的抑制作用则较弱,但其对MCF-7细胞的抑制作用明显高于出发菌株EZG0807。
Objective:Endophytic fungus EZG0807 isolated from Curcuma wenyujin was mutagenized through the simulating space environment of micro-gravity and high magnetic field, aiming to screen the mutant strain with higher bioactivity. And it was compared with the starting strain EZG0807 at the aspect of cell growth, morphology and bioactivity of its metabolites.
Methods:The mutant strains were screened by the microdiluted method. To screen the active mutant, filter paper method and MTT assay were utilized to investigate its metabolites activity against the test bacteria Staphylococcus aureus, Proteus vulgaris, Escherichia coli, Bacillus subtilis and tumour MCF-7, respectively. The screened mutant was then compared with the starting strain EZG0807 at the aspect of morphology through observing the morphology of the colony, mycelium and spores with visual and microscope. Using cross method to measure the colony diameter on PDA medium and dry weight method to determine the growth curve in Czapek Dox medium to contrast the growth rate between the two strains. The antibacterial and antitumor activity of metabolites of the two strains was contrasted by filter paper method and MTT assay.
Results:
1.129 strains were isolated from the endophytic fungus EZG0807 from Curcuma wenyujin treated by the simulating space environment. Among these,15 strains were get under the condition of 24 h,0 g; 24 h,1 g 14; 24 h,2 g 16; 48 h,0 g 17; 48 h,1 g 6; 48 h,2 g 22; 72 h,0 g13;72h, 1g14and72h,2g12;
2. The result of the initial screening by filter paper method showed that the metabolites of 17 strains among the 129 strains had good inhibition effect to Staphylococcus aureus, Proteus vulgaris, Escherichia coli and Bacillus subtilis;
3. The result of the secondary screening by MTT assay manifested that the inhibition rate of metabolites of the mutant 72 2-6 to MCF-7 cells was 58.08% higher than the starting strain EZG0807;
4. At the aspect of the colony morphology, the mycelium of mutant 72 2-6 was upright shape, slender and more bulking than strain EZG0807, the substrate color was dimmer; the morphology of mycelium and spores of the two strains were almost the same, but sporangia were found in the strain EZG0807 only;
5. Finding by comparing the colony diameter and growth curve, the growth rate of mutant 72 2-6 was slower than strain EZG0807 on PDA medium, also in Czapek Dox medium, but the dry weight of cell of the two strains after stabilization period was almost the same;
6. At the aspect of bioactivity, the inhibition of metabolites of mutant 72 2-6 was almost the same with the starting strain EZG0807 to Proteus vulgaris, and lower than it to the other three instructed bacteria. But to the inhibition to MCF-7 cells, it was higher than the strain EZG0807.
模拟空间微重力、高磁场环境对温莪术内生真菌EZG0807进行诱变,筛选出抗生活性更高的正突变株。研究比较突变株与出发菌株EZG0807的菌体形态、细胞生长及其代谢产物的抗生活性。
方法：
采用稀释涂布平板法对诱变得到的菌株进行分离纯化；通过滤纸片扩散法和MTT法分别研究诱变株代谢产物对大肠杆菌、普通变形杆菌、枯草芽孢杆菌、金黄色葡萄球菌的抗菌活性和抗乳腺癌细胞MCF-7的活性,筛选出正突变株；将得到的正突变株与出发菌株EZG0807通过肉眼和显微观察法比较两者的菌落、菌丝及孢子形态；十字交叉法测量菌落直径和干重法测定生长曲线比较两者的生长速度；通过滤纸片扩散法抑菌实验和MTT抗肿瘤实验比较其抑菌及抗肿瘤细胞活性。
结果：
1.温莪术内生真菌EZG0807经模拟空间环境诱变后共分离得到129株菌,其中24 h,0 g条件下得到15株菌；24 h,1 g 14株；24 h,2 g 16株；48 h,0 g 17株；48 h,1 g 6株；48 h,2 g 22株；72 h,0 g 13株；72 h,1 g 14株；72 h,2 g 12株；
2.滤纸片扩散法初筛结果显示,129株菌中17株菌的代谢产物对金黄色葡萄球菌、普通变形杆菌、大肠杆菌、枯草芽孢杆菌有较好的抑制作用；
3.MTT法复筛结果表明：初筛的17株菌中诱变菌722-6的代谢产物对MCF-7细胞的抑制率比出发菌株EZG0807高58.08%；
4.菌落形态上,正突变株722-6的菌丝较出发菌株EZG0807纤长,膨松,且菌丝呈直立状,基内颜色偏暗；两株菌的菌丝及孢子形态相似,第4天时出发菌株EZG0807在光学显微镜下观察到孢子囊,而正突变株菌722-6没有；
5.通过比较菌落直径大小和生长曲线发现：正突变株722-6在PDA培养基上的菌落生长速度比出发菌株EZG0807慢,在查氏液体培养基中的生长速度也较慢,但进入稳定期后,两株菌的菌体干重基本相同；
6.在生物活性方面,正突变株722-6的代谢产物对普通变形杆菌的抑制作用与出发菌株EZG0807相当,对其它三种供试菌的抑制作用则较弱,但其对MCF-7细胞的抑制作用明显高于出发菌株EZG0807。
Objective:Endophytic fungus EZG0807 isolated from Curcuma wenyujin was mutagenized through the simulating space environment of micro-gravity and high magnetic field, aiming to screen the mutant strain with higher bioactivity. And it was compared with the starting strain EZG0807 at the aspect of cell growth, morphology and bioactivity of its metabolites.
Methods:The mutant strains were screened by the microdiluted method. To screen the active mutant, filter paper method and MTT assay were utilized to investigate its metabolites activity against the test bacteria Staphylococcus aureus, Proteus vulgaris, Escherichia coli, Bacillus subtilis and tumour MCF-7, respectively. The screened mutant was then compared with the starting strain EZG0807 at the aspect of morphology through observing the morphology of the colony, mycelium and spores with visual and microscope. Using cross method to measure the colony diameter on PDA medium and dry weight method to determine the growth curve in Czapek Dox medium to contrast the growth rate between the two strains. The antibacterial and antitumor activity of metabolites of the two strains was contrasted by filter paper method and MTT assay.
Results:
1.129 strains were isolated from the endophytic fungus EZG0807 from Curcuma wenyujin treated by the simulating space environment. Among these,15 strains were get under the condition of 24 h,0 g; 24 h,1 g 14; 24 h,2 g 16; 48 h,0 g 17; 48 h,1 g 6; 48 h,2 g 22; 72 h,0 g13;72h, 1g14and72h,2g12;
2. The result of the initial screening by filter paper method showed that the metabolites of 17 strains among the 129 strains had good inhibition effect to Staphylococcus aureus, Proteus vulgaris, Escherichia coli and Bacillus subtilis;
3. The result of the secondary screening by MTT assay manifested that the inhibition rate of metabolites of the mutant 72 2-6 to MCF-7 cells was 58.08% higher than the starting strain EZG0807;
4. At the aspect of the colony morphology, the mycelium of mutant 72 2-6 was upright shape, slender and more bulking than strain EZG0807, the substrate color was dimmer; the morphology of mycelium and spores of the two strains were almost the same, but sporangia were found in the strain EZG0807 only;
5. Finding by comparing the colony diameter and growth curve, the growth rate of mutant 72 2-6 was slower than strain EZG0807 on PDA medium, also in Czapek Dox medium, but the dry weight of cell of the two strains after stabilization period was almost the same;
6. At the aspect of bioactivity, the inhibition of metabolites of mutant 72 2-6 was almost the same with the starting strain EZG0807 to Proteus vulgaris, and lower than it to the other three instructed bacteria. But to the inhibition to MCF-7 cells, it was higher than the strain EZG0807.
引文
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