| 摘要: |
| 【目的】建立野生型罗伊乳酸杆菌的高效电转化方法，为乳酸杆菌工程菌的研究提供参考。【方法】采用醋酸锂(LiAc)和二硫苏糖醇(DTT)对不同浓度(0.5×109,1.0×109,2.5×109,5.0×109,10.0×109 mL-1)的罗伊乳酸杆菌分别预处理0，10，20，30 min，制备罗伊乳酸杆菌感受态细胞，用于电转化，采用PCR和EcoRⅠ酶切鉴定阳性转化子，建立罗伊乳酸杆菌高效电转化方法。【结果】罗伊乳酸杆菌转化效率随LiAc和DTT处理时间的延长和细胞密度的升高而提高，在本试验条件下罗伊乳酸杆菌电转化的最佳菌体浓度为10.0×109 mL-1,LiAc和DTT的最佳处理时间为20 min，在该条件下转化效率达(32.60±7.12)×107 cfu/μg，比未经LiAc 和DTT处理的细胞转化效率高104倍以上。【结论】建立了罗伊乳酸杆菌高效电转化方法，电转化的最佳菌体浓度为10.0×109 mL-1,LiAc和DTT的最佳处理时间为20 min。 |
| 关键词: 电转化 乳酸杆菌 转化效率 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金面上项目“无抗性标记纤维素酶基因重组乳酸杆菌表达系统的构建”（31072060） |
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| Establishment of high efficiency electrotransformation method of Lacto bacillus reuteri cells pretreated with lithium acetate and dithiothreitol |
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SUN Lin, HU Xiongbing, WANG Lei
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College of Animal Science and Technology, Northwest A&F University
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| Abstract: |
| 【Objective】This study was to develop a high transformation efficiency method of wild-type Lactobacillus reuteri in order to meet the genetic manipulation of L.reuteri.【Method】The pretreatment of L.reuteri cells was performed with lithium acetate (LiAc) and dithiothreitol (DTT) at different time(0,10,20,30 min) of pretreatment and different cell concentrations (0.5×109,1.0×109,2.5×109,5.0×109,10.0×109 mL－1) of electrotransformation.The positive transformats with PCR and enzyme digestion were assayed.【Result】The transformation efficiency improved as time and cell concentration increased.The optimizing time was 20 min and cell concentration was 10.0×109 mL－1.At this condition the transformation efficiency reached （32.60±7.12）×107 transformants/μg DNA,which was 104 times more than untreated cells.【Conclusion】We established a new and high efficient transformation method and its transformation efficiency is （32.60±7.12）×107 transformants/μg DNA at the condition of 10.0×109 mL－1 cell concentration and 20 min pretreatment. |
| Key words: electroporation Lactobacillus transformation efficiency |
