【作者】 张力；

【导师】 鲁明波； 余龙江；

【作者基本信息】 华中科技大学 ， 微生物学， 2011， 硕士

【摘要】 乳酸是一种天然的有机酸,乳酸及乳酸衍生物广泛应用于食品、医药、酿造、纺织等领域,其中以L-乳酸为原料合成的聚乳酸被认为是最有前途的可生物降解高分子材料。目前,因传统的乳酸菌育种中主要存在筛选周期较长、多价载体构建受限和难以获得全局最优目标表型等问题,限制了乳酸菌遗传育种的发展。为此,本研究基于细胞全局转录水平,采用融合PCR技术获得RNA聚合酶（RANP）的首要亚基-σ因子的表达序列,并采用易错PCR技术引入突变,获得转录突变库,筛选获得性能显著提升的细胞株。（1）克隆获得鼠李糖乳酸杆菌σ因子及其本源启动子;采用融合PCR方法获,得σ因子的表达序列;采用定点突变结合融合PCR的方法,在不改变阅读框架的前提下,成功突变了σ因子中的EcoRI酶切位点;采用易错PCR方法在σ因子的表达序列中引入随机突变并与乳酸菌组成型的表达载体pMG36e连接,转化大肠杆菌DH5α感受态细胞,获得突变文库。（2）不仅确定了噬淀粉乳酸杆菌B0112为本研究的最适受体菌株,还确定了最佳电转化条件:采用5%甘氨酸和0.5 mol/L蔗糖预处理细胞壁,待菌体生长到OD₆₀₀达到0.6左右时收获细胞,并采用电转缓冲液EB2清洗三次后,制备成电转化感受态,在电压为1.5 kv的条件下,得到10⁴数量级的阳性转化子。（3）在乳酸筛选压力下,得到优势菌株LA5,其生长速度快于对照C1和C2,推测可能是由于氨基酸的替换及C末端27个氨基酸的缺失,导致了σ因子的空间结构发生了变化,产生了一定的调控效果。更多还原

【Abstract】 Lactic acid, an important natural organic acid, are widely used in food, medicine, brewing, textiles and other fields. What’s more, L-lactic acid as the raw material of synthesing polylactic acid is known as the most promising biodegradable high molecular material. Presently, there are some diffculties in Lactic acid bacteria traditional breeding, such as longer time in screening cycle, difficulties in constructing multivalent vector and obtaining global optimal target phenotype which limits the development of Lactic acid bacteria’s genetical breeding. Therefore, based on the level of global transcription, the study is described as follows: Firstly, complete expression sequences ofσfactor, the primary subunit of RNA polymerase（RANP）, was constructed by fusion PCR; Secondly, then transcriptional mutant library was constructed by error-prone PCR; Lastly, destinated strains with significantly improved cell phenotype were selected.（1）Theσfactor and native promoter region was amplified from Lactobacillus rhamnosus; Fusion primers were designed to obtain the complete expression sequences ofσfactor by fusion PCR, and the EcoRI restriction site inσfactor was mutated in the premise of unchanging the open reading frame; Then the complete expression sequence ofσfactor was subjected to error-prone PCR and inserted into the plasmid pMG36e which is the Lactic acid bacteria constitutive expression vector, and then transformed into Escherichia coli DH5αcompetent cells to obtain the mutant library.（2）It showed that Lactobacillus amylovorus B0112 was the best recipient strain; Highest electrotransformation efficiency of 10⁴ transformants could be obtained when Lactobacillus amylovorus B0112 was pretreated under 5% of glycine/0.5 mol/L sucrose, and washed three times with electrotransformation buffer of EB2, as well as the 1.5 kV of field strength and one time of pulse with the OD₆₀₀ nm value of 0.6～0.65.（3）LA5 strain was obtained under the pressure of Lactic acid, of which the growth rate faster than control 1 and control 2 strains. It is speculated that substitution of amino acids and the lost of 27 amino acids ofσfactor in C terminal led to a change in spatial structure, and resulted in a certain regulatory effect.更多还原