甲基营养菌Methylobacterium sp.MB200中与L-丝氨酸耐受性相关基因的克隆与分析

摘要

Ten strains of E rw in ia carotovora bacteriophages were iso lated from the law sew age. R esults of their hot stability and pH-stab ility showed that the phages nam ed Erj2, Erb1 and Erc2 had relatively high stability w ith in the tem perature of -20~40℃ and pH value of 4 .0~9 .0 . Their biobgical characteristics analysis showed their optm al m ultip licity of infection was 0 .0001 , 0.001, 0.0001 in turn; They were doub le-stranded D NA bacterbphage; The glow th curve of the page E rj2 show ed that the incubation period w as 15 m in , the tin e of burstphase w as 13 m in and the burst size was 442 ; E lection m icrographics showed thatphage ErE and Erc2 exhibited a cubic sym m etry , and E rj2 had a bng tail, Erc2 had a short tail. Co-culture of phage E rj2 and Erc2 w ith E . Am ylovora, resulted in a decrease in the absorbance, w hereas the 0 D 600 plots for the E rj2 and E rc2 ccrcultures w ere less than 0 .5 ; ccrculture w ith E rj2 resulted in a decrease at 0 D600 after 4 h nearly to zero . This result showed that the phage could inhibit the grow th of host bacteria, indicating a biocontrol effect and application prospects.%甲基营养菌MB200,能利用非C-C键低碳化合物生长并合成L-丝氨酸,对L-丝氨酸耐受浓度低,提高其在静息细胞反应系统中对L-丝氨酸的耐受能力可更好的提高L-丝氨酸的产量.利用质粒转座子pTnMod-RKm'构建了甲基营养菌MB200的突变体库,往培养基中添加20 mg/mL L-丝氨酸对突变体库中的突变体进行平板筛选,共筛选到177株耐受L-丝氨酸的突变体,以10 mg/mL的浓度梯度复筛,发现某些突变体能最高耐受40 mg/mL L-丝氨酸.分别提取突变体总DNA,用BamHI酶随机酶切后连接到克隆载体pMD18-T上,导入到大肠杆菌感受态细胞DH5α中,利用双抗性在(转座子抗性与T载体上的抗性)平板上挑取转化子,并提取质粒,酶切验证后进一步测序.共克隆到了17个基因,为提高甲基营养菌对L-丝氨酸的耐受能力提供了可研究的基因资源.