摘要
bkdF基因和nsdA基因是在阿维链霉菌(Streptomyces avermitills)基因组中发现的两个与阿维链霉菌次生代谢和分化发育密切相关的基因。bkdF的基因产物作为支链α酮酸脱氢酶(branched-chain α keto acid dehydrogenase,BCDH)复合体的Elα亚基参与了avermectin起始单元的生物合成。bkdF基因缺失会导致阿维链霉菌产阿维菌素能力的丧失。nsdA基因(negative regulator of streptomyces differentiation)是一个在链霉菌基因组中广泛存在的全局性负调控因子,天蓝色链霉菌的nsdA基因中断菌株能够提前且超量产生放线紫红素、钙依赖抗生素和次甲基霉素,同时产孢量也相应增多。本研究的目的是利用基因置换技术中断阿维链霉菌的bkdF基因和nsdA基因,研究它们对阿维链霉菌次级代谢的影响。
本研究采用了一种崭新的PCR介导的基因中断技术用于bkdF基因中断菌株的构建。合成一对长度分别为58,59nt的引物,其5’端的39nt序列分别与bkdF基因两侧同源,3’端则分别与安普霉素抗性标记盒(aac(3)Ⅳ+oriT)两侧序列一致。以该引物扩增的PCR产物电转化能表达入Red重组酶且含有目标质粒的Escherichia.coli菌株BW25113/pIJ790/pXW1224,获得了阳性重组质粒pXW1230。将该质粒中的约4kbBglⅡ酶切片段插入基因置换载体pHZ1351,再接合转移至阿维链霉菌BIB9903,筛选得到表型为Apra~RThio~S的接合子BIB0423。PCR验证bkdF基因已被正确中断。对BIB0423的发酵分析以及HPLC和MS分析显示,重组菌的阿维菌素合成途径被完全阻断,发酵产物为单一组分寡霉素A。
以pHL214为nsdA基因置换载体,接合转移至阿维链霉菌BIB9903中,筛选得到nsdA中断菌株BIB0515,PCR验证BIB0515中nsdA基因已被正确中断。形态观察发现,中断菌株BIB0515的表型发生了较大改变。在YMS平板上培养数天,BIB0515产孢量较出发菌株明显减少,并伴有大量黑色素生成。对中断菌株发酵产物的HPLC分析表明中断菌株的阿维菌素效价较出发菌株大幅度下降。该实验结果与预期结果不符,具体原因有待进一步研究探明。
bkdF and nsdA gene found in Streptomyces avermitilis genome are both closely related to secondary metabolism and morphological differentiation. bkdF gene encoding E1a subunit of branched-chain a keto acid dehydrogenase(BCDH) is involved in the biosynthesis of avermectin starter unit. Deletion of bkdF resulted in a S.avermitilis bkd mutant which lacked the ability to produce natural avermectins. nsdA gene is a globally negative regulator gene extensively existent in streptomyces genome. S.coelicolor nsdA null mutants produce more actinorhodin, CD A, methyleonomycin and spores than wild type strain. The goal of the study is to disrupt bkdF and nsdA gene by using gene replacement method, and investigate their effect on secondary metabolism of 5. avermitilis.In this study, a novel PCR-mediated gene replacement protocol was used to construct bkdF gene disruption strain. Firstly, a pair of long (58,59nt) primers were prepared, which have at 5'end 39nt matching the flanking sequence of bkdF, and the 3'sequence matching the right or left end of the apramycin resistant cassettes ( aac(3)IV+oriT ) .The linear DNA PCR-amplified by these primers was electro-transformed to the strain BW25113/ pIJ790/ pXW1224 which expresses λRed enzyme and contains target plasmid, then a positive recombined plasmid (pXW1230) was obtained. The 4kb fragment from pXW1230 restricted by Bgl II was inserted into pHZ1351, and introduced into S. avermitilis BIB9903 by conjugal transfer. A ApraRThios isolate BIB0423 was obtained and bkdF disruption was confirmed by PCR. HPLC and MS analysis of fermentation culture indicated that the avermectins production was blocked and the only product of BIB9903 was oligomycin A.Using as gene replacement vector, pHL214 was introduced into S. avermitilis BIB9903 by conjugal transfer. NsdA null mutant BIB0515 was obtained by screening and nsdA disruption was confirmed by PCR analysis. The phenotype of the mutant changed greatly. After cultivated on YMS plate, it produced less spores and more melanin pigment than wild strain. HPLC analysis of fermentation culture indicated that the avermectins production of nsdA disruption strain decreased sharply. The result doesn't accord with the anticipation, and the reason remains to elucidate.
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