【作者】 庞秀华；

【导师】 邓子新；

【作者基本信息】 华中农业大学 ， 分子生物学， 2000， 博士

【摘要】 链霉菌的染色体一般为线性结构，大小约为8Mb，相当于大肠杆菌染色体的1.5倍，并且在染色体的末端结合有末端蛋白。本实验以吸水链霉菌应城变种10-22为材料，首先对它的基因组的性质进行了探索性研究。脉冲电泳实验揭示10-22的染色体为一线性结构，可被限制性内切酶AseⅠ切割成11条片段，分子量总和约为7.35Mb。这与已报道的链霉菌基因组的大小接近。但经蛋白酶K和SDS处理的10-22染色体DNA的电泳行为并没有象天蓝色链霉菌A3(2)或变铅青链霉菌1326经同样处理的样品一样表现出差异。该结果暗示10-22染色体的末端可能与迄今已报道的染色体末端结构有所差别，但其分子细节尚不清楚。 链霉菌的共性之一是基因组的不稳定性，主要表现为染色体的缺失。诱变剂处理，原生质体再生及其它一些未知因素的刺激可以加剧链霉菌基因组固有的不稳定性。对10-22来说，染色体的不稳定区包括了170-，210-A，210-B，280-，300-，500-，680-，900-和1300-A共九条AseⅠ片段所在的区域。通过对出发菌株10-22及N-103，T157，Q303，N-1011，T106，T155，Q1513，WH-27和WH-14等衍生菌株酶切带谱的分析将上述各片段在染色体的相对位置确定下来，并进而给出了一个近乎完整的染色体的环形物理图谱。在检测染色体末端片段的尝试中，由于经SDS和蛋白酶K处理的基因组DNA样品的泳动行为未表现出差异，相应地经限制性内切酶切割后，基因组酶切片段的泳动行为也没有观察到明显的差异，目前，尚无法确定染色体的两个末端片段。 一般地，链霉菌遗传表型的变化与基因组的不稳定密切相关，在10-22中也是如此。与野生型菌株相比，10-22的缺失突变菌株均表现了不同程度的形态及生理生化特征的变化，具体表现为孢子颜色及色素颜色的丧失或变浅，抗生素生物合成能力的丧(?)。通过对5102-Ⅳ号抗生素生物合成阻断突变株的研究，揭示了5102-Ⅳ号抗生素(?)成能力的丧失与染色体的300kb片段的缺失有关，暗示了在300kb的片段上可能(?)负责该抗生素合成的基因簇。以此片段为探针钓出了位于该区域的文库克隆，(?)用指纹印迹和杂交技术将这些文库克隆排列起来，进而以位于该区域的不同位(?)片段做臂构建了用于转化和接合转移用的基因置换质粒，并试图通过基因置换(?)域置换下来，但尚未得到最终结果。更多还原

【Abstract】 Streptomyces species has a linear chromosome with a size of c.8Mb, which is almost one and half times that of E. coli, and terminal proteins at its ends. Genome mapping and structure of Streptomyces hygroscopicus 10-22 is the prime interest of the present study. PFG1E (Pulsed Field Gel Electrophoresis) revealed a linear chromosome with a size of 7.35Mb. and 11 fragments could be generated with Asel digestion. No obvious difference couM be observed with SDS-treated and PK-treated 10-22 samples, which seems to be opposite of 5. coelicolor A3(2) and S. lividans 1326 (which were known to possess terminal proteins and thus will retard in the slot during PFGE), indicating that the terminal structure of 10-22 might be different from the ones described so far in Streptomyces, whose exact property still remains unknown.Oae of the common features of Streptomyces is its instability, which was manifested by frequently occurring chromosomal deletions, as is the same case with 10-22. In 10-22, the unstable region encompasses the 170-, 210-A, 210-B, 280-, 300-, 500-, 680-, 900- and 1300-A Asel fragments. By the analysis of the restriction banding patterns of 10-22 and its variant mutants N-103, T157, Q303, N-1011, T106, Q1513, T155, WH-14, WH-27, etc, the above mentioned fragments could be located on chromosome and thus make it possible to draw an almost complete chromosomal physical map of 10-22, though stil! circuilar. The attempt of detecting the terminal fragments was failed as there is no difference observed with SDS and PK treated samples digested with Asel.Normally, the changes of phenotype are closely related to the genome instabilities. The sarnss phenomenon could also be observed in S. hygroscopicus 10-22. Notable changes in spotre color, pigments production and antibiotics biosynthesis have been observed in 10-22 derivatives in which large chromosomal deletions had occurred. 30 blocked strains for 5102-IV antibiotic production were obtained in a screening project. PFGE analysis of these 30 blocked strains revealed a common chromosomal deletion in the 300-kb Asel fragment which might be responsible for 5102-IV antibiotic biosynthesis. So this 300kb fragment was recovered and used as probe to hybridize with 10-22 genomic library. Cosmids within this region were aligned and suitable fragments in this region were selected and used further for the construction of plasmids for gene replacement. At last, 16 mespmcement plasmids for two different parts were obtained and have been introduced intou 1022. but final results had not obtained yet.更多还原