委内瑞拉链霉菌秦岭变种属间接合转移系统的构建及透明颤菌血红蛋白的表达

Construction of conjugal transfer system of Streptomyces venezuelae var.Qinlingensis and the expression of vitreoscilla hemoglobin

【目的】构建委内瑞拉链霉菌秦岭变种属间接合转移系统及透明颤菌血红蛋白的表达。【方法】以链霉菌广泛使用的整合型质粒pSET152和复制型pHZ1358为出发质粒,通过供体大肠杆菌(Escherichia coli)ET12567(pUZ8002)进行属间接合转移委内瑞拉链霉菌秦岭变种。【结果】确定了该变种的最佳接合转移条件;通过SOE-PCR(Splicing by overlap extension PCR)技术构建含PermE和vhb结构基因融合片段的整合型表达载体pJD100,转化ET12567(pUZ8002)后属间接合转移委内瑞拉链霉菌秦岭变种。通过PCR和CO结合差光谱验证了vhb基因在委内瑞拉链霉菌秦岭变种中的整合表达。【结论】本文首次探索了委内瑞拉链霉菌秦岭变种接合转移系统,确定了委内瑞拉链霉菌秦岭变种的最佳接合转移条件,并采用基因工程手段使vhb基因在委内瑞拉链霉菌秦岭变种中获得表达。

[Objective]To Construct of conjugal transfer system of Streptomyces venezuelae var. Qinlingensis and express of vitreoscilla hemoglobin. [Methods] Intergeneric genetic transfer system was based upon plasmid pSET152 and pHZ1358 from Donor E.coli ET12567（pUZ8002） to Streptomyces venezuelae var. Qinlingensis [Results] Our data showed that Intergeneric genetic transfer system was demonstrated and optimized. Integrating plasmid pJD100 containing Perm Eand construction gene of vhb,constructed by SOE-PCR,was transformed to ET12567（pUZ8002）,and then transfered to Streptomyces venezuelae var. Qinlingensis by conjugation. The integrating expression of vhb gene in Streptomyces venezuelae var. Qinlingensis was verified by PCR and CO binding diference spectrum. [Couclusion] In summary,intergeneric genetic transfer system was demonstrated and optimized,and this is the first report to express vhb gene in Streptomyces venezuelae var. Qinlingensis.