潮霉素B抗性为选择标记的整合型表达载体的构建及在米根霉中的遗传转化

Construction of an integration vector carrying hygromycin B resistance gene and its genetic transformation in Rhizopus oryzae

摘要:

为了建立适合米根霉的遗传转化体系，应用重叠延伸PCR的方法构建了以潮霉素B抗性为选择标记的单交换整合型表达载体pBS-hygro-ldhA；分别采用PEG/CaCl2介导的原生质体转化、原生质体电转化及萌发孢子电转化的方法将表达载体pBS-hygro-ldhA转化入米根霉AS 3.819菌株中，并研究了菌丝酶解时间、孢子萌发时间以及电转化电场强度对于转化效率的影响；通过荧光定量PCR (qPCR) 对米根霉转化子基因组中质粒整合拷贝数进行了检测，并研究了其对米根霉转化子抗性稳定性的影响。实验结果表明成功获得整合了表达载体pBS-hygro-ldhA的米根霉转化子。菌丝酶解140 min产生的原生质体其再生率和转化率最高，原生质体电转化最佳电场强度为13 kV/cm，孢子萌发2.5 h转化率最高，萌发孢子电转化最佳电场强度为14 kV/cm。萌发孢子电转化方法转化率要高于原生质体转化的方法。荧光定量PCR检测结果表明，在一定范围内，高质粒整合拷贝数的米根霉转化子比较稳定。研究建立了用于工业米根霉菌株的遗传转化体系，为米根霉代谢调控研究以及菌种改造工作提供了基础与支持。

Abstract:

To construct a system of genetic transformation suitable for Rhizopus oryzae, we constructed a single-exchange vector pBS-hygro carrying hygromycin B resistance gene (hph) as its selective marker using gene splicing by overlap extension PCR (SOE PCR) technique. We introduced this recombinant vector into Rhizopus oryzae AS 3.819 by PEG/CaCl2-mediated transformation of protoplast, electroporation of protoplast and germinated spores; and we studied the effects of hydrolysis time, field strength and spore germination time on transformation frequency. We conducted quantitative real-time PCR (qPCR) assay to determine the gene copy number of ldhA integrated in the genome of R. oryzae transformants and its effect on the stability of transformants. We successfully achieved R. oryzae transformants integrated with pBS-hygro-ldhA vector. The optimal hydrolysis time for protoplast production was 140 min, and the optimal field strength of electroporation pulse for protoplast was 13 kV/cm. The optimal germination time of spores for electroporation was 2.5 h, and the optimal field strength of electroporation pulse was 14 kV/cm. The transformation frequency of method based on germinated spores was generally higher than the methods based on protoplast. The qPCR test results suggested that transformants with high copy number of integration in a certain range were relatively stable. Our results provided basis and support for metabolic?regulation and genetic engineering breeding of R. oryzae.