吸水链霉菌谷氨酰胺转胺酶基因的阻断及其对细胞分化的影响

Effect of transglutaminase-gene disruption in Streptomyces hygroscopicus on cell differentiation

【目的】通过对吸水链霉菌(Streptomyces hygroscopicus)中谷氨酰胺转胺酶基因的阻断,以期深入了解谷氨酰胺转胺酶生理功能,并为谷氨酰胺转胺酶发酵优化提供新的研究思路。【方法】以温敏型质粒pKC1139为出发质粒,构建阻断吸水链霉菌谷氨酰胺转胺酶编码基因的重组质粒pKC1139-TG1,转化吸水链霉菌原生质体,通过抗性筛选和PCR验证,成功得到一株谷氨酰胺转胺酶阻断菌株,命名为S.h-△TG。【结果】以原始菌株为对照,重组子基内菌丝生长不受影响,但是由基内菌丝分化形成气生菌丝的过程受到影响,重组子基本不产气生菌丝。【结论】谷氨酰胺转胺酶对吸水链霉菌气生菌丝的形成有着重要的影响,参与链霉菌气生菌丝的形成。

[Objective]To provide theoretical basis for understanding the physiological function of transglutaminase in Streptomyces,which would benefit for improving the production of this enzyme,transglutaminase gene was disrupted in Streptomyces hygroscopicus.[Methods ]First,the interruption plasmid pKC1139-TG1 was constructed by inserting a segment of transglutaminase gene in the temperature-sensitive vector pKC1139.Second,pKC1139-TG1 was transformed into S.hygroscopicus protoplast and the interruption plasmid was inserted into the chromosome by homogenous recombination.Third,the strain with disrupted transglutaminase gene was screened by apramycin resistance and named as S.h-△TG.[Results]Compared with the parent strain,S.h-△TG was able to grow as substrate mycelium,but not able to form aerial hyphae.[Conclusion]Transglutaminase may be involved in the aerial hyphae formation of S.hygroscopicus.