摘要
选用从雷州半岛近岸海域分离鉴定的2株海洋红酵母(胶红酵母J6和黏红酵母J2)作为出发菌株,进行亚硝基胍(NTG)和紫外线(UV)复合诱变,以期筛选出高产类胡萝卜素的突变株。先用NTG处理出发菌株的菌悬液60min,再用UV照射120s,且要连续诱变3次;每次复合诱变后的菌悬液经短时间的液体培养后涂布于红酵母琼脂平板培养,从中筛选出一定数量的第1、2和3代突变菌株;将突变菌株接种于红酵母发酵培养基,经培养、离心和烘干,制备出干菌体细胞;用分光光度计法测定干菌体细胞中的类胡萝卜素含量,筛选出高产类胡萝卜素的突变株,并进行遗传稳定性试验。结果表明,胶红酵母J6的正向突变率为56.57%(56/99),6株高产类胡萝卜素的突变株中,第3代3株,第2代2株,第1代1株,类胡萝卜素含量最高的突变株是胶红酵母J6-82,比出发菌株提高了99.56%;黏红酵母J2的正向突变率为53.01%(44/83),6株高产类胡萝卜素的突变株中,第3代4株,第2代2株,类胡萝卜素含量最高的突变株是黏红酵母J2-75,比出发菌株提高了93.64%。本试验采用的NTG和UV 3次复合诱变的方法有利于海洋红酵母的变异及其类胡萝卜素含量的提升,且突变菌株有良好的遗传稳定性。
In order to screen out mutant strains with high-yield carotenoid,2 strains of marine Rhodotorula(Rhodotorula mucilaginosa J6 and Rhodotorila glutinis J2)separated and identificated from the coastal area near the Leizhou Peninsula were selected as the starting strains for the combined mutagenesis of nitroso-guanidin(NTG)and ultraviolet(UV)for three times continuously.Initially,the bacterial suspension of the starting strains was treated with NTG for 60 min,and then irradiated with UV for 120 s.After a short time of liquid culture,the suspension of each compound mutation was coated on the agar plate of red yeast,and a certain number of the first,second and third mutant strains were screened out.The mutant strains were inoculated in the red yeast fermentation medium for culturing.After cultured,centrifuged and dried,stem cells were obtained;Mutant strains with high yield carotenoids were separated out in the base of the carotenoid content in stem cells which was determined by adopting spectrophotometry.Eventually,genetic stability test was designed and executed to evaluate the prior combined mutagenesis.The results showed that the forward mutation rate of Rhodotorula mucilaginosa J6 was 56.57%(56/99),there were 3 strains in the third generation,2 strains in the second generation and only one in the first generation among the 6 high-yield carotenoid mutants.The mutant with highest carotenoid content was Rhodotorula mucilaginosa J6-82 by 99.56% higher than that of the staring strain.Furthermore,in regarding of the Rhodotorula glutinis J2,the forward mutation rate was53.01%(44/83).In the whole 6 high-yield carotenoid mutants,4 strains were derived from the third generation and the others were obtained in the second generation.The mutant with the highest carotenoid content was Rhodotorula glutinis J2-75 by 93.64% higher than that of the starting strain.It was worthwhile to conclude that the methods of NTG and UV combined mutagenesis for three times implemented in this study was beneficial to the variation of marine Rhodotorula and significantly increases carotenoid content.Dramatically,the resulted mutant strains had a good genetic stability as mentioned in this paper.
引文
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