Effects of orf5 Gene Over-expressed in Monascus on Lovastatin Content

doi:10.11924/j.issn.1000-6850.casb2022-0585

Abstract

Abstract:

In order to analyze the effect of orf5 gene on lovastatin content in Monascus and to enrich the regulatory mechanisms of lovastatin metabolism, the over-expressing vector with the gus-orf5 fusion gene was transformed into M. purpureus by ATMT and REMI methods. The co-culture conditions of ATMT and the endonuclease of REMI were optimized. The transformed strains were identified by GUS staining and PCR, and the lovastatin content in positive strains was detected. The results showed that the culture medium supplemented with 20 μg/mL hygromycin could be used as the screening concentration for transgenic Monascus strain. The co-culture medium of Co-IM appended with 1/10 LB was optimal for ATMT, and XbaI-mediated transformation was better in REMI method. Hygromycin resistant colonies were then obtained by both ATMT and REMI. PCR showed that orf5 and hyg genes had been transferred into the Monascus, and GUS staining was blue. The content of lovastatin in the overexpressed strain was significantly higher than that in the control, up to 36.89%. Over-expression of orf5 in Monascus can increase the accumulation of lovastatin, which provides reference for further study of the function of orf5 gene in Monascus.

Key words: Monascus purpureus, orf5 gene, Agrobacterium tumefaciens mediated genetic transformation (ATMT), restrictive enzyme-mediated integration (REMI), lovastatin

WANG Jiao, GUI Yanling, HAN Jie, ZHAO Jiehong. Effects of orf5 Gene Over-expressed in Monascus on Lovastatin Content[J]. Chinese Agricultural Science Bulletin, 2023, 39(12): 93-98.

Figures/Tables 7

References 25

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靶标基因	引物序列(5'-3')	靶标长度/bp
orf5	CTACAGAATGGGTCCGTCGT	316
orf5	CCCAGTCCACTCCCAACTTA	316
hyg	GTGCTTGACATTGGGGAGTT	383
hyg	ACATTGTTGGAGCCGAAATC	383

靶标基因	引物序列(5'-3')	靶标长度/bp
orf5	CTACAGAATGGGTCCGTCGT	316
orf5	CCCAGTCCACTCCCAACTTA	316
hyg	GTGCTTGACATTGGGGAGTT	383
hyg	ACATTGTTGGAGCCGAAATC	383

培养基	生长状态
培养基	农杆菌		红曲霉		农杆菌和红曲霉共生长
1/4 LB	＋		－		－
1/10 LB	＋		＋		－
Co-IM＋1/10 LB	＋		＋		＋
Co-IM＋0.01% FeSO₄	－		＋		－
Co-IM＋1 mol/L MES	－		＋		－
Co-IM＋0.1 mol/L AS		－		＋		－
Co-IM＋0.01% FeSO₄+1 mol/L MES+0.1 mol/L AS		－		＋		－
Co-IM+9 g/L Glucose		－		－		－
Co-IM		－		＋		－

培养基	生长状态
培养基	农杆菌		红曲霉		农杆菌和红曲霉共生长
1/4 LB	＋		－		－
1/10 LB	＋		＋		－
Co-IM＋1/10 LB	＋		＋		＋
Co-IM＋0.01% FeSO₄	－		＋		－
Co-IM＋1 mol/L MES	－		＋		－
Co-IM＋0.1 mol/L AS		－		＋		－
Co-IM＋0.01% FeSO₄+1 mol/L MES+0.1 mol/L AS		－		＋		－
Co-IM+9 g/L Glucose		－		－		－
Co-IM		－		＋		－