红曲霉过表达orf5基因对洛伐他汀含量的影响

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

中国农学通报 ›› 2023, Vol. 39 ›› Issue (12): 93-98.doi: 10.11924/j.issn.1000-6850.casb2022-0585

红曲霉过表达orf5基因对洛伐他汀含量的影响

王娇(), 桂艳玲, 韩洁, 赵杰宏()

贵州中医药大学药学院，贵州省中药生药学重点实验室，贵阳 550025

收稿日期:2022-07-12 修回日期:2022-11-27 出版日期:2023-04-25 发布日期:2023-04-21
通讯作者: 赵杰宏，男，1978年出生，山西长治人，教授，博士，主要从事中药生物技术研究。通信地址：550025 贵州省贵安大学城贵州中医药大学，Tel：0851-88233089，E-mail：105798711@qq.com。
作者简介:
王娇，女，1998年出生，贵州遵义人，硕士研究生，研究方向：真菌生物技术研究。通信地址：550025 贵州省贵安大学城贵州中医药大学，Tel：0851-88233089，E-mail：2771037249@qq.com。
基金资助:
贵州省科技支撑项目“药食两用红曲中桔霉素调控网络挖掘及CRISPR基因编辑菌种研发”(黔科合支撑[2019]2776号); 国家自然科学基金“蝉花有性型的基源再鉴定及其形成机制研究”(81960692)

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

WANG Jiao(), GUI Yanling, HAN Jie, ZHAO Jiehong()

Key Lab of Pharmacognostics of Guizhou Province/College of Pharmacy, Guizhou University of Traditional Chinese Medicine, Guiyang 550025

Received:2022-07-12 Revised:2022-11-27 Online:2023-04-25 Published:2023-04-21

摘要/Abstract

摘要：

为研究红曲霉过表达orf5基因对洛伐他汀含量的影响，丰富洛伐他汀代谢调控机制。用ATMT和REMI法将gus-orf5融合基因转化红曲霉，并对ATMT法的共培养条件和REMI法的内切酶进行优化筛选，对获得的潮霉素抗性菌株进行GUS染色和PCR鉴定，并检测阳性菌株的洛伐他汀含量变化。结果筛选出20 μg/mL潮霉素的培养基作为转基因红曲霉的抗性筛选浓度，补充1/10 LB的Co-IM共培养基有利于ATMT法转化，在REMI法中XbaI介导的转化效果较好。用ATMT和REMI法均获得抗性菌落，经PCR检测表明orf5和hyg基因均已导入红曲霉，GUS染色呈蓝色。过表达菌株的洛伐他汀含量比对照明显提高，高达36.89%。红曲霉过表达orf5有利于增加洛伐他汀的积累，为深入研究红曲霉orf5基因的功能提供参考。

关键词: 紫色红曲霉, orf5基因, 农杆菌介导遗传转化(ATMT), 限制性内切酶介导基因整合(REMI), 洛伐他汀

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

王娇, 桂艳玲, 韩洁, 赵杰宏. 红曲霉过表达orf5基因对洛伐他汀含量的影响[J]. 中国农学通报, 2023, 39(12): 93-98.

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.

图/表 7

图1. pgus-orf5过表达载体结构图

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

表1. PCR检测引物

图2. 潮霉素浓度筛选和转化农杆菌检测 A：潮霉素浓度筛选；1~5：30 μg/mL、25 μg/mL、20μg/mL、15 μg/mL、10 μg/mL；B：hyg的PCR检测；C：农杆菌GUS染色；M：DL1000 DNA marker

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		－		＋		－

表2. 培养基对农杆菌和红曲霉共培养的影响

图3. REMI法转化红曲霉获得的抗性菌落 A：红曲霉原生质体(400×)；B：KpnI处理；C：HindIII处理；D：XbaI处理

图4. 红曲霉转化子的PCR检测与GUS染色 A：PCR检测hyg序列；1~4：不同转化子菌株；5~6：质粒对照；B：PCR检测orf5序列；7~10：不同转化子菌株；11~12：质粒对照；C：菌丝球GUS染色；上：阴性对照；下：转化子；M：DL1000 Marker

图5. 过表达菌株洛伐他汀和色素的含量变化 A：洛伐他汀含量比较；B：红曲色素含量比较；WT：野生型菌株

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红曲霉过表达orf5基因对洛伐他汀含量的影响

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

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