利用CRISPR/Cas 9编辑红曲霉pyrG基因对其次生代谢的影响

doi:10.13560/j.cnki.biotech.bull.1985.2021-1482

摘要/Abstract

摘要：

为研究红曲霉pyrG基因对其次生代谢的影响,通过农杆菌介导遗传转化获得了转Cas 9基因的红曲霉底盘菌株,然后设计并体外转录合成pyrG基因的定向编辑sgRNA,对底盘菌株的原生质体遗传转化,经5-FOA抗性筛选、PCR扩增、T7EI酶切和测序验证,获得pyrG基因定向编辑的红曲霉4个菌株,其中2株单碱基缺失,2株2碱基缺失。比较分析表明,野生型红曲霉在添加尿嘧啶核苷的培养基中会极显著增加洛伐他汀含量,并极显著降低桔霉素含量,而经CRISPR/Cas 9失活pyrG的变异菌株与野生型相比,洛伐他汀和桔霉素含量明显增加。表明尿嘧啶核苷合成关键酶基因pyrG和外源尿嘧啶核苷对红曲霉的次生代谢有协同调控作用,为红曲霉功能基因研究和次生代谢调控提供参考。

关键词: CRISPR/Cas 9, 紫色红曲霉, pyrG变异株, 原生质体, 底盘菌株

Abstract:

In order to study the effects of pyrG gene of monascus on its secondary metabolism,the monascus chassis strain with Cas9 was obtained by agrobacterium-mediated genetic transformation. Then the directed-editing sgRNA of pyrG gene were designed and synthesized in vitro. After genetic transformation of protoplast of chassis strain,4 mutant strains were screened out,including 2 strains of single-base deleted mutation and 2 strains of double-base deleted mutation,analyzed by resistant on 5-FOA,PCR amplification,T7EI digestion and sequencing. Comparative analysis showed that the content of lovastatin of the wild-type strain significantly increased,and the content of citrinin significantly decreased when cultured in the medium supplemented with uracil. The contents of lovastatin and citrinin of pyrG mutants increased. The results showed that uracil nucleoside and pyrG gene had regulatory effects on secondary metabolism of monascus. It provides reference for functional gene research and secondary metabolism regulation of monascus.

Key words: CRISPR/Cas 9, Monascus purpureus, PyrG mutant strain, protoplast, chassis strain

唐光甫, 桂艳玲, 满海乔, 赵杰宏. 利用CRISPR/Cas 9编辑红曲霉pyrG基因对其次生代谢的影响[J]. 生物技术通报, 2022, 38(8): 198-205.

TANG Guang-fu, GUI Yan-ling, MAN Hai-qiao, ZHAO Jie-hong. Editing pyrG Gene of Monascus by CRISPR/Cas 9 and Its Effects on Secondary Metabolism[J]. Biotechnology Bulletin, 2022, 38(8): 198-205.

图/表 8

表1 PCR检测引物和sgRNA序列

Table 1 Primers and sgRNA sequences for PCR detection

引物名称 Primer name	引物或sgRNA序列 Primer or sgRNA sequence（5'-3'）	退火温度 Annea-ling temperature/℃	靶标序列长度 Target sequence length/bp
Cas9-F	GTTTAAGGTCCTGGGCAACA	55	312
Cas9-R	ATCGTGGGGTACTTCTCGTG	55	312
PyrGgr-F	GTTGAAACGAACCCCAGCAC	57	380
pyrGgr-R	CCACATCGACATCCTCTCCG	57	380
sgRNA	TTAATACGACTCACTATAGGGggcttgaagttcctgcgttgGTTTTAGAGCTAGAAATA	/	/

图1 pyrG基因sgRNA靶标序列箭头表示pyrGgr引物对的位置

Fig. 1 Target sequence of sgRNA for gene pyrG The arrow indicates the position of the pyrGgr primer pair

图2 桔霉素标准曲线

Fig. 2 Standard curve of citrinin

图3 农杆菌介导Cas 9转化红曲霉 A：农杆菌PCR检测;B：农杆菌和红曲霉共培养;C：红曲霉Cas 9底盘菌株PCR检测;1-8：不同菌株;M：DL1000 DNA Marker

Fig. 3 Transformation of Cas 9 into monascus mediated by A. tumefaciens A：PCR detection of A. tumefaciens. B：Co-culture of A. tumefaciens and monascus. C：PCR detection of the Cas 9 of monascus chassis strain. 1-8：Different strains. M：DL1000 DNA marker

图4 体外sgRNA转化红曲霉原生质体 A：5-FOA对红曲霉的抑制作用;B：sgRNA电泳检测;C：制备的原生质体;D：抗性红曲霉筛选

Fig. 4 Transformation of monascus protoplasts by sgRNA in vitro A：Inhibition of 5-FOA on monascus. B：Electrophoresis of the sgRNA. C：Prepared protoplasts. D：Screening resistant monascus

图5 红曲霉突变株pyrG基因的变异分析 A：局部套峰序列;B：部分菌株pyrG基因的PCR检测;C：T7EI酶切PCR产物;D：部分菌株pyrG基因的变异位点。MU：突变株;WT：野生型;“+”：有WT DNA;“-”：无WT DNA;方框：PAM;灰底色：sgRNA靶标序列

Fig. 5 Variation analysis of pyrG gene of monascus mutant A：Part nested peak sequence. B：PCR detection of pyrG gene in some strains. C：PCR product cutted by T7EI. D：Mutation loci of pyrG gene in some strains. MU：Mutant. WT：Wild-type. “+”：With DNA. “-”：Without DNA. Black box：Refers to the PAM site. Grey background：Refers to the sgRNA target sequence

图6 部分突变株的菌落形态和显微特征显微图片放大倍数400×

Fig. 6 Colony morphology and microscopic characteristics of partial mutant strains Magnification of micrographs 400×

图7 培养基中添加尿嘧啶核苷对不同菌株化学成分含量的影响 A：洛伐他汀吸光度;B：红曲色素含量;C：桔霉素含量;“-”：表示未添加;“+”：表示添加;WT：野生型;MU：变异菌

Fig. 7 Effect of uracil riboside on the chemical composition content of different strains in medium A：Lovastatin absorbance. B：Monascus pigment content. C：Citrinin content. “-”：Not added. “+”：Added. WT：Wild type strain. MU：Mutant strain

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