敲除Spt7对黑曲霉生长及菌落形态的影响

Effects of Spt7 knockout on growth and morphology of Aspergillus niger

摘要:

在酵母中Spt7作为一种多功能蛋白复合物Spt-Ada-Gcn5-乙酰转移酶(Spt-Ada-Gcn5- acetyltransferase, SAGA)复合体的核心蛋白，其不仅负责维持SAGA复合物的稳定，还负责细胞内10%以上的基因转录。除此之外，丝状真菌中关于Spt7功能的研究很少。【目的】探究Spt7在黑曲霉Aspergillus niger CGMCC 1062中的功能。【方法】以黑曲霉A. niger CGMCC 1062为出发菌株，通过农杆菌转化法将敲除spt7基因的质粒转入黑曲霉中；并分别将Δspt7菌株与对照组点种在CM培养基、不同碳源及含H₂O₂培养基上进行生长观察；通过实时定量聚合酶链反应(real-time quantitative polymerase chain reaction, qRT-PCR)分析糖酵解关键基因、产孢相关基因的相对转录水平。【结果】成功获得spt7基因敲除菌株Δspt7；通过实验发现Δspt7菌株较对照菌株生长缓慢、菌落变白且产孢延迟；spt7基因的敲除显著影响菌株对不同碳源的利用；但Δspt7菌株同对照组均能在20 mmol/L H₂O₂的平板上正常生长。Δspt7菌株中糖酵解关键酶fbp、pfk、trk、pks、fda和gsdA基因的转录水平较对照组分别下调了2.65、4.46、6.05、4.90、3.20和3.20倍；产孢相关基因wetA、abaA和brlA的转录水平相较于对照组分别下调了529.93、172.40倍和9.61倍。【结论】spt7基因的缺失影响菌株的正常生长、菌落形态及分生孢子产生。

关键词:黑曲霉;基因敲除;Δspt7菌株;菌落形态;抗氧化胁迫

Abstract:

As the core protein of a multifunctional protein complex Spt-Ada-Gcn5- acetyltransferase (SAGA) in yeast, Spt7 is not only responsible for maintaining the stability of the SAGA complex, but also responsible for the transcription of more than 10% of genes. However, there were few studies on the functions of Spt7 in filamentous fungi. [Objective] To investigate the effects of Spt7 on Aspergillus niger CGMCC 1062. [Methods] In this study, the plasmid with spt7 gene knockout was transferred into Aspergillus niger CGMCC 1062 by Agrobacterium tumefaciens transformation method. The colony morphology of Δspt7 strain and control group was observed, which grew on CM medium, different carbon sources, and H₂O₂-containing mediums. The relative transcription levels of glycolysis key genes and sporin-producing related genes were analyzed by real-time quantitative polymerase chain reaction (qRT-PCR). [Results] The Δspt7 strain was successfully obtained. It was found that the growth of Δspt7 strain was slow, the colony became white, and the sporulation was delayed. The knockout of the spt7 gene significantly affected the use of different carbon sources by the strain. However, Δspt7 strain and the control group grew normally on the plate with 20 mmol/L H₂O₂. The transcriptional levels of fbp, pfk, trk, pks, fda, and gsdA genes in the Δspt7 strain were 2.65 times, 4.46 times, 6.05 times, 4.90 times, 3.20 times, and 3.20 times lower than those in the control group, respectively. The transcriptional levels of wetA, abaA, and brlA were down-regulated by 529.93 times, 172.40 times, and 9.61 times, respectively, as compared with the control group. [Conclusion] The deletion of spt7 gene affects the normal growth, colony morphology, and conidial production of the strain.

Key words:Aspergillus niger;gene knockout;Δspt7 strain;colony morphology;anti-oxidative stress