过表达Spt7对黑曲霉生长及抗逆性影响

Effect of Spt7 Overexpression of on the Growth and Stress Resistance of Aspergillus niger

SAGA复合体是一种多功能蛋白复合物,负责细胞内10%以上的基因转录。Spt7作为SAGA复合体的核心蛋白,维持SAGA复合体的稳定。除酵母之外的真菌中并未有Spt7相关的研究报道。本研究对不同黑曲霉、丝衣霉等丝状真菌来源的Spt7的氨基酸序列进行多序列比对及蛋白结构的分析,发现Spt7在不同物种一致性较低,但均存在保守型的Bromo结构域。以黑曲霉1062为出发菌株,通过农杆菌转化法将过表达spt7的质粒转入1062菌株中,获得黑曲霉OE spt7转化子。通过对OE spt7转化子及对照组在CM培养基生长形态进行观察分析发现,过表达Spt7有益于菌体的生长及分生孢子的生成,在72 h时OE spt7转化子和对照组的菌落直径分别达到2.9 cm和2.8 cm,且转化子的孢子数量达到(5.8-6.3)×10^(7)/cm^(2),较对照组(2.3×10^(7)/cm^(2))提高1.5-1.7倍,且转化子菌丝分支较对照组多。另外,与对照组相比,OE spt7转化子在15 mmol/L H_(2)O_(2)条件下孢子萌发更快,菌落直径是对照组4倍,且在15%NaCl高渗以及39℃高温的条件下较对照组生长更加鲜活。通过qRT-PCR分析参与菌体抗氧化胁迫的过氧化物酶及耐高温的热休克蛋白基因的转录水平,发现除了CatR转录水平下调3.56倍,其他的过氧化物酶SOD、CpeB、GPX分别上调了3.8、1.89、3.56倍,除Hsp40及Hsp70较对照组无显著差异,Hsp90的转录水平上调19倍。

The SAGA complex is a multifunctional protein complex responsible for more than 10%of intracellular gene transcription.Spt7 serves as the core protein and maintains the stability of the SAGA complex.While no Spt7-related studies have been reported in fungi except for yeast.In this study,the amino acid sequences of Spt7 from Aspergillus niger,Cladosporium mitis and other filamentous fungal sources were compared via multiple sequences alignment and the protein structures analysis,it was found that Spt7 had low consistency in different species,but all of them had the Bromo structural domains.Aspergillus niger 1062 was used as the original strain,the OE spt7 transformants of A.niger were obtained by transferring the plasmid overexpressing spt7 into strain 1062 by Agrobacterium transformation method.By observing and analyzing the growth morphology of OE spt7 transformants and control strain in CM medium,it was found that overexpression of Spt7 was beneficial to the growth and conidial production,and the colony diameters of OE spt7 transformant and control strain reached 2.9 and 2.8 cm,respectively,at 72 h.The spores number of transformants reached(5.8-6.3)×10^(7)/cm^(2) compared with the control strain(2.3×10^(7)/cm^(2))by 1.5-1.7 times,and the mycelial branches of transformant was more than those of the control strain.In addition,the spores of OE spt7 transformants germinated faster under 15 mmol/L H_(2)O_(2) and their colony diameters were about 4 times larger than those of control.Furthermore,OE spt7 transformants grew more vividly under 15% NaCl hyperosmotic as well as 39℃ high temperature than the control ones.The transcript levels of key genes involved in antioxidant stress and high temperature in the bacterium were analyzed by qRT-PCR,and the results showed that the antioxidant enzymes SOD,CpeB,and GPX were up-regulated by 3.8,1.89,and 3.56-fold,respectively among all peroxidase transcript levels analyzed,except for CatR transcript level was down-regulated by 3.56-fold.The transcript level of Hsp90 was up-regulated 19-fold while Hsp40 and Hsp70 were not significantly different compared to the control.