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

doi:10.13560/j.cnki.biotech.bull.1985.2021-0946

Abstract

Abstract:

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）×10⁷/cm² compared with the control strain（2.3×10⁷/ cm²）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 H₂O₂and 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.

Key words: Aspergillus niger, Spt7, colony morphology, anti-oxidative stress, high-temperature and hyperosmotic tolerance

XUE Xian-li, WANG Jing-ran, BI Hang-hang, WANG De-pei. Effect of Spt7 Overexpression of on the Growth and Stress Resistance of Aspergillus niger[J]. Biotechnology Bulletin, 2022, 38(5): 112-122.

Figures/Tables 8

Table 1 Primers used in the experiment

名称Name	引物序列Sequence of primers（5'-3'）	引物作用Functions of primers
Pgla-F	TCACTACAGATACAAGGATCCTGCCATTGGCGGAGGGGT	pOEspt7质粒构建引物（pgla-spt7通过重组酶连接到p66质粒）
Pgla-R	AGCGACATTGCTGAGGTGTAATGATGCTGG	pOEspt7质粒构建引物（pgla-spt7通过重组酶连接到p66质粒）
spt7-F	TACACCTCAGCAATGTCGCTCGGACACCACC
spt7-R（含终止子）	TGTGATCCGCCTGGAGGATCCCAAGGCAAGTACACTGAACAAGGA
YZ-Ku70LL-F	CCGTCCGCCATAGTAGAATGT	pOE spt7质粒验证引物
YZ-spt7-5R	GTTCTTACGAACCTCGCTCAT
YZ-spt7-3R	AAACAACAACGAGCCTTTGGTC
YZ-ku70RR-R	TTGTTGGATTCGCTAGTGCGCT
RT-actin-F	ACCACCGACTCCCTACTA	pOE spt7 qRT-PCR引物
RT-actin-R	AGTCAAGAGAGAGATGGGAT
RT-hsp90-F	TCTTCCCTCTCTCTCTACCTC
RT-hsp90-R	AAAACGGTTTGAAAGGTGTG
RT-hsp70-F	GTGGGTGTCTTCCGTGATGACCGCA
RT-hsp70-R	GGACCTCAGCGTCGGCAAA
RT-hsp40-F	TGTTCAAGAAACCCAACATCCT
RT-hsp40-R	TGTGGGAGACGACGGCCATG
RT-GPX-F	CGCCTTCTCAATCTCCTTCA
RT-GPX-R	AGCGCATCAAGTGGAACTTT
RT-SOD-F	TTATCGCTCACAATGGCTGCTT
RT-SOD-R	GACAGGTCAGGGAGAGTAGC
RT-catR-F	CTTGTCACCGAGTGCCCGTTT
RT-catR-R	GTAATCCGGACCCTCCTGTTGGG
RT-cpeB-F	AACGGTTCGCTCCTCTTAAC
RT-cpeB-R	AGGAAATCTTTGCTCCGTACTT

Fig.1 Structural and amino acid multiple sequence alignment of Spt7 protein and phylogenetic tree analysis A: Structure of Spt7 protein in the yeast and Aspergillus niger. B: Amino acid multiple sequence alignment of Spt7 protein in the yeast and Aspergillus niger. C: Phylogenetic tree of the filamentous fungus Spt7

Fig. 2 Growth morphology observation and spore count of control and transformants at 30℃ A: Control group and OE spt7 colony morphology. B: Count of spores. C: Mycelial morphology of control and OE spt7 transformants under 40× microscope

Fig. 3 Growths of control group and OE spt7 transform-ants in CM medium containing different concen-trations of H2O2

Fig. 4 Growths of control group and OE spt7 transforma-nts in CM medium containing 15% NaCl

Fig. 5 Growths of control group and OE spt7 transforma-nts at 39℃ of high temperature

Fig. 6 Transcript levels of antioxidant enzymes in the control and OE spt7 transformants

Fig. 7 Transcript levels of intracellular heat shock proteins in the control and OE spt7 transformants under heat stress conditions at 42℃ A：Transcript levels of heat shock protein after 3 h of heat stress. B：Transcript levels of heat shock protein after 5 h of heat stress

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