过表达Spt7对黑曲霉生长及抗逆性影响

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

摘要/Abstract

摘要：

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）×10⁷/cm²,较对照组（2.3×10⁷/cm²）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 H₂O₂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。

关键词: 黑曲霉, Spt7, 菌落形态, 抗氧化胁迫性, 高温高渗耐受性

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

薛鲜丽, 王静然, 毕杭杭, 王德培. 过表达Spt7对黑曲霉生长及抗逆性影响[J]. 生物技术通报, 2022, 38(5): 112-122.

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.

图/表 8

表1 试验中用到引物

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

图1 Spt7蛋白结构和氨基酸多序列比对及系统进化树分析 A：酵母及黑曲霉Spt7蛋白结构;B：酵母及黑曲霉中Spt7氨基酸多蛋白序列比对;C：丝状真菌Spt7的系统进化树

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

图2 30℃下对照组及转化子的生长形态观察及孢子计数 A：对照组及OE spt7菌落形态;B：孢子计数;C：40倍镜下对照组及OE spt7转化子菌丝形态

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

图3 对照组及OE spt7转化子在含有不同浓度H2O2 CM培养基的生长情况

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

图4 对照组及OE spt7转化子在含有15%NaCl的CM培养基生长情况

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

图5 对照组及OE spt7转化子在39℃高温下生长情况

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

图6 对照组及OE spt7转化子抗氧化物酶的转录水平

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

图7 对照组及OE spt7转化子在42℃热激条件下胞内热休克蛋白的转录水平 A：热激3 h后热休克蛋白转录水平;B：热激5 h后热休克蛋白转录水平

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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