生物技术通报 ›› 2022, Vol. 38 ›› Issue (5): 112-122.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0946
收稿日期:
2021-07-24
出版日期:
2022-05-26
发布日期:
2022-06-10
作者简介:
薛鲜丽,女,博士,讲师,研究方向:丝状真菌遗传改造;E-mail: 基金资助:
XUE Xian-li1(), WANG Jing-ran1, BI Hang-hang1, WANG De-pei1,2()
Received:
2021-07-24
Published:
2022-05-26
Online:
2022-06-10
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。
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.
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 | |
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 |
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 | |
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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