SUN Tingting,MA Yisha,LIU Ruipeng,et al.Molecular Cloning and Expression Analysis of Mn-SOD Gene From Auricularia heimuer[J].Northern Horticulture,2022,(04):110-117.[doi:10.11937/bfyy.20213549]
黑木耳Mn-SOD基因的分子克隆与表达分析
- Title:
- Molecular Cloning and Expression Analysis of Mn-SOD Gene From Auricularia heimuer
- 关键词:
- 黑木耳; 锰超氧化物歧化酶基因; 克隆; 原核表达; 荧光定量PCR
- Keywords:
- Auricularia heimuer; Mn-SOD gene; cloning; prokaryotic expression; quantitative real-time PCR
- 文献标志码:
- A
- 摘要:
- 以黑木耳DL202为试材，采用RT-PCR技术克隆了Mn-SOD基因全长，并对其进行生物信息学分析和表达分析，以期为进一步研究Mn-SOD基因在黑木耳抗逆过程中的功能奠定基础。结果表明：Mn-SOD基因cDNA全长为609 bp，编码202个氨基酸，命名为Ah-MnSOD；蛋白具有锰超氧化物歧化酶（Mn-SOD）家族的保守结构域，不具有跨膜结构和信号肽；亚细胞定位主要位于过氧化物酶体中；系统进化分析表明Ah-MnSOD与Auricularia subglabra亲缘关系最近。此外，构建了Ah-MnSOD基因的原核表达载体并成功诱导出目的蛋白。荧光定量PCR结果显示Ah-MnSOD基因在不同发育阶段均有表达，且在原基中表达最高。
- Abstract:
- Taking Auricularia heimuer DL202 as the test material,Mn-SOD gene was cloned by RT-PCR,and its bioinformatics analysis and expression analysis were studied,in order to lay the foundation for further study of its function in the stress resistance of A.heimuer.The results showed that Mn-SOD cDNA contained 609 bp and encoded 202 amino acids,named Ah-MnSOD.The amino acid sequence had Mn-SOD conserved domains and had no transmembrane structure and signal peptide.Subcellular localization showed that Ah-MnSOD was modified and synthesized in peroxisome.Phylogenetic analysis indicated that Ah-MnSOD had the closest relationship with Auricularia subglabra.In addition,the prokaryotic expression vector pET-32a-Ah-MnSOD was constructed and the target protein was successfully expressed by induction with IPTG.The results of qRT-PCR showed that Ah-MnSOD gene was all expressed during different stages of A.heimuer,and reached the highest level at the primordium stage.
参考文献/References:
[1]YUAN Y,WU F,SI J,et al.Whole genome sequence of Auricularia heimuer(Basidiomycota,Fungi),the third most important cultivated mushroom worldwide[J].Genomics,2019,111(1):50-58.[2]孙健,孙婷婷,王旭彤,等.黑木耳内切葡聚糖酶基因克隆与原核表达[J].吉林农业大学学报,2019,41(3):308-331.[3]李晶晶,姚方杰,鲁丽鑫.黑木耳自交菌株菌丝体的遗传多样性分析[J/OL].吉林农业大学学报,https://doi.org/10.13327/j.jjlau.2021.1220.(2021-08-02)[2021-08-16].[4]马银鹏,陈鹤,孔祥辉,等.黑龙江省黑木耳主栽品种SSR遗传多样性初探[J].食用菌,2016,38(5):17-18.[5]MURGIA I,TARANTINO D,VANNINI C,et al.Arabidopsis thaliana plants overexpressing thylakoidal ascorbate peroxidase show increased resistance to paraquat-induced photooxidative stress and to nitric oxide-induced cell death[J].The Plant Journal,2004,38(6):940-953.[6]BUBLIY O A,LOESCHKE V.Correlated responses to selection for stress resistance and longevity in a laboratory population of Drosophila melanogaster[J].Journal of Evolutionary Biology,2005,18(4):789-803.[7]GALE E M,NARENDRAPURAPU B S,SIMMONETT A C,et al.Exploring the effects of H-bonding in synthetic analogues of nickel superoxide dismutase (Ni-SOD):Experimental and theoretical implications for protection of the Ni-SOD bond[J].Inorganic Chemistry,2010,49(15):7080-7096.[8]GANGWAR R,KUMARI P,CHATRATH A,et al.Characterisation of recombinant thermostable manganese-superoxide dismutase (NeMnSOD) from Nerium oleander[J].Molecular Biology Reports,2020,47(5):3251-3270.[9]陈莉.麝香百合和仙客来转Mn-SOD基因植株的获得及其耐热性鉴定[D].杨凌:西北农林科技大学,2007.[10]KUMAR R R,DUBEY K,GOSWAMI S,et al.Heterologous expression and characterization of novel manganese superoxide dismutase (Mn-SOD),a potential biochemical marker for heat stress-tolerance in wheat (Triticum aestivum)[J].International Journal of Biological Macromolecules,2020,161:1029-1039.[11]高建华,郭艳翔,刘佳宾,等.杏鲍菇菌丝体中SOD提取方法研究[J].食用菌,2007(2):50-51.[12]侯玉艳,吴素蕊,张丽,等.黑脉羊肚菌SOD的纯化及特性研究[J].食品工业科技,2015,36(15):147-151.[13]李丽,袁建琴.杏鲍菇菌丝体胞内和胞外SOD提取条件与活力变化研究[J].中国食品添加剂,2020(10):6-10.[14]YIN C M,ZHAO W X,ZHU J H,et al.Cloning and characterization of a differentially expressed mitochondrial manganese superoxide dismutase gene from Pleurotus ostreatus[J].Annals of Microbiology,2015,65(3):1597-1606.[15]杨加亮,田云恒,马爱民.虎奶菇MnSOD提取、鉴定及基因的克隆[J].食品工业科技,2020,41(15):150-157.[16]杨焕玲,游华芳,赵妍,等.草菇锰超氧化物歧化酶基因异源表达条件优化及其耐胁迫功能分析[J].食用菌学报,2020,27(4):51-58.[17]SUN J,WANG S X,WANG X T,et al.Cloning and expression analyses of a cellobiohydrolase gene from Auricularia heimuer[J].Biotechnology & Biotechnological Equipment,2019,33(1):1327-1334.[18]YUAN J S,REED A,CHEN F,et al.Statistical analysis of real-time PCR data[J].BMC Bioinformatics,2006(7):85-96.[19]KROGH A,LARSSON B,HEIJNE G,et al.Predicting transmembrane protein topology with a hidden Markov model:Application to complete genomes[J].Journal of Molecular Biology,2001,305(3):567-580.[20]ALMAGRO A J J,TSIRIGOS K D,SONDERBY C K,et al.SignalP 5.0 improves signal peptide predictions using deep neural networks[J].Nat Biotechnol,2019,37(4):420-423.[21]CHI S,QIANG C,XIANG L W,et al.Heat stress induces apoptotic-like cell death in two Pleurotus species[J].Current Microbiology,2014,69(5):611-616.[22]MIRIYALA S,SPASOJEVIC I,TOVMASYAN A,et al.Manganese superoxide dismutase,MnSOD and its mimics[J].BBA-Molecular Basis of Disease,2012,1822(5):794-814.[23]武欢,卢珍红,郝向阳,等.非洲菊GjMnSOD基因的克隆及表达分析[J].生物技术通报,2021,37(11):1-10.
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备注/Memo
第一作者简介：孙婷婷（1988-），女，博士，讲师，现主要从事食药用菌分子育种等研究工作。E-mail:stt_fighting@126.com.责任作者：邹莉（1966-），女，博士，教授，现主要从事资源微生物等研究工作。E-mail:shyj@nefu.edu.cn.基金项目：中央高校基本科研业务费资助项目（2572020DF06）；哈尔滨学院青年博士科研启动基金资助项目（HUDF2018105）。收稿日期：2021-08-28