生物技术通报 ›› 2023, Vol. 39 ›› Issue (11): 340-349.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0624
刘媛媛1(), 魏传正1, 谢永波1, 仝宗军2, 韩星2, 甘炳成2, 谢宝贵1(), 严俊杰2()
收稿日期:
2023-06-30
出版日期:
2023-11-26
发布日期:
2023-12-20
通讯作者:
谢宝贵,男,博士,教授,研究方向:食用菌生物学;E-mail: mrcfafu@163.com;作者简介:
刘媛媛,女,博士,研究方向:食用菌生物学;E-mail: lyylyy0815@163.com
基金资助:
LIU Yuan-yuan1(), WEI Chuan-zheng1, XIE Yong-bo1, TONG Zong-jun2, HAN Xing2, GAN Bing-cheng2, XIE Bao-gui1(), YAN Jun-jie2()
Received:
2023-06-30
Published:
2023-11-26
Online:
2023-12-20
摘要:
II类过氧化物酶(Class II peroxidase,CIIp)是活性氧代谢的重要抗氧化物酶,参与机体氧化应答过程。基于金针菇全基因组数据鉴定到两个II类过氧化物酶基因,并分别命名为FfCIIp1、FfCIIp2,采用在线网站软件对基因进行生物信息学分析,利用实时荧光定量PCR技术分析基因在金针菇不同子实体组织和胁迫应答中的表达模式。结果显示,FfCIIp1全长1 638 bp,包含一个1 131 bp的完整开放阅读框,编码376个氨基酸。FfCIIp2全长1 410 bp,包含一个1 032 bp的完整开放阅读框,编码343个氨基酸。保守结构域及进化树分析表明,FfCIIp1和FfCIIp2分别归属于锰过氧化物酶和通用过氧化物酶家族。RT-qPCR结果表明,两个FfCIIp基因均在伸长期菌柄中高表达,且在快速伸长区段显著上调;损伤和氧化胁迫处理均能诱导FfCIIp1和FfCIIp2上调表达,FfCIIp2的上调幅度明显高于FfCIIp1。以上结果表明,金针菇两个CIIp家族基因参与菌柄伸长与胁迫应答过程,且FfCIIp2对损伤和氧化胁迫更为敏感。
刘媛媛, 魏传正, 谢永波, 仝宗军, 韩星, 甘炳成, 谢宝贵, 严俊杰. 金针菇II类过氧化物酶基因在子实体发育与胁迫应答过程的表达特征[J]. 生物技术通报, 2023, 39(11): 340-349.
LIU Yuan-yuan, WEI Chuan-zheng, XIE Yong-bo, TONG Zong-jun, HAN Xing, GAN Bing-cheng, XIE Bao-gui, YAN Jun-jie. Characteristics of Class II Peroxidase Gene Expression During Fruiting Body Development and Stress Response in Flammulina filiformis[J]. Biotechnology Bulletin, 2023, 39(11): 340-349.
引物名称Primer name | 引物序列Primer sequence(5'-3') |
---|---|
qFfGAPDH-F qFfGAPDH-R | CCTCTGCTCACTTGAAGGGT |
GCGTTGGAGATGACTTTGAA | |
qFfCIIp1-F qFfCIIp1-R | TGACTTTCGCATTCCCCGAA |
ACAGAGAGTCAAGGCAACGG | |
qFfCIIp2-F | AAGTCGAAAGGCGGGACAAT |
qFfCIIp2-R | CCGTCAGGATTGATGGTCGT |
表1 引物序列
Table 1 Sequences of primers
引物名称Primer name | 引物序列Primer sequence(5'-3') |
---|---|
qFfGAPDH-F qFfGAPDH-R | CCTCTGCTCACTTGAAGGGT |
GCGTTGGAGATGACTTTGAA | |
qFfCIIp1-F qFfCIIp1-R | TGACTTTCGCATTCCCCGAA |
ACAGAGAGTCAAGGCAACGG | |
qFfCIIp2-F | AAGTCGAAAGGCGGGACAAT |
qFfCIIp2-R | CCGTCAGGATTGATGGTCGT |
图3 金针菇FfCIIp与其他担子菌CIIp的系统进化树 标尺:进化距离;分支处数字:自检置信度(Bootstrap=1 000);分支名称:数据库的ID号、物种拉丁名和序列名称组成;正方形:金针菇FfCIIp序列
Fig. 3 Phylogenetic tree of F. filiformis FfCIIp and CIIp from other basidiomycetes The bar: Evolutionary distance. The values above branches: The degree of bootstrap support from a 1000 replicate analysis. The taxon names of branches: composed of ID in database, Latin name of species and sequence name. Square: F. filiformis FfCIIp sequence
图4 金针菇与其他真菌CIIp蛋白的多序列比对与保守结构分析 碱基的背景色:颜色越深位点保守性越高;下划线:过氧化物酶的保守结构域;FfCIIp1、FfCIIp2:金针菇II类过氧化物酶序列;黑色实心倒三角:MnP和VP家族中酸性氨基酸残基对Mn2+氧化起关键作用的位点;空心倒三角:LiP和VP家族中色氨基酸残基对酚类化合物氧化起关键作用的位点;GP家族:无以上4个催化位点
Fig. 4 Multiple sequence alignment and conserved domain analysis of CIIp amino acid sequences from F. filiformis and other fungi Base background color: The darker the color, the more conservative the site. The transverse line: Peroxidase domain. FfCIIp1, FfCIIp2: Class II peroxidase sequences of F. filiformis. Black solid inverted triangle: Three acidic amino acid residues in the MnP and VP families are crucial for Mn2+ oxidation. Hollow inverted triangle: Tryptophan residue in the LiP and VP families is responsible for oxidation of phenolic compounds. GP: No above 4 catalytic sites
图5 金针菇FfCIIp基因在不同子实体组织中的差异表达 A:FfCIIp1在子实体样品中的差异表达(PR:原基;DI:分化期;EL-stipe:伸长期菌柄;EL-pileus:伸长期菌盖;MA-stipe:成熟期菌柄、MA-pileus:成熟期菌盖);B:FfCIIp2在子实体样品中的差异表达;C:FfCIIp1在菌柄不同区段中的差异表达(elo:为快速伸长区;sta:为稳定区,此区段不再伸长);D:FfCIIp2在菌柄不同区段中的差异表达。不同字母代表差异显著,P<0.05,n=3
Fig. 5 Expression patterns of FfCIIp during fruiting body tissues of F. filiformis A: Differential expressions of FfCIIp1 in fruiting body samples(PR: Primordium stage. DI: Differentiation stage. EL-stipe: The stipe of elongation stage. EL-pileus: The pileus of elongation stage. MA-stipe: The stipe of maturation stage. MA-pileus: The pileus of maturation stage). B: Differential expression of FfCIIp2 in fruiting body samples. C: Differential expression of FfCIIp1 in different segment of stipe(elo: Fastest elongating region. sta: Stable region, this region no longer elongated). D: Differential expression of FfCIIp2 in different segment of stipe. The different letters over the columns within the same graph denote significant differences. P<0.05, n=3
图6 金针菇菌丝损伤胁迫后FfCIIp基因的差异表达 A:金针菇Fv01菌丝损伤胁迫处理;B:FfCIIp1在应答损伤胁迫的差异表达;C:FfCIIp2在应答损伤胁迫的差异表达(*号代表与0 h样品的差异显著,*:P<0.05,**:P<0.01,***:P<0.001,n=3)
Fig. 6 Expression pattern of FfCIIp gene after mechanical injury to F. filiformis hyphae A: Fv01 mycelial injury stress induction. B: The expression pattern of FfCIIp1 in response to mechanical injury of F. filiformis. C: The expression pattern of F. filiformis FfCIIp2 in response to mechanical injury of F. filiformis(The ‘*’ over the columns denotes significant differences with 0 h. *: P<0.05, **: P<0.01, ***: P<0.001, n=3)
图7 金针菇菌丝H2O2胁迫后FfCIIp基因的差异表达 A:FfCIIp1在应答H2O2胁迫的差异表达;B:FfCIIp2在应答H2O2胁迫的差异表达(*号代表与0 mmol/L样品的差异显著,*:P<0.05,**:P<0.01,***:P<0.001,n=3)
Fig. 7 Expression pattern of FfCIIp gene after H2O2 stress of F. filiformis hyphae A: The expression pattern of FfCIIp1 in response to H2O2 stress. B: The expression pattern of FfCIIp2 in response to H2O2 stress.(The ‘*’ over the columns denotes significant differences with 0 mmol/L. *: P<0.05,**: P<0.01,***: P<0.001,n=3)
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