<i>StCAT1</i>与<i>StCAT3</i>抗马铃薯早疫病的分子调控机制

doi:10.7668/hbnxb.20195821

摘要/Abstract

摘要：

过氧化氢酶(CAT)在植物抵抗外界生物胁迫中发挥着重要作用。为了探究StCAT1与StCAT3基因在马铃薯抗早疫病菌侵染过程中的作用,通过RT-PCR方法克隆得到了StCAT1与StCAT3的cDNA,测序结果表明,这2个基因的全长均为1 479 bp,均编码493个氨基酸。通过构建系统发育树发现,StCAT1与番茄的SlCAT1有高度同源性,StCAT3与番茄SlCAT3还有彭氏番茄的SpCAT3有高度同源性。茄链格孢侵染马铃薯6 d后,StCAT1显著上调约3.9倍,StCAT3显著上调约8.7倍。进一步构建了StCAT1与StCAT3共沉默载体,利用VIGS技术对StCAT1与StCAT3进行瞬时共沉默,利用qRT-PCR筛选获得了StCAT1与StCAT3有效共沉默马铃薯株系VIGS-3。对马铃薯沉默株系接种早疫病菌,沉默株系的叶片病斑面积显著大于对照组,其与对照组相比增大42%,表明StCAT1与StCAT3共沉默后可降低马铃薯植株的早疫病抗性。同时,利用DAB染色法测定了共沉默株系叶片的H₂O₂水平,发现沉默株系的H₂O₂含量显著高于对照组,共沉默株系叶片的DAB染色强度是对照组的3.8倍。上述结果表明,StCAT1与StCAT3可通过调节H₂O₂水平介导马铃薯对早疫病的抗性,为揭示StCAT1与StCAT3在马铃薯抗早疫病中的分子调控机制奠定理论基础。

关键词: 马铃薯早疫病, StCAT1, StCAT3, VIGS, H₂O₂

Abstract:

Catalase(CAT)plays a critical role in plant resistance against biotic stresses.To investigate the functions of StCAT1 and StCAT3 in potato defense against Alternaria solani infection,this study cloned the cDNA sequences of StCAT1 and StCAT3 via RT-PCR.Sequencing results demonstrated that both genes were 1 479 bp in length,encoding 493 amino acids.Phylogenetic tree construction revealed that StCAT1 shared high homology with SlCAT1 from tomato,while StCAT3 exhibited high homology with both SlCAT3(tomato)and SpCAT3 from wild tomato.Following A.solani infection for 6 days,the expression of StCAT1 and StCAT3 was significantly upregulated by approximately 3.9-fold and 8.7-fold,respectively.A co-silencing vector targeting both StCAT1 and StCAT3 was constructed,and transient co-silencing was achieved using virus-induced gene silencing(VIGS).qRT-PCR screening identified an effective co-silenced potato line(VIGS-3)with suppressed StCAT1 and StCAT3 expression.Upon A.solani inoculation,the silenced line exhibited 42% larger lesion areas compared to the control,indicating that co-silencing StCAT1/StCAT3 compromised potato resistance to early blight.DAB(3,3'-diaminobenzidine)staining further demonstrated that H₂O₂ accumulation in co-silenced leaves was significantly elevated,with staining intensity being 3.8-fold higher than that in the control.These results demonstrate that StCAT1 and StCAT3 regulate H₂O₂ homeostasis to mediate potato resistance against A.solani,providing a theoretical foundation for elucidating the molecular mechanisms of CAT genes in potato-pathogen interactions.

Key words: Early Blight in Potatoes, StCAT1, StCAT3, VIGS, H₂O₂

中图分类号:

刘鑫, 李倩, 刘芳明, 丁明亚, 杨志辉, 朱杰华. StCAT1与StCAT3抗马铃薯早疫病的分子调控机制[J]. 华北农学报, 2025, 40(5): 171-178. doi: 10.7668/hbnxb.20195821.

LIU Xin, LI Qian, LIU Fangming, DING Mingya, YANG Zhihui, ZHU Jiehua. The Molecular Regulatory Mechanism of StCAT1 and StCAT3 in Resistance to Early Blight in Potatoes[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(5): 171-178. doi: 10.7668/hbnxb.20195821.

http://www.hbnxb.net/CN/Y2025/V40/I5/171

图/表 8

表1 本研究所用的引物

Tab.1 Primers used in this study

引物名称 Primer name	序列(5'—3') Sequence(5'—3')
StCAT1-F	ATGGATCCCTCTAAGTATCGC
StCAT1-R	TCACATTGTGGGCTTCACA
StCAT3-F	ATGGATCCTTACAAGTATCGTCC
StCAT3-R	TCACATTGTAGGCCTTATATTGAGAC
V-CAT1-F	CG GAATTCCTGGAAACTATCCTGAGTGG
V-CAT1-R	CACCGTATTGGACCAAACTATAGGTACCCC
V-CAT3-F	TATTGGACCAAACTATAATGGATCC
	TTACAAGTATCGTCCTTCAAG
V-CAT3-R	CTCCTGGGGTCCAGACACCTGTTATT GGTACCCC
qStCAT1-F	CCGTGATGGAATGAAGTT
qStCAT1-R	GCCTGTAATCTTGTGGAA
qStCAT3-F	CCTTCTTCTACGATGATG
qStCAT3-R	TACACAAGACTCTAATAG

图1 StCAT1与StCAT3基因PCR扩增 M.DL2000 DNA Marker;1.StCAT1的PCR产物;2.StCAT3的PCR产物。

Fig.1 PCR amplification of the StCAT1 and StCAT3 genes M.DL2000 DNA Marker;1.PCR product of StCAT1; 2.PCR product of StCAT3.

图2 StCAT1和StCAT3的系统发育树构建

Fig.2 Construction of the phylogenetic tree of StCAT1 and StCAT3

图3 StCAT1与StCAT3在早疫病侵染期间的表达模式分析不同小写字母表示差异显著(P<0.05, t检验)。图5—7同。

Fig.3 Analysis of the expression patterns of StCAT1 and StCAT3 during the early blight infection period Different lowercase letters indicate significant differences (P<0.05,Student's t-test).The same as Fig.5—7.

图4 StCAT1与StCAT3共沉默载体构建 A.StCAT1与StCAT3的沉默基因序列PCR扩增:M.DL2000 DNA Marker,1.StCAT1沉默片段,2.StCAT3沉默片段;B.StCAT1与StCAT3沉默融合片段PCR扩增:M.DL5000 DNA Marker;C.StCAT1与StCAT3基因的共沉默载体双酶切验证:M.DL5000 DNA Marker.

Fig.4 Construction of the co-silencing vector for StCAT1 and StCAT3 A.PCR amplification of the silenced gene sequences of StCAT1 and StCAT3:M.DL2000 DNA Marker,1.StCAT1 silenced fragment,2.StCAT3 silenced fragment;B.PCR amplification of the silenced fusion fragments of StCAT1 and StCAT3:M.DL5000 DNA Marker;C.Double enzyme digestion verification of the co-silencing vector of StCAT1 and StCAT3 genes:M.DL5000 DNA Marker.

图5 StCAT1和StCAT3基因共沉默马铃薯阳性株qRT-PCR鉴定

Fig.5 qRT-PCR identification of positive potato plants with co-silenced StCAT1 and StCAT3 genes

图6 StCAT1与StCAT3共沉默株的早疫病抗性鉴定 A.早疫病抗性鉴定表型图。

Fig.6 Resistance evaluation of co-silenced StCAT1 and StCAT3 plants to early blight disease A.Phenotype of early blight resistance evaluation.

图7 发病叶片的DAB染色与强度定量 A.沉默组与对照组植株叶片接种早疫病后的DAB染色。

Fig.7 DAB staining and intensity quantification of infected leaf tissue A.DAB staining of leaves from silenced and control plants after inoculation with early blight.

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StCAT1与StCAT3抗马铃薯早疫病的分子调控机制

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