马铃薯miR7997家族靶基因鉴定及其在晚疫病侵染下的表达分析

doi:10.7668/hbnxb.20195974

摘要/Abstract

摘要： 为探究马铃薯miR7997家族响应晚疫病原菌侵染的分子机制,以马铃薯Desiree为试验材料,对马铃薯miR7997家族序列特征、靶基因预测、表达模式和Stu-miR7997与靶基因在晚疫病胁迫下的表达特性进行分析。结果表明:马铃薯miR7997家族由3个成员Stu-miR7997a/b/c组成,分布于2条染色体上,成熟体序列高度保守,其中Stu-miR7997a/b成熟体序列完全相同;Stu-miR7997s前体序列均可形成典型的茎环二级结构,最小折叠自由能为-37.00~-49.50 kcal/mol,成熟体均位于前体5'端臂上,Stu-miR7997c在序列长度和功能元件分布上与Stu-miR7997a/b明显不同;启动子分析显示,Stu-miR7997s含光响应、激素响应、转录因子响应及胁迫防御相关元件。靶基因预测结果显示,Stu-miR7997家族共鉴定出19条靶基因,绝大多数靶基因受Stu-miR7997家族共同调控;组织特异性表达测定结果显示,Stu-miR7997s在马铃薯的各组织部位中均有不同程度的表达,Stu-miR7997a/b在茎中表达量最高,Stu-miR7997c在根部表达量最高。晚疫病致病疫霉侵染后,Stu-miR7997家族均显著下调表达,其靶基因转录因子MYB92、NAD(P)H-醌氧化还原酶及果胶裂解酶基因的表达显著上调,Stu-miR7997家族可能通过负调控靶基因表达,间接影响马铃薯的抗病反应,为进一步研究马铃薯miR7997基因家族在马铃薯抗晚疫病中的作用机制提供理论基础。

关键词: 马铃薯晚疫病, miR7997家族, 生物信息学, 靶基因, 表达模式

Abstract:

To investigate the molecular mechanisms of the potato miR7997 family in response to Phytophthora infestans infection,this study analyzed the sequence characteristics,target gene prediction,expression patterns,and stress-responsive expression dynamics of Stu-miR7997 and its targets using the potato cultivar Desiree.The results revealed that the potato miR7997 family comprised three members(Stu-miR7997a/b/c)distributed across two chromosomes,with Stu-miR7997a/b sharing identical mature sequences.All precursor sequences formed canonical stem-loop secondary structures,with minimum folding free energies ranging from -37.00 to -49.50 kcal/mol,and mature sequences were located on the 5' arm.Stu-miR7997c exhibited distinct sequence length and functional element distribution compared to Stu-miR7997a/b.Promoter analysis identified light-responsive,hormone-responsive,transcription factor-binding,and stress defense-related cis-regulatory elements;target prediction identified 19 genes predominantly co-regulated by the miR7997 family.Tissue-specific expression profiling showed that Stu-miR7997a/b were highly expressed in stems,while Stu-miR7997c accumulated predominantly in roots.Upon P.infestans infection,all miR7997 members were significantly downregulated,whereas their target genes-including the transcription factor MYB92 gene,NAD(P)H-quinone oxidoreductase gene,and pectin lyase gene-were markedly upregulated.These findings suggest that the Stu-miR7997 family may indirectly modulate potato disease resistance by negatively regulating target genes.This study provides a theoretical foundation for further exploration of the miR7997 family in potato late blight resistance.

Key words: Potato late blight, miR7997 family, Bioinformatics, Target genes, Expression patterns

中图分类号:

曹梦琦, 池明, 唐达, 杨恒照, 陈婧婷. 马铃薯miR7997家族靶基因鉴定及其在晚疫病侵染下的表达分析[J]. 华北农学报, 2025, 40(5): 179-187. doi: 10.7668/hbnxb.20195974.

CAO Mengqi, CHI Ming, TANG Da, YANG Hengzhao, CHEN Jingting. Identification of Target Genes of the Potato miR7997 Family and Their Expression Dynamics During Late Blight Infection[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(5): 179-187. doi: 10.7668/hbnxb.20195974.

http://www.hbnxb.net/CN/Y2025/V40/I5/179

图/表 12

表1 引物信息

Tab.1 Primer information

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')
Stu-7997c-RT	CTCAACTGGAGCTAGTTTCGTCGTAGGGCAGTTGAGATTTTTGA
TY-Stu-7997-R	CTGGAGCTAGTTTCGTCGTAGGG
Q-Stu-7997c-F	CCAGCTGGGATATTGCTCGGACTCT
Stu-7997a/b-RT	CTCAACTGGAGCTAGTTTCGTCGTAGGGCAGTTGAGTTTGAAGA
Q-Stu-7997a/b-F	CCAGCTGGGATGCTGCTCGGAC
U6-F	CGGGGACATCCGATAAAA
U6-R	TTGGACCATTTCTCGATTTG
RT-U6	CTCAACTGGAGCTAGTTTCGTCGTAGGGCAGTTGAGTTGGACCA

表2 靶基因荧光定量引物

Tab.2 Target genes RT-qPCR primers

基因名称 Gene name	引物序列(5'—3') Primer sequence(5'—3')
Stu-β-tublin	ATGTTCAGGCGCAAGGCTT
	TCTGCAACCGGGTCATTCAT
PGSC0003DMT400054374	TGCAGCTAGCTTGAGGAGAG
	TACTGTTGTGTGGGATCTTGGGA
PGSC0003DMT400003790	AAGCTGCTCAAGAAGAGAGGG
	ACCATAAGTTGGCGAGTGCG
PGSC0003DMT400060373	CCGATGTTGGTATGCAGGTGA
	GTCCAATCCCTCTCGTCCAC
PGSC0003DMT400014983	AAATGCCAAAGAGGTGACGAA
	GCTCCATGCTTGATGCCAA
PGSC0003DMT400039552	GACACGCACCCCTTGACATT
	CACCTGAAATGTGGGCAACT
PGSC0003DMT400026391	TGGCTGAGGCACTTGATGAT
	TTTTGAGCGACGGATGGACG

图1 Stu-miR7997家族成员序列比对 A.Stu-miR7997家族成员成熟体序列比对;B.Stu-miR7997家族成员前体序列比对。

Fig.1 Sequence alignment of Stu-miR7997 family members A.Mature sequence alignment of Stu-miR7997 family members;B.Stu-miR7997 family member precursor sequence alignment.

表3 Stu-miR7997家族前体序列位置信息

Tab.3 Position information of Stu-miR7997 family precursor sequence

前体miRNA ID Pre-miRNA ID	染色体ID Chr ID	起始 Start	终止 End	长度/nt Length
Stu-MIR7997a	2	1 313 823	1 313 940	118
Stu-MIR7997b	5	3 479 983	3 480 100	118
Stu-MIR7997c	5	266 441	266 560	120

图2 Stu-pre-MIR7997前体二级结构预测箭头标注位置为成熟体序列。

Fig.2 Secondary structure prediction of Stu-pre-MIR7997 precursor The position of the arrow is marked as a mature sequence.

图3 Stu-miR7997家族顺式作用元件预测

Fig.3 Prediction of Stu-miR7997 family cis-acting elements

图4 Stu-miR7997家族成员靶基因调控网络图三角形代表Stu-miR7997家族成员;圆形代表靶基因;箭头表示靶向关系;靶基因ID中PGSC0003DMTXXXXXX 缩写为 DMTXXXXXX(如DMT400039552代表PGSC0003DMT400039552)。

Fig.4 Target gene regulatory network of Stu-miR7997 family members Triangles represent members of the Stu-miR7997 family;Circles represent target genes;Arrows indicate targeting relationships;Target gene ID PGSC0003DMTXXXXXX is abbreviated as DMTXXXXXX(e.g.,DMT400039552 represents PGSC0003DMT400039552).

图5 Stu-miR7997家族靶基因GO 富集图

Fig.5 GO enrichment map of Stu-miR7997 family target genes

图6 Stu-miR7997家族在马铃薯不同组织部位中的表达模式不同小写字母表示差异显著(P<0.05);不同的大写字母表示差异极显著(P<0.01)。图7—8同。

Fig.6 The expression pattern of Stu-miR7997 family in different tissues of potato Different lowercase letters denote significant differences (P< 0.05), and different uppercase letters indicate highly significant differences(P<0.01).The same as Fig.7—8.

图7 Stu-miR7997家族在感染晚疫病前后的表达模式

Fig.7 The expression pattern of Stu-miR7997 family before and after late blight infection

表4 Stu-miR7997家族的靶基因

Tab.4 Target gene of Stu-miR7997 family

靶基因 Target gene	匹配区域 Alignment area	靶基因功能 Function of target gene	期望值 Expectation	机制 Inhibition
PGSC0003DMT400039552	miRNA 21 AAACUUCUCAGGCUCGUCGUA 1 ::::::::::::::::: ::: Target 223 UUUGAAGAGUCCGAGCAACAU 243	NAD(P)H- 醌氧化还原酶	1.5	剪切
PGSC0003DMT400054374	miRNA 21 AAACUUCUCAGGCUCGUCGUA 1 ::::::::::::::::: ::: Target 952 UUUGAAGAGUCCGAGCAACAU 972	核酸结合蛋白	1.5	剪切
PGSC0003DMT400060373	miRNA 21 AAACUUCUCAGGCUCGUCGUA 1 ::::::::::.::::.: ::: Target 1089 UUUGAAGAGUUCGAGUAACAU 1109	果胶裂解酶	2.5	剪切
PGSC0003DMT400014983	miRNA 21 AAACUUCUCAGGCUCGUCGUA 1 ::::::::: ::::::: ::: Target 693 UUUGAAGAGCCCGAGCAACAU 713	MYB转录因子MYB92	3.0	剪切

图8 Stu-miR7997靶基因感染晚疫病后上调表达靶基因

Fig.8 Stu-miR7997 target genes upregulated after infection with late blight

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