小麦叶锈菌效应因子Pt6647 ORF克隆及其表达分析

doi:10.7668/hbnxb.20195780

摘要/Abstract

摘要：

为明确小麦叶锈菌效应因子Pt6647的表达特性及在寄主致病中的作用,以前期筛选获得的高表达候选效应子Pt6647为研究对象,采用PCR技术进行基因克隆,利用NCBI数据库进行序列比对分析,通过信号肽预测与功能验证分析其分泌特性,借助亚细胞定位技术确定其细胞内分布,并应用实时荧光定量PCR技术检测其在侵染过程中的表达模式。结果显示,成功克隆获得长度为765 bp的Pt6647开放阅读框(ORF);该基因在8个小麦叶锈病菌生理小种中高度保守,仅存在2个氨基酸变异;该效应子由254个氨基酸组成,其N端1—18 位氨基酸被证实为具有分泌功能的信号肽;亚细胞定位显示,全长的Pt6647定位于细胞膜,而去除信号肽的截短体(Pt6647^Δsp)则定位于细胞核、细胞质和细胞膜;表达模式分析表明,Pt6647在叶锈菌侵染小麦后36 h表达量达到高峰。成功克隆并鉴定了小麦叶锈菌效应子Pt6647,其特异性的亚细胞定位和侵染早期的表达高峰表明效应子Pt6647在病原菌侵入寄主植物早期发挥关键作用。

关键词: 小麦叶锈菌, 效应因子, 基因克隆, 荧光定量PCR, 亚细胞定位

Abstract:

To elucidate the expression characteristics of the effector protein Pt6647 from Puccinia triticina and further reveal its role in host pathogenicity,the candidate effector protein Pt6647,which was previously identified as highly expressed during host-pathogen interaction,was selected for analysis.We performed gene cloning via PCR,sequence alignment using the NCBI database,signal peptide prediction and functional validation to assess its secretory activity,subcellular localization to determine its cellular distribution,and Quantitative Real-time PCR(qRT-PCR)to examine its expression profile during infection.The open reading frame(ORF)of Pt6647,765 bp in length,was successfully cloned.The gene was highly conserved across eight different physiological races of P.triticina,with only two amino acid variations observed.The Pt6647 protein consisted of 254 amino acids,with its N-terminal 1—18 amino acids confirmed as a functional signal peptide for secretion.Subcellular localization analysis revealed that the full-length Pt6647 protein localized to the cell membrane,while the signal peptide-truncated variant(Pt6647^Δsp)was distributed in the nucleus,cytoplasm,and cell membrane.Expression profiling demonstrated that the transcription of Pt6647 peaked at 36 hours post-inoculation(hpi).The effector Pt6647 was successfully cloned and characterized.Its specific subcellular localization and early infection expression peak suggest that Pt6647 plays a critical role in the initial stages of pathogen invasion.

Key words: Puccinia triticina, Effector, Gene cloning, Quantitative Real-time PCR, Subcellular localization

中图分类号:

李若琳, 刘文泽, 常佳迎, 张娜, 杨文香. 小麦叶锈菌效应因子Pt6647 ORF克隆及其表达分析[J]. 华北农学报, 2026, 41(2): 187-193. doi: 10.7668/hbnxb.20195780.

LI Ruolin, LIU Wenze, CHANG Jiaying, ZHANG Na, YANG Wenxiang. Cloning and Expression Analysis of Puccinia triticina Effector Pt6647 ORF[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(2): 187-193. doi: 10.7668/hbnxb.20195780.

http://www.hbnxb.net/CN/Y2026/V41/I2/187

图/表 10

表1 本试验所用引物

Tab.1 Primers used in the research

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	备注 Note
Pt6647-F	ATGAAGTTTCTCATCTTCGC	Pt6647克隆引物
Pt6647-R	CTAGTAGGTGACAGGGACAG
qPt6647-F	CCAACATCTCGGCTACGCACAG	荧光定量引物
qPt6647-R	CACATGATGACATACGGGACTGGAC
pSU-Pt6647-F	CCGGAATTCATGAAGTTTCTCATCTTCGC	信号肽分泌检测
pSU-Pt6647-R	CCGCTCGAGCGCGTCAATGACTGGGAGAA
pGR-Pt6647-F	CCCATCGATATGAAGTTTCTCATCTTCGCATT	亚细胞定位引物
pGR-Pt6647-R	CCCCCGGGGTAGGTGACAGGGACAGGACG
pGR-Pt6647^Δsp-F	CCCATCGATATGCAGCCAATTTCAAGAGCAC
pGR-Pt6647^Δsp-R	CCCCCGGGGTAGGTGACAGGGACAGGAC

图1 Pt6647 ORF克隆产物 M.DNA Marker;1—4.Pt6647 ORF 全长。

Fig.1 Pt6647 ORF cloning M.DNA Marker;1—4.The total length of Pt6647 ORF.

图2 Pt6647信号肽预测及结构 A.Pt6647信号肽结构;B.Pt6647信号肽预测。

Fig.2 Prediction and structure of Pt6647 signal peptide A.Pt6647 signal peptide structure; B.Pt6647 signal peptide prediction.

表2 Pt6647氨基酸含量

Tab.2 Amino acid content of Pt6647

氨基酸 Amino acid	数量 Quantity	百分比/% Percentage	氨基酸 Amino acid	数量 Quantity	百分比/% Percentage
Ala (A)	22	8.7	Lys (K)	7	2.8
Arg (R)	13	5.1	Met (M)	4	1.6
Asn (N)	13	5.1	Phe (F)	7	2.8
Asp (D)	8	3.1	Pro (P)	14	5.5
Cys (C)	14	5.5	Ser (S)	32	12.6
Gln (Q)	13	5.1	Thr (T)	13	5.1
Glu (E)	16	6.3	Trp (W)	5	2.0
Gly (G)	22	8.7	Tyr (Y)	9	3.5
His (H)	6	2.4	Val (V)	13	5.1
Ile (I)	11	4.3	Leu (L)	12	4.7

图3 Pt6647蛋白三级结构预测 A.小麦叶锈菌蛋白模型;B.Pt6647蛋白预测模型;C.模型构建信息。

Fig.3 Tertiary structure prediction of Pt6647 A.P.triticina protein model;B.Pt6647 protein prediction model; C.Model construction information.

图4 Pt6647系统发育树分析

Fig.4 Phylogenetic tree analysis of Pt6647

图5 Pt6647 在叶锈菌中氨基酸序列多态性比对(仅显示存在差异的部分菌株)

Fig.5 Sequence polymorphism comparison of Pt6647 in races of P.triticina (Only some strains with differences are shown)

图6 Pt6647信号肽分泌功能验证

Fig.6 Secretion function verification of Pt6647 signal peptide

图7 Pt6647 在烟草叶片中的亚细胞定位

Fig.7 Subcellular localization of Pt6647 in N.benthamiana leaves

图8 Pt6647 在叶锈菌 THSN 侵染小麦Thatcher不同时间的表达模式不同小写字母表示在P<0.05水平差异显著。

Fig.8 Expression patterns of Pt6647 in wheat Thatcher infected by THSN at different time points Different lowercase letters indicate significant differences at the 0.05 level.

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