马铃薯StCYP83基因家族鉴定及其抗晚疫病的功能分析

doi:10.3864/j.issn.0578-1752.2023.16.007

摘要/Abstract

摘要：

【目的】通过鉴定马铃薯StCYP83基因家族成员，分析其响应致病疫霉（Phytophthora infestans）侵染的表达模式，挖掘具有抗晚疫病功能的StCYP83并解析其免疫机制，为马铃薯抗晚疫病分子育种提供新型抗性基因资源。【方法】利用双向BLAST法鉴定StCYP83基因家族成员，通过ExPASy Prot Param、Cell-Ploc 2.0、ESPript等软件分析StCYP83蛋白序列基本信息、亚细胞定位情况、保守基序；利用qRT-PCR技术分析StCYP83响应致病疫霉侵染的表达模式；利用农杆菌介导的本氏烟瞬时表达体系和马铃薯过表达（OE）稳定转化植株分析候选基因影响寄主植物抗致病疫霉的免疫功能及作用机理。【结果】在马铃薯基因组中共鉴定到10个StCYP83家族基因，分别命名为StCYP83B1—StCYP83B10，编码蛋白长度介于387—503 aa，分子量介于44—57 kDa，亚细胞定位预测结果显示StCYP83蛋白均定位于内质网膜。qRT-PCR结果表明，StCYP83家族成员在不同程度上均可响应致病疫霉侵染而诱导表达，暗示StCYP83家族基因可能在马铃薯与致病疫霉互作中发挥作用。基于此，从中选取明显响应致病疫霉侵染而诱导上调表达、且与拟南芥AtCYP83B1同源性最高的StCYP83B1用于后续的免疫功能解析。本氏烟瞬时过表达的接菌测试结果表明，StCYP83B1具有抗致病疫霉的生物学功能；同时，过表达StCYP83B1可显著促进PTI标记基因（NbWRKY7、NbWRKY8）、SA信号通路标记基因（NbPR1和NbPR2）和JA信号通路标记基因（NbPR3和NbLOX）的上调表达，并提高flg22诱发的活性氧迸发。此外，StCYP83B1编码蛋白保守基序中的半胱氨酸位点为其抗性功能所必需。StCYP83B1过表达（StCYP83B1-OE）株系对致病疫霉的抗性有所增强，且呈现出增强的PTI免疫反应，包括flg22诱发的活性氧水平升高以及PTI标记基因（StWRKY7、StWRKY8和StACRE31）的显著诱导上调表达。此外，马铃薯SA信号通路相关基因（StPR1、StPR2、StPR5和StPAL2）和JA信号通路相关基因（StLOX、StAOS和StOPR3）也被诱导上调表达。【结论】共鉴定到10个StCYP83家族成员，StCYP83家族成员在不同程度上均可响应致病疫霉的侵染而诱导表达。StCYP83B1通过激活PTI、SA和JA信号通路调控植物对致病疫霉的抗性；而StCYP83B1血红素结合域中的半胱氨酸位点为其抗性功能所必需。

关键词: 马铃薯, StCYP83基因家族, 致病疫霉, 植物抗病性, 晚疫病

Abstract:

【Objective】The objective of this study is to identify StCYP83 gene family in potato and analyze their expression patterns in response to Phytophthora infestans infection, so as to mine the StCYP83 genes with potential function in resistance to late blight and provide novel resistance gene resources for molecular resistance breeding in potato.【Method】The members of StCYP83 gene family were identified by bidirectional BLAST method. The basic information of StCYP83 protein sequence, subcellular localization and conserved motifs were analyzed by ExPASy Prot Param, Cell-Ploc 2.0 and ESPript, etc. The qRT-PCR was used to analyze the expression pattern of StCYP83 genes in response to P. infestans infection. The immune function of candidate gene StCYP83B1 against P. infestans was analyzed in either Agrobacterium tumefaciens-mediated transient transformation in leaves of Nicotiana benthamiana or stably transformed potato lines with overexpression (OE) of StCYP83B1.【Result】A total of 10 StCYP83 genes were identified in the potato genome, which were named StCYP83B1-StCYP83B10, respectively, with the encoded protein lengths ranging from 387 to 503 aa and molecular weights ranging from 44 to 57 kDa. The subcellular localization of StCYP83 proteins was predicted in the endoplasmic reticulum. The qRT-PCR confirmed that members of StCYP83 could be induced in response to P. infestans infection, suggesting that StCYP83 genes might play a role in the interaction between potato and P. infestans. Accordingly, StCYP83B1 with the highest homology to AtCYP83B1 was selected as a candidate gene for subsequent immune functional analysis. The pathogenicity assay on N. benthamiana leaves showed that overexpression of StCYP83B1 could enhance plant resistance against P. infestans. In accordance with this, overexpression of StCYP83B1 could significantly promote the up-regulation expression of PTI marker genes (NbWRKY7 and NbWRKY8), SA signaling marker genes (NbPR1 and NbPR2), JA signaling marker genes (NbPR3 and NbLOX) and enhance the reactive oxygen species (ROS) burst induced by flg22. In addition, cysteine site in the conserved motif of StCYP83B1 protein was required for its immune function. StCYP83B1 overexpression (StCYP83B1-OE) lines showed enhanced resistance to P. infestans. In accordance with this, StCYP83B1-OE could enhance PTI immune responses, including the increased level of ROS induced by flg22 and the significantly up-regulated expression of PTI marker genes (StWRKY7, StWRKY8 and StACRE31) as well as SA-mediated signaling marker genes (StPR1, StPR2, StPR5 and StPAL2) and JA-mediated signaling marker genes (StLOX, StAOS and StOPR3) in response to P. infestans infection.【Conclusion】A total of 10 members of StCYP83 family were identified, which could be induced by P. infestans infection in different degrees. StCYP83B1 regulates plant resistance to P. infestans by activating PTI, SA and JA signaling pathways. The cysteine site in the heme binding domain of StCYP83B1 is required for its immune function.

Key words: potato (Solanum tuberosum), StCYP83 gene family, Phytophthora infestans, plant resistance, late blight

孔乐辉, 宗德乾, 史青尧, 殷盼盼, 巫文玉, 田鹏, 单卫星, 强晓玉. 马铃薯StCYP83基因家族鉴定及其抗晚疫病的功能分析[J]. 中国农业科学, 2023, 56(16): 3124-3139.

KONG LeHui, ZONG DeQian, SHI QingYao, YIN PanPan, WU WenYu, TIAN Peng, SHAN WeiXing, QIANG XiaoYu. Identification of StCYP83 Gene Family in Potato and Analysis of Its Function in Resistance Against Late Blight[J]. Scientia Agricultura Sinica, 2023, 56(16): 3124-3139.

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基因名 Gene name	基因ID Gene ID	蛋白长度 Length (aa)	分子量 Molecular weight (Da)	等电点 pI
StCYP83B1	PGSC0003DMG400016616	496	56706.30	9.00
StCYP83B2	PGSC0003DMG400031519	491	56113.44	8.53
StCYP83B3	PGSC0003DMG401031520	497	57164.85	8.85
StCYP83B4	PGSC0003DMG402031520	496	56749.34	9.10
StCYP83B5	PGSC0003DMG400011325	476	54658.53	8.79
StCYP83B6	PGSC0003DMG401021057	420	47673.35	5.45
StCYP83B7	PGSC0003DMG400026778	503	57072.21	7.12
StCYP83B8	PGSC0003DMG402021057	461	52325.37	9.00
StCYP83B9	PGSC0003DMG400006024	473	54683.80	7.56
StCYP83B10	PGSC0003DMG400019899	387	44732.72	6.63