中国农业科学 ›› 2023, Vol. 56 ›› Issue (16): 3124-3139.doi: 10.3864/j.issn.0578-1752.2023.16.007
孔乐辉(), 宗德乾, 史青尧, 殷盼盼, 巫文玉, 田鹏, 单卫星(
), 强晓玉(
)
收稿日期:
2023-06-08
接受日期:
2023-06-14
出版日期:
2023-08-16
发布日期:
2023-08-18
通信作者:
联系方式:
孔乐辉,E-mail:542909995@qq.com。
基金资助:
KONG LeHui(), ZONG DeQian, SHI QingYao, YIN PanPan, WU WenYu, TIAN Peng, SHAN WeiXing(
), QIANG XiaoYu(
)
Received:
2023-06-08
Accepted:
2023-06-14
Published:
2023-08-16
Online:
2023-08-18
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。
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.
表1
StCYP83基因家族的理化特性"
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 |
图4
过表达StCYP83B1可增强植物对致病疫霉的抗性 A:Western blot检测烟草叶片中StCYP83B1蛋白表达,蛋白质载量用丽春红染色表示The protein expression of StCYP83B1 in N. benthamiana leaves was examined by Western blot, and protein loading was indicated by Ponceau staining。B:农杆菌介导的本氏烟叶片瞬时转化:左侧注射GFP对照,右侧注射转有StCYP83B1的农杆菌,表达2 d后接种致病疫霉游动孢子,于5 dpi记录病斑,并进行台盼蓝染色A. tumefaciens-mediated transient transformation in N. benthamiana leaves. The left and right sides were expressed with GFP and StCYP83B1 genes, respectively. After two days, P. infestans zoospores were inoculated followed by photographing of disease symptom and trypan blue staining at 5 dpi。C:病斑面积统计。用于接菌测试的烟草叶片不少于10片(n=15)The lesion area was statistically analyzed. At least 10 N. benthamiana leaves were used for inoculation test (n=15)。D:侵染叶片中菌丝定殖量分析,以本氏烟管家基因NbF-box为内参,利用qRT-PCR检测致病疫霉管家基因PiUBC的表达,将过表达GFP叶片中的致病疫霉生物量设为1。3次独立试验均获得相似结果Quantification of P. infestans biomass in infected N. benthamiana leaves. The expression level of housekeeping gene PiUBC of P. infestans was detected by qRT-PCR with housekeeping gene NbF-box of N. benthamiana as internal reference. The P. infestans biomass in leaves overexpressing GFP was set to 1. Similar results were obtained from three individual experiments"
图5
StCYP83B1过表达可增强植物PTI免疫反应 A:将过表达GFP和StCYP83B1的本氏烟叶片用1 μmol·L-1 flg22处理后,利用鲁米诺化学发光法检测活性氧迸发水平。结果表示为6个生物学重复的平均值±标准误ROS burst was measured using a luminol-based chemiluminescence assay in the overexpression of GFP and StCYP83B1 leaves treated with 1 μmol·L-1 flg22. Results were the mean±SE of 6 biological replicates。B:在致病疫霉侵染不同时期,NbWRKY7和NbWRKY8在过表达GFP和StCYP83B1的本氏烟叶片的表达量,将过表达GFP叶片中0 hpi的PTI标记基因的表达量设为1 The expression levels of NbWRKY7 and NbWRKY8 in leaves of N. benthamiana overexpressed with GFP and StCYP83B1, respectively, during P. infestation infection. The expression level of PTI marker genes at 0 hpi in GFP-expressed leaves was set to 1"
图7
StCYP83B1血红素结合域中的半胱氨酸位点为其抗性功能所必需 A:Western blot检测烟草叶片中StCYP83B1(C438S)蛋白表达,蛋白质载量用丽春红染色表示The protein expression of StCYP83B1(C438S) in N. benthamiana leaves was examined by Western blot, and protein loading was indicated by Ponceau staining。B:农杆菌介导的本氏烟叶片瞬时转化,表达2 d后接种致病疫霉游动孢子,于5 dpi记录病斑,并进行台盼蓝染色The N. benthamiana leaves were transiently expressed with either StCYP83B1 or StCYP83B1(C438S) for 2 days prior to the inoculation with P. infestans zoospores, followed by photographing of disease symptom and trypan blue staining at 5 dpi。C:病斑面积统计。用于接菌测试的烟草叶片不少于10片(n=26)The lesion area was statistically analyzed. At least 10 N. benthamiana leaves were used for the pathogenicity assay (n=26)。D:侵染叶片中菌丝定殖量分析,以本氏烟管家基因NbF-box为内参,利用qRT-PCR检测致病疫霉管家基因PiUBC的表达,将过表达StCYP83B1(C438S)叶片中的致病疫霉生物量设为1。3次独立试验均获得相似结果Quantification of P. infestans biomass in infected N. benthamiana leaves. The expression level of housekeeping gene PiUBC of P. infestans was detected by qRT-PCR with housekeeping gene NbF-box of N. benthamiana as internal reference. The P. infestans biomass in leaves overexpressing StCYP83B1(C438S) was set to 1. Similar results were obtained from three individual experiments"
图8
StCYP83B1过表达可增强马铃薯对致病疫霉的抗性 A:接种致病疫霉4 d后马铃薯叶片病斑面积观察以及台盼蓝染色结果Lesion area of potato leaves at 4 dpi and the leaves were stained by trypan blue。B:病斑面积统计。用于接菌测试的马铃薯叶片不少于10片(n=10)The lesion area was statistically analyzed. At least 10 potato leaves were used for the pathogenicity assay (n=10)。C:侵染叶片中菌丝定殖量分析,以马铃薯管家基因StEF1α为内参,利用qRT-PCR检测致病疫霉管家基因PiUBC的表达,将大西洋中的致病疫霉生物量设为1 Quantification of P. infestans biomass in infected potato leaves. The expression level of housekeeping gene PiUBC of P. infestans was detected by qRT-PCR with housekeeping gene StEF1α of potato as internal reference. The P. infestans biomass in Atlantic was set to 1"
图9
StCYP83B1-OE可增强马铃薯的PTI免疫反应 A:将大西洋和StCYP83B1-OE株系的叶片用1 μmol·L-1 flg22处理后,利用鲁米诺化学发光法检测活性氧迸发水平。结果表示为6个生物学重复的平均值±标准误ROS burst was measured using a luminol-based chemiluminescence assay in the Atlantic and StCYP83B1-OE leaves treated with 1 μmol·L-1 flg22. Results were the mean±SE of 6 biological replicates。B:用40 µmol·L-1 flg22处理马铃薯叶片后提取总RNA,以马铃薯管家基因StEF1α为内参,利用qRT-PCR检测StCYP83B1-OE株系中StWRKY7、StWRKY8和StACRE31的表达量,将大西洋中0 h的PTI标记基因表达量设为1(n=3)Total RNA was extracted from 40 µmol·L-1 flg22-treated potato leaves. The expression levels of StWRKY7, StWRKY8 and StACRE31 of StCYP83B1-OE were detected by qRT-PCR with housekeeping gene StEF1α of potato as internal reference. The expression level of PTI marker genes at 0 h in Atlantic was set to 1 (n=3)"
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