中国农业科学 ›› 2021, Vol. 54 ›› Issue (19): 4110-4120.doi: 10.3864/j.issn.0578-1752.2021.19.007
曲潇玲(),焦裕冰,罗健达,宋丽云,李莹,申莉莉(
),杨金广(
),王凤龙
收稿日期:
2021-02-22
接受日期:
2021-04-14
出版日期:
2021-10-01
发布日期:
2021-10-12
通讯作者:
申莉莉,杨金广
作者简介:
曲潇玲,E-mail: 基金资助:
QU XiaoLing(),JIAO YuBing,LUO JianDa,SONG LiYun,LI Ying,SHEN LilLi(
),YANG JinGuang(
),WANG FengLong
Received:
2021-02-22
Accepted:
2021-04-14
Online:
2021-10-01
Published:
2021-10-12
Contact:
LilLi SHEN,JinGuang YANG
摘要:
【目的】马铃薯Y病毒(potato virus Y,PVY)是危害我国烟草生产的最重要病毒之一,NAC转录因子与植物的抗病、抗逆密切相关,本论文克隆NbNAC062进行生物信息学分析,并研究其在PVY侵染过程中的作用,为烟草抗病毒药剂的开发提供靶标。【方法】以本氏烟(Nicotiana benthamiana)为材料克隆NbNAC062,利用MEGA、UniProt、SMART、TMHMM Server 2.0、Sol Genomics Network、PlantCARE等技术进行生物信息学分析;利用激光共聚焦与实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)明确PVY侵染前后NbNAC062蛋白定位及mRNA表达量变化;基于病毒介导的基因沉默(virus-induced gene silencing,VIGS)和过表达技术,构建pTRV::NbNAC062沉默载体与pEarleyGate100::RFP::NbNAC062过表达载体,采用qRT-PCR和Western blot检测NbNAC062在本氏烟中沉默与过表达后,PVY的积累量变化及未折叠蛋白应答(unfolded protein response,UPR)相关基因BiP的表达差异。【结果】NbNAC062编码646个氨基酸,N端28—179 aa为NAC结构域,129—185 aa为DNA结合区域,C末端621—643 aa为疏水跨膜结构,系统进化树与蛋白序列分析表明本氏烟NbNAC062与渐狭叶烟草NaNAC062亲缘关系最近。NbNAC062启动子中包含脱落酸、茉莉酸甲酯、水杨酸以及逆境响应相关的多种顺式作用元件。PVY侵染激活NbNAC062从细胞膜转移至细胞核,且诱导NbNAC062上调表达。PVY侵染本氏烟5、7 d,处理组NbNAC062 mRNA水平分别为对照组的2.52、1.95倍;PVY侵染3 d,BiP mRNA表达量为对照组的2.39倍,PVY侵染7 d,BiP表达量极显著低于对照组,下调表达56.77%。本氏烟沉默NbNAC062并接种PVY,接种后3、5、7 d,与对照组相比,沉默组PVY CP mRNA上调表达,分别为对照组的2.12、2.41、1.38倍,BiP mRNA表达量则下调,分别下调28.19%、58.11%、10.77%,接种后5、7 d沉默组PVY CP蛋白含量亦显著高于对照组。过表达NbNAC062并接种PVY,接种后24、48、72 h,与对照组相比,过表达组PVY CP mRNA分别下调22.60%、34.51%、36.21%,接种48、72 h,BiP mRNA上调表达,分别为对照组的1.56、1.35倍,过表达组PVY CP蛋白含量亦低于对照组。【结论】NbNAC062属于NAC类膜结合转录因子,可被PVY侵染激活转移至细胞核,可能通过调控UPR相关基因BiP的表达,促进细胞生存,抑制PVY早期侵染。
曲潇玲,焦裕冰,罗健达,宋丽云,李莹,申莉莉,杨金广,王凤龙. 本氏烟NbNAC062的克隆及对马铃薯Y病毒侵染的抑制作用[J]. 中国农业科学, 2021, 54(19): 4110-4120.
QU XiaoLing,JIAO YuBing,LUO JianDa,SONG LiYun,LI Ying,SHEN LilLi,YANG JinGuang,WANG FengLong. Cloning of Nicotiana benthamiana NAC062 and Its Inhibitory Effect on Potato Virus Y Infection[J]. Scientia Agricultura Sinica, 2021, 54(19): 4110-4120.
表1
本试验所用引物"
引物Primer | 序列Sequence |
---|---|
NbNAC062 F | ATGATGGCAGTACTTCCTGG |
NbNAC062 R | TACTCGCACTCTAAAGTATTCCC |
TRV-NbNAC F | TAAGGTTACCGAATTCTTGGATGGATCACACCCTGGC |
TRV-NbNAC R | AGACGCGTGAGCTCGGTACCTTCTGTATCATCAGCAATACAGC |
Fu-NbNAC F | CTTTAGATCTTCTAGAATGATGGCAGTACTTCCTGG |
Fu-NbNAC R | AGGAGGCCATGAATTCTACTCGCACTCTAAAGTATTCCC |
PVY-CP-F | GATGAATGGGCTTATGGTTTGGTG |
PVY-CP-R | GATTTGCCTAAGGGTTGGTTTCG |
Actin-F | CAAGGAAATCACCGCTTTGG |
Actin-R | AAGGGATGCGAGGATGGA |
qPCR-NbNAC062 F | TGGACAAGAATTGGCATCGC |
qPCR-NbNAC062 R | AACACCTCGGGCTCAAAGAAG |
qPCR-BiP F | GCCACAGAAGAAGCTACCAAGTTG |
qPCR-BiP R | GGTCCTCTCTGGGTTAACAGCG |
图4
PVY侵染前后NbNAC062蛋白亚细胞定位 A:NbNAC062蛋白亚细胞定位,BBcellProbe M01为细胞膜绿色染料,激发波长/发射波长为488 nm/500 nm Subcellular localization of NbNAC062 protein, BBcellProbe M01 is cell membrane green dye, excitation wavelength/emission wavelength is 488 nm/500 nm;B:PVY侵染后NbNAC062蛋白亚细胞定位,DAPI为细胞核蓝色染料,激发波长/发射波长为358 nm/461 nm Subcellular localization of NbNAC062 protein after PVY infection, DAPI is nuclear blue dye, excitation wavelength/emission wavelength is 358 nm/461 nm"
图5
沉默NbNAC062表型分析与沉默效率 A:沉默第7天处理组pTRV::NbNAC062、阴性对照组pTRV00、阳性对照组pTRV::PDS表型 On the 7th day of silence, the phenotype of pTRV::NbNAC062 treatment group, pTRV00 negative control group and pTRV::PDS positive control group;B:沉默NbNAC062第15天沉默效率 Silencing efficiency of NbNAC062 on the 15th day;C:NbNAC062沉默植株接种PVY-GFP第5天叶片荧光情况 Fluorescence of leaves on the 5th day after silencing NbNAC062 inoculated with PVY-GFP"
图6
沉默NbNAC062后PVY与BiP积累量变化 A:沉默NbNAC062后PVY侵染1、3、5、7 d,qRT-PCR检测PVY CP mRNA变化Changes of PVY CP mRNA are detected by qRT-PCR when PVY infection was 1, 3, 5, 7 days after silencing NbNAC062;B:沉默NbNAC062后PVY侵染1、3、5、7 d,qRT-PCR检测BiP变化Changes of BiP are detected by qRT-PCR when PVY infection was 1, 3, 5, 7 days after silencing NbNAC062;C:沉默NbNAC062后PVY侵染1、3、5、7 d,Western blot检测PVY蛋白量变化。每天取样组中左侧为对照右侧为处理Changes of PVY protein are detected by Western blot when PVY infection was 1, 3, 5, 7 days after silencing NbNAC062. In the daily sampling group, the left side is the control and the right side is the treatment;D:C对应的Actin Western blot蛋白杂交图The Western blot protein hybridization map of Actin corresponding to C"
图7
过表达NbNAC062后PVY与BiP积累量变化 A:过表达NbNAC062后PVY侵染1、3、5、7 d,qRT-PCR检测PVY CP mRNA变化Changes of PVY CP mRNA are detected by qRT-PCR when PVY infection was 1, 3, 5, 7 days after overexpression NbNAC062;B:过表达NbNAC062后PVY侵染1、3、5、7 d,qRT-PCR检测BiP变化Changes of BiP are detected by qRT-PCR when PVY infection was 1, 3, 5, 7 days after overexpression NbNAC062;C:过表达NbNAC062后PVY侵染1、3、5、7 d,Western blot检测PVY蛋白量变化。每天取样组中左侧为对照右侧为处理Changes of PVY protein are detected by Western blot when PVY infection was 1, 3, 5, 7 days after overexpression NbNAC062. In the daily sampling group, the left side is the control and the right side is the treatment;D:C对应的Actin Western blot蛋白杂交图The Western blot protein hybridization map of Actin corresponding to C"
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