烟草Hsc70-2的克隆及对马铃薯Y病毒侵染烟草的促进作用

doi:10.3864/j.issn.0578-1752.2020.04.009

中国农业科学 ›› 2020, Vol. 53 ›› Issue (4): 771-781.doi: 10.3864/j.issn.0578-1752.2020.04.009

烟草Hsc70-2的克隆及对马铃薯Y病毒侵染烟草的促进作用

龚明月¹,段啸天¹,余婷婷¹,王杰²,申莉莉²,李莹²,刘明宏³,李永亮⁴,吕洪坤⁵,章松柏¹(),杨金广²()

1 长江大学农学院,湖北荆州434025
2 中国农业科学院烟草研究所,山东青岛266101
3 贵州省烟草公司遵义市公司,贵州遵义563000
4 云南省烟草公司保山市公司,云南保山678000
5 中国烟草总公司海南省公司海南雪茄研究所,海口571100

收稿日期:2019-08-19 接受日期:2019-09-18 出版日期:2020-02-16 发布日期:2020-03-09
联系方式: 龚明月,E-mail：576092384@qq.com。
基金资助:
烟草绿色防控重大专项(110101601024LS-04);云南省烟草公司科技项目(2018530000241014);海南省烟草公司科技项目(201846000024056)

Cloning of Hsc70-2 and Its Promoting Effect on Potato virus Y Infection in Nicotiana benthamiana

MingYue GONG¹,XiaoTian DUAN¹,TingTing YU¹,Jie WANG²,LiLi SHEN²,Ying LI²,MingHong LIU³,YongLiang LI⁴,HongKun LÜ⁵,SongBai ZHANG¹(),JinGuang YANG²()

1 College of Agronomy, Yangtze University, Jingzhou 434025, Hubei
2 Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, Shandong
3 Zunyi City Company, Guizhou Tobacco Company, Zunyi 563000, Guizhou
4 Baoshan City Company, Yunnan Tobacco Company, Baoshan 678000, Yunnan
5 Hainan Cigar Research Institute, Hainan Provincial Corporation, China National Tobacco Corporation, Haikou 571100

Received:2019-08-19 Accepted:2019-09-18 Published:2020-02-16 Online:2020-03-09

摘要/Abstract

摘要：

【目的】马铃薯Y病毒（Potato virus Y,PVY）是危害烟草、马铃薯、番茄、辣椒等主要农作物的重要病毒,给农业生产带来巨大损失。论文旨在克隆Hsc70-2蛋白在本氏烟（Nicotiana benthamiana）中的基因序列并分析其生物学信息,研究NbHsc70-2蛋白对PVY侵染烟草的影响,为进一步解析PVY的侵染机制提供理论依据。【方法】以本氏烟为材料,克隆NbHsc70-2蛋白的基因编码序列（coding sequence,CDS）,利用MEGA 6.0 进行多序列比对并构建系统进化树;利用实时荧光定量PCR（qRT-PCR）分析NbHsc70-2在本氏烟各组织中的表达水平;采用在线软件BaCeILo和SignalP 4.0对NbHsc70-2进行生物信息学分析;构建NbHsc70-2-RFP融合蛋白确定该蛋白的亚细胞定位;研究PVY处理对本氏烟叶片中NbHsc70-2的影响;利用激光共聚焦观察PVY-GFP处理对NbHsc70-2-RFP定位的影响;构建NbHsc70-2 VIGS沉默体系及瞬时表达载体,采用qRT-PCR比较NbHsc70-2在沉默/过表达后对PVY表达的影响。【结果】NbHsc70-2编码649个氨基酸,系统进化树分析表明,NbHsc70-2与普通烟NaHsc70-2序列相似性最高,亲缘关系最近,属于热激蛋白家族,C端具有热激蛋白家族的高度保守基序结构;qRT-PCR分析表明,NbHsc70-2在叶中表达量最高,在根和茎中的表达量较低;BaCeILo预测及激光共聚焦显微镜观察均显示NbHsc70-2定位在细胞质中,并且PVY-GFP侵染本氏烟后NbHsc70-2-RFP部分转移至细胞核,与PVY-GFP共定位在细胞质及细胞核中;将NbHsc70-2基因沉默载体pTRV::NbHsc70-2导入本氏烟中7 d后相比于对照组,沉默组出现心叶皱缩及矮化现象;接种PVY-GFP 7 d后通过手持紫外灯观察到沉默组中无明显绿色荧光出现,而对照组中PVY-GFP系统侵染使得非接种叶片呈现荧光现象,沉默组荧光点数约为对照组28%;接种PVY后,利用qRT-PCR检测沉默NbHsc70-2后1、3、5 d的PVY CP基因积累量,结果表明沉默NbHsc70-2后PVY CP基因积累量下降,其中3 d和5 d与对照相比差异显著,基因表达水平分别为对照的14%和0.004%;将NbHsc70-2过表达载体pEarleyGate100::GWC::NbHsc70-2与PVY同时导入本氏烟后,结果表明相比于对照组,过表达组48 h和72 h 其PVY CP基因积累量显著上升,基因表达水平分别为对照组的2.31、2.56倍。【结论】PVY侵染会引起NbHsc70-2表达量上升;NbHsc70-2是PVY侵染本氏烟重要的组成成分,沉默该基因显著抑制PVY的表达,过表达NbHsc70-2则显著提高PVY的表达,即NbHsc70-2的表达水平与PVY复制呈正相关,NbHsc70-2蛋白促进PVY对烟草的侵染。

关键词: 本氏烟, NbHsc70-2蛋白, 马铃薯Y病毒, 基因沉默, 过表达, 侵染复制

Abstract:

【Objective】Potato virus Y (PVY) is an important virus that harms major crops such as tobacco, potato, tomato, pepper and so on, and brings great losses to agricultural production. The objective of this study is to clone NbHsc70-2, analyze the bioinformatics of NbHsc70-2 protein, research the effect of NbHsc70-2 protein on PVY infection in Nicotiana benthamiana, and to provide a theoretical basis for further analyzing the infection mechanism of PVY.【Method】The gene coding sequence (CDS) of Hsc70-2 protein from N. benthamiana was cloned in pEarleyGate100 vector. Multi-sequence alignment and phylogenetic analysis were performed using MEGA 6.0 software. Fluorescence quantitative RT-PCR (qRT-PCR) analysis of NbHsc70-2 expression level was performed in different tissues of N. benthamiana. Bioinformatics analysis using online software BaCeILo and SignalP 4.0.was performed for construction of NbHsc70-2-RFP fusion protein and determination of the subcellular localization of the identified NbHsc70-2 protein. PVY-GFP was used to analyze the effect of PVY infection on NbHsc70-2 in N. benthamiana leaves and localization of this protein. Furthermore, NbHsc70-2 VIGS silencing system and transient expression vector were constructed, and the effect of the protein on PVY expression after silencing/overexpression was analyzed by comparing the results with qRT-PCR assay.【Result】NbHsc70-2 encodes a total of 649 amino acids. The phylogenetic analysis indicated that NbHsc70-2 belongs to the heat shock protein (HSP) family and has the highest similarity with the NaHsc70-2. The C-terminal has the highly conserved motif structure of the HSP family. qRT-PCR analysis showed that NbHsc70-2 had the highest expression level in leaves and low expression in roots and stems. Moreover, BaCeILo prediction and laser confocal microscopy depicted that NbHsc70-2 was localized in the cytoplasm, and after PVY-GFP infection to N. benthamiana, NbHsc70-2-RFP was partially transferred to the nucleus and colocalized with virus in the cytoplasm and nucleus. The induction of NbHsc70-2 gene silencing vector pTRV::NbHsc70-2 into N. benthamiana resulted in stunted growth of infected plants compared to the control at 7 dpi. Whilst, inoculation of silent plants with PVY-GFP didn’t reflect green fluorescence, whereas PVY-GFP inoculation resulted in the green fluorescence production from the leaves of plants kept as control, the number of fluorescent spots in the silent group was about 28% of that in control group. qRT-PCR analysis showed that further inoculation of PVY resulted in accumulation of PVY CP at 1, 3, and 5 d after silencing NbHsc70-2. Moreover, the level of PVY CP decreased after silencing NbHsc70-2, and the difference between 3 and 5 dpi was significant compared to the control, the gene expression level was 14% and 0.004% of that in the control, respectively. Contrarily, high level of PVY CP was observed when N. benthamiana plants were inoculated with NbHsc70-2 overexpression vector along with PVY. The detailed analysis showed that the accumulation of PVY CP was significantly increased at 48 h and 72 h, and the gene expression level was 2.31 and 2.56 times of that in the control group, respectively.【Conclusion】PVY infection causes increased expression of NbHsc70-2. NbHsc70-2 is an important component of PVY infection to N. benthamiana, silencing NbHsc70-2 significantly inhibited PVY expression, and overexpression of NbHsc70-2 significantly increased PVY expression, i.e., the expression level of NbHsc70-2 was positively correlated with PVY replication. NbHsc70-2 protein promoted PVY infection in tobacco.

Key words: Nicotiana benthamiana, NbHsc70-2 protein, Potato virus Y (PVY), gene silencing, overexpression, infection and replication

龚明月,段啸天,余婷婷,王杰,申莉莉,李莹,刘明宏,李永亮,吕洪坤,章松柏,杨金广. 烟草Hsc70-2的克隆及对马铃薯Y病毒侵染烟草的促进作用[J]. 中国农业科学, 2020, 53(4): 771-781.

MingYue GONG,XiaoTian DUAN,TingTing YU,Jie WANG,LiLi SHEN,Ying LI,MingHong LIU,YongLiang LI,HongKun LÜ,SongBai ZHANG,JinGuang YANG. Cloning of Hsc70-2 and Its Promoting Effect on Potato virus Y Infection in Nicotiana benthamiana[J]. Scientia Agricultura Sinica, 2020, 53(4): 771-781.

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引物 Primer	序列 Sequence (5′ to 3′)	限制性内切酶 Restriction enzyme
NbHsc70-2 F	ATGCGTATTATTAACGAGCCTAC
NbHsc70-2 R	TTAATCAACTTCCTCAATCTTAGGAC
NbHsc70-2-XbaI F	CTTTAGATCTTCTAGAATGCGTATTATTAACGAGCCTACTG	XbaI
NbHsc70-2-KpnI R	ATATTAATGTCGACGGTACCATCAACTTCCTCAATCTTAGGACC	KpnI
RNAi NbHsc70-2 F	TAAGGTTACCGAATTCATTCTTTCAGGTGAGGGTAATGAG	EcoRI
RNAi NbHsc70-2 R	AGACGCGTGAGCTCGGTACCCAGGAGGAATACCAGAAAGTTC	KpnI
NbHsc70-2-AhdI F	AGCAGGCTTTGACTTTAGGTCATGCGTATTATTAACGAGCCTACTG	AhdI
NbHsc70-2-AhdI R	TGGGTCTAGAGACTTTAGGTCTTAATCAACTTCCTCAATCTTAGGA	AhdI
NbHsc70-2qRT-F	AGGCTCCACTAGGATTCCGAAGG
NbHsc70-2qRT-R	AGTGACATCCAACAGCAACAGGTC
PVY-CP-F	GATGAATGGGCTTATGGTTTGGTG
PVY-CP-R	GATTTGCCTAAGGGTTGGTTTCG
Actin-F	CAAGGAAATCACCGCTTTGG
Actin-R	AAGGGATGCGAGGATGGA

烟草Hsc70-2的克隆及对马铃薯Y病毒侵染烟草的促进作用

Cloning of Hsc70-2 and Its Promoting Effect on Potato virus Y Infection in Nicotiana benthamiana

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