酵母双杂交筛选与小麦黄花叶病毒P2互作的寄主因子

doi:10.3969/j.issn.1004-1524.2021.03.15

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (3): 497-505.DOI: 10.3969/j.issn.1004-1524.2021.03.15

酵母双杂交筛选与小麦黄花叶病毒P2互作的寄主因子

韩晓蕾¹^,²^,³, 高仕祺¹^,²^,³, 张帆³, 羊健³, 刘芃³, 姜鸿明¹, 李林志¹^,^*()

1. 烟台市农业科学研究院,山东烟台 265500
2.烟台大学生命科学学院,山东烟台 265500
3.宁波大学/农产品质量安全危害因子与风险防控国家重点实验室,浙江宁波 315000

收稿日期:2020-10-27 出版日期:2021-03-25 发布日期:2021-03-25
作者简介:*, 李林志,E-mail: linzhili2002@163.com
韩晓蕾(1994—),女,黑龙江牡丹江人,硕士,主要从事植物病毒学研究。E-mail: hanxiaolei123@yeah.net
通讯作者: 李林志
基金资助:
山东省农业良种工程(2019LZGC001);山东省重大科技创新工程(2018YFJH0602);国家重点研发计划(2017YFD0100600);农业部财政部项目(CARS-3-2-23);丘陵旱地小麦-玉米抗旱减灾技术集成与示范

Screening of host factors interacting with wheat yellow mosaic virus P2 by yeast two-hybrid system

HAN Xiaolei¹^,²^,³, GAO Shiqi¹^,²^,³, ZHANG Fan³, YANG Jian³, LIU Peng³, JIANG Hongming¹, LI Linzhi¹^,^*()

1. Yantai Academy of Agricultural Sciences, Yantai 265500, China
2. College of Life Sciences, Yantai University, Yantai 265500, China
3. Ningbo University/State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Ningbo 315000, China

Received:2020-10-27 Online:2021-03-25 Published:2021-03-25
Contact: LI Linzhi

摘要/Abstract

摘要：

小麦黄花叶病毒(wheat yellow mosaic virus,WYMV)隶属马铃薯Y病毒科(Potyviridae),大麦黄花叶病毒属(Bymovirus)。该病毒基因组是由两条正义单链RNA1和RNA2构成,共编码10个蛋白,其编码的P2在病毒的复制过程中发挥重要功能。文章构建了小麦cDNA酵母文库,通过共转试验共筛选获得若干个候选互作寄主因子,包括水杨酸信号通路因子、E3泛素化连接酶、防御反应、氧化还原酶活性、光系统II组件等18种蛋白,研究P2与寄主因子的互作。进一步试验证实,其中的TaTIFY 10A、Ta14-3-3与WYMV-P2存在互作关系。结果表明,WYMV-P2与小麦的E3泛素化类转录因子、水杨酸信号转录因子、植物光系统的稳定组件及叶绿体的形成关键因子等可能存在互作关系,P2可能参与了寄主多种信号途径,为明确WYMV与寄主的互作机制提供了研究基础。

关键词: 小麦黄花叶病毒, 小麦, P2, cDNA文库, 酵母双杂交

Abstract:

Wheat yellow mosaic virus (WYMV) is classified in the genus Bymovirus, family Potyviridae. The genome is composed of two sense RNA single-stranded RNA1 and RNA2 and encodes 10 proteins, among which P2 plays an important role in virus replication. In order to identify the targets of P2 in host, co-transformation of cDNA library vector and pGBKT7-P2 was constructed, eighteen candidate host targets of P2 were screened out, which were divided into several types based on their functions, including salicylic acid signal pathway factor, E3 ubiquitin ligase, defense response, oxidoreductase activity and photosystem II assembly. Additionally, the interaction of P2 and TaTIFY or Ta14-3-3 was further verified by Y2H system. Based on the results, it could be speculated that P2 may interact with E3 ubiquitin transcription factor, salicylic acid signal transcription factor, or the factors associated with plant photosystem and chloroplasts formation. In conclusion, P2 may participate in multiple signaling pathways during viral infection, and provide an insight into molecular interaction mechanism between WYMV and the host.

Key words: wheat yellow mosaic virus, wheat, P2, cDNA library, yeast two-hybrid

中图分类号:

S435.12

韩晓蕾, 高仕祺, 张帆, 羊健, 刘芃, 姜鸿明, 李林志. 酵母双杂交筛选与小麦黄花叶病毒P2互作的寄主因子[J]. 浙江农业学报, 2021, 33(3): 497-505.

HAN Xiaolei, GAO Shiqi, ZHANG Fan, YANG Jian, LIU Peng, JIANG Hongming, LI Linzhi. Screening of host factors interacting with wheat yellow mosaic virus P2 by yeast two-hybrid system[J]. Acta Agriculturae Zhejiangensis, 2021, 33(3): 497-505.

图/表 7

表1 酵母重组质粒构建引物

Table 1 Primers for construction of yeast recombinant plasmid

编号 Number	引物名称 Primers name	引物序列 Primers sequence(5'-3')	酶切位点 RE site
1	pGADT7 forward primer	TAATACGACTCACTATAGGGCGA	SamⅠ
	pGADT7 reverse primer	CTGTGCATCGTGCACCATCT	BamHⅠ
2	pGBKT7 forward primer	GTAATACGACTCACTATAGGGCGA	NotⅠ
	pGBKT7 reverse primer	ATTCTCAGTGAAATTTTAAACATA	PstⅠ
3	Ta14-3-3 forward primer	TCCCCCGGGATGGCCTTCTTCTCCCACCAC	SamⅠ
	Ta14-3-3 reverse primer	CGCGGATCCTCACATATATATCTCCATGCC	BamHⅠ
4	WYMV-P2 forward primer	AAAGCGGCCGCATGTCAGCTGAGGAAACAGGT	NotⅠ
	WYMV-P2 reverse primer	GCACTGCAGATGGCCCGGCCTCCTGAGGAA	PstⅠ
5	TaTIFY 10A forward primer	TCCCCCGGGATGGCGGCTTCCGCGAGGCAG	SamⅠ
	TaTIFY 10A reverse primer	CGCGGATCCTTATTGGTTTGGCTTCAGGGC	BamHⅠ

图1 四十七个单克隆的PCR检测 M,DL8000 对照;1~47,重组子。

Fig.1 Picture of 47 clones M, DL8000 DNA marker; 1-47, Recombinants.

图2 酵母重组质粒双酶切电泳图 M,DL8000;泳道1~2,NdeⅠ/PstⅠ双酶切pGBKT7-P2;泳道3~4,NdeⅠ/PstⅠ双酶切pGBKT7。

Fig.2 Double digestion patterns of recombinant plasmid M, DL8000; Lane 1-2, pGBKT7-P2 enzyme digestion by NdeⅠ/PstⅠ; Lane 3-4, pGBKT7 enzyme digestion by NdeⅠ/PstⅠ.

图3 pGBKT7-P2、pGBKT7载体自激活验证及毒性验证 A,模式图,pGADT7-RECT与pGBKT7-Lam互作为阴性对照,pGADT7-RECT与pGBKT7-53互作为阳性对照,pGBKT7-P2与pGBKT7互作为自激活验证;B,SD/-Trp/-Leu培养基;C,SD/-Trp/-Leu/-Ade/-His+X-α-Gal培养基。

Fig.3 Autoactivation and toxicity testing of pGBKT7-P2 or pGBKT7 A, Mode pattern, pGADT7-RECT interact with pGBKT7-Lam served as negative control, pGADT7-RECT interact with pGBKT7-53 served as positive control, pGBKT7-P2 interact with pGBKT7 served as auto-activation tests; B, SD/-Trp/-Leu medium; C, SD/-Trp/-Leu/-Ade/-His+X-α-Gal medium.

表2 P2-pGBKT7为诱饵筛选到的互作基因信息

Table 2 The information of the genes interaction with P2-pGBKT7 screening by yeast two-hybrid

编号 Number	序列名 Sequence ID	基因名 Gene name	功能 Function
1	TraesCS2A02G407700.1	TapTOM75	Aquaporin PIP-type pTOM75
2	TraesCS5A02G322500.1	TaCB36	Chlorophyll a-b binding protein 36
3	TraesCS6A02G245100.1	TaRS31	Serine/arginine-rich splicing factor RS31-like
4	TraesCS2D02G248400.2	TaOEE1	Oxygen-evolving enhancer protein 1
5	TraesCS5A02G164600.1	TaLIP	Light-induced protein
6	TraesCS3D02G055500.1	Ta14-3-3	14-3-3-like protein C
7	TraesCS2B02G195600.1	TaTIFY 10A	Protein TIFY 10A-like
8	TraesCS3D02G247300.2	TaID31	Protein IQ-DOMAIN 31-like
9	TraesCS7A02G235200.3	TaAGWD	Alpha-glucan water dikinase
10	TraesCS7D02G276300.1	TaCBP21	Chlorophyll a-b binding protein 21
11	TraesCS3D02G232300.1	TaUBA2c	UBP1-associated protein 2C-like
12	TraesCS2B02G126500.1	TaRL19-3	60S ribosomal protein L19-3-like
13	TraesCS3D02G079600.2	TaBAG6	Large proline-rich protein BAG6
14	TraesCS4A02G159700.2	TaCGS1	Cystathionine gamma-synthase 1
15	TraesCS2D02G253900.1	TaPSP	Photosystem II 10 kDa polypeptide
16	TraesCS3A02G309800.1	TaCNR8	Cell number regulator 8-like
17	TraesCS5D02G169600.2	TaRBCSC	Ribulose bisphosphate carboxylase small chain
18	TraesCS4D02G215600.2	TaBOI	BOI-related E3 ubiquitin-protein ligase 3

图4 利用酵母双杂交检测pGBKT7-P2与TaTIFY10A-pGADT7、Ta14-3-3-pGADT7相互作用

Fig.4 Interaction analysis among pGBKT7-P2 and TaTIFY10A-pGADT7 or Ta14-3-3-pGADT7 in the yeast two-hybrid system

图5 WYMV-P2与TaTIFY 10A-pGADT7、Ta14-3-3-pGADT7在酵母互作验证 pGADT7-RECT与pGBKT7-Lam互作为阴性对照;pGADT7-RECT与pGBKT7-53互作为阳性对照;A,SD/-Trp/-Le培养基和SD/-Trp/-Leu/-Ade/-His培养基;B,SD/-Trp/-Leu/-Ade/-His+X-α-Gal培养基。

Fig.5 Interaction of WYMV-P2 with and TaTIFY10A-pGADT7/Ta14-3-3-pGADT7 in a yeast two-hybrid analysis pGADT7-RECT interact with pGBKT7-Lam served as negative control; pGADT7-RECT interact with pGBKT7-53 served as positive control; A, SD/-Trp/-Leu medium and SD/-Trp/-Leu/-Ade/-His medium; B, SD/-Trp/-Leu/-Ade/-His+X-α-Gal medium.

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酵母双杂交筛选与小麦黄花叶病毒P2互作的寄主因子

Screening of host factors interacting with wheat yellow mosaic virus P2 by yeast two-hybrid system

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