利用Y2H筛选西方烟中与苹果茎痘病毒CP互作的蛋白

doi:10.16420/j.issn.0513-353x.2022-0507

摘要/Abstract

摘要：

苹果茎痘病毒（apple stem pitting virus，ASPV）是苹果和梨树上普遍发生的潜隐病毒之一，西方烟（Nicotiana occidentalis）是其重要的草本寄主之一。在本研究中，构建了酵母双杂交诱饵载体pGBKT7-CP（CP-BD），并从西方烟cDNA文库中筛选与ASPV CP互作的蛋白。经通过酵母双杂交回转验证和NCBI BLAST比对，获得30种可能与ASPV CP互作的西方烟蛋白质，为研究ASPV侵染致病的分子机制及CP在侵染过程中的功能打下基础。

关键词: 酵母双杂交, 外壳蛋白, 苹果茎痘病毒, 蛋白互作

Abstract:

Apple stem pitting virus（ASPV）is a latent virus on apple and pear trees worldwide. Nicotiana occidentalis is an important herbaceous host plant for it. In this study，ASPV coat protein（CP）was used as bait to screen a cDNA library of N. occidentalis. Thirty proteins that interact with ASPV CP were obtained by yeast two-hybrid co-transformation and NCBI BLAST. This study could be useful for understanding the molecular mechanism of ASPV infection in N. occidentalis and the function of CP in the process of virus infection.

Key words: yeast two-hybrid, coat protein, apple stem pitting virus, protein interaction

丁磊, 孙平平, 张磊, 李正男. 利用Y2H筛选西方烟中与苹果茎痘病毒CP互作的蛋白[J]. 园艺学报, 2023, 50(11): 2509-2515.

DING Lei, SUN Pingping, ZHANG Lei, LI Zhengnan. Screening Proteins of Nicotiana occidentalis That Interact with Coat Protein of Apple Stem Pitting Virus Using Yeast Two-hybrid System[J]. Acta Horticulturae Sinica, 2023, 50(11): 2509-2515.

图/表 5

参考文献 25

[1]	Adams M J, Antoniw J F, Bar-Joseph M, Brunt A A, Candresse T, Foster G D, Martelli G P, Milne R G, Zavriev S K, Fauquet C M. 2004. The new plant virus family Flexiviridae and assessment of molecular criteria for species demarcation. Archives of Virology, 149:1045-1060. doi: 10.1007/s00705-004-0304-0 pmid: 15098118
[2]	Adams M J, Candresse T, Hammond J, Kreuze J F, Martelli G P, Namba S, Pearson M N, Ryu K H, Saldarelli P, Yoshikawa N. 2012. Betaflexiviridae//King A M Q,AdamsMJ,Carstens E B,lefkowitz E J. Virus taxonomy:classification and nomenclature of viruses:Ninth Report of the International Committee on Taxonomy of Viruses. San Diego: Elsevier:920-941.
[3]	Arntjen A. 2009. Construction of an infectious full-length cDNA clone and investigations on variability of apple stem pitting virus. Braunschweig:Mitteilungen Aus Dem Julius Kühn-Institut.
[4]	Dhir S, Tomar M, Thakur P D, Ram R, Zaidi A A. 2010. Molecular evidence for apple stem pitting virus infection in India. Plant Pathology, 59 (2):393-393. doi: 10.1111/j.1365-3059.2009.02164.x
[5]	Edelmann M J, Kessler B M. 2008. Ubiquitin and ubiquitin-like specific proteases targeted by infectious pathogens: emerging patterns and molecular principles. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1782 (12):809-816. doi: 10.1016/j.bbadis.2008.08.010 URL
[6]	Feng L, Ning H, Ling W, Hui L, Sun T, Waqar A, Khushi M, Guo J, Xu L, Gao S, Que Y, Su Y, Liu F, Huang N, Wang L, Ling H, Sun T, Ahmad W, Muhammad K, Guo J, Xu L, Gao S, Que Y. 2018. A novel l-ascorbate peroxidase 6 gene, scapx6, plays an important role in the regulation of response to biotic and abiotic stresses in sugarcane. Frontiers in Plant Science,Doi:10.3389/fpls.2017.02262.
[7]	Fields S, Song O. 1989. A novel genetic system to detect protein:protein interactions. Nature, 340:245-246. doi: 10.1038/340245a0
[8]	Gao De-hang, He Cheng-yong, Xing Fei, Hou Wan-ying, Li Shi-fang, Zhan Bin-hui, Wang Hong-qing. 2021. Identification and sequence analysis of apple stem pitting virus from hawthorn tree. Acta Phytopathologica Sinica, 51 (4):515-524. (in Chinese)
	高德航, 何成勇, 邢飞, 侯婉莹, 李世访, 战斌慧, 王红清. 2021. 苹果茎痘病毒在山楂中的首次鉴定与序列分析. 植物病理学报, 51 (4):515-524.
[9]	Ivanov K I, Mäkinen K. 2012. Coat proteins, host factors and plant viral replication. Current Opinion in Virology, 2:712-718. doi: 10.1016/j.coviro.2012.10.001 pmid: 23122854
[10]	Jelkmann W. 1994. Nucleotide sequences of Apple stem pitting virus and of the coat protein gene of a similar virus from pear associated with vein yellows disease and their relationship with potex- and carlaviruses. Journal of General Virology, 75 (7):1535-1542. doi: 10.1099/0022-1317-75-7-1535 URL
[11]	Jones R. 2009. Plant virus emergence and evolution:origins,new encounter scenarios,factors driving emergence,effects of changing world conditions,and prospects for control. Virus Research, 141:113-130. doi: 10.1016/j.virusres.2008.07.028 URL
[12]	Klerks M M, Leone G, Lindner J L, Schoen C D, van den Heuvel J F. 2001. Rapid and sensitive detection of Apple stem pitting virus in apple trees through RNA amplification and probing with fluorescent molecular beacons. Phytopathology, 91:1085-1091. doi: 10.1094/PHYTO.2001.91.11.1085 pmid: 18943445
[13]	Koßmann J, Sonnewald U, Willmitzer L. 1994. Reduction of the chloroplastic fructose-1,6-bisphosphatase in transgenic potato plants impairs photosynthesis and plant growth. Plant Journal, 6 (5):637-650. doi: 10.1046/j.1365-313X.1994.6050637.x URL
[14]	Kushwaha N K, Mansi, Sahu P P, Prasad M, Chakrabroty S. 2019. Chilli leaf curl virus infection downregulates the expression of the genes encoding chloroplast proteins and stress-related proteins. Physiology and Molecular Biology of Plants, 25 (23):1185-1196. doi: 10.1007/s12298-019-00693-1
[15]	Li Li-li, Dong Ya-feng, Zhang Zun-ping, Zhang Zhi-hong, Fan Xu-dong, Pei Guang-qian. 2010. RT-PCR detection and molecular variability of Apple stem pitting virus. Acta Horticulturae Sinica, 37 (1):9-16. (in Chinese)
	李丽丽, 董雅凤, 张尊平, 张志宏, 范旭东, 裴光前. 2010. 苹果茎痘病毒RT-PCR检测及分子变异分析. 园艺学报, 37 (1):9-16.
[16]	Ma X, Hong N, Moffett P, Zhou Y, Wang G. 2019. Functional analysis of apple stem pitting virus coat protein variants. Virology Journal,Doi:10.1186/s12985-019-1126-8.
[17]	Martelli G P, Jelkmann W. 1998. Foveavirus, a new plant virus genus. Archives of Virology, 143 (6):1245-1249. pmid: 9687881
[18]	Mathioudakis M M, Maliogka V I, Dovas C I, Vasilakakis M, Katis N I. 2006. First record of the apple stem pitting virus(ASPV)in quince in Greece. Journal of Plant Pathology, 88:225.
[19]	Morán M, Quilis C C, Olmos A, Ruiz-García A B. 2020. Loquat(Eriobotrya japonica)is a new natural host of apple stem pitting virus. Plants, 9 (11):Doi:10.3390/plants9111560.
[20]	Sun Pingping, Ju Mingxiu, Ma Qiang, Li Zhengnan. 2019. Genome and biology of an isolate of Apple stem pitting virus from Inner Mongolia,China. Acta Horticulturae Sinica, 46 (10):2037-2046. (in Chinese) doi: 10.16420/j.issn.0513-353x.2018-1074 URL
	孙平平, 鞠明岫, 马强, 李正男. 2019. 苹果茎痘病毒内蒙古分离物基因组序列和生物学特征研究. 园艺学报, 46 (10):2037-2046. doi: 10.16420/j.issn.0513-353x.2018-1074 URL
[21]	Wang X, Ren Y, Gong C, Chen Y, Ge X, Kong J, Sun W, Du X. 2021. 40S ribosomal protein S18 is a novel maternal peptidoglycan-binding protein that protects embryos of zebrafish from bacterial infections. Developmental & Comparative Immunology,Doi:10.1016/ j.dci.2021.104212.
[22]	Weber P H, Bujarski J J. 2015. Multiple functions of capsid proteins in (+) stranded RNA viruses during plant-virus interactions. Virus Research, 196:140-149. doi: 10.1016/j.virusres.2014.11.014 pmid: 25445337
[23]	Wu Z B, Ku H M, Su C C, Chen I Z, Jan F J. 2010. Molecular and biological characterization of an isolate of Apple stem pitting virus causing pear vein yellows disease in Taiwan. Journal of Plant Pathology, 92:721-728.
[24]	Ye Chaoyan, Zhang Yang, Wang Maojun, Wu Jun, Chen Weiliang. 2015. Screening and fungistatic activity analysis of acetolactate synthase inhibitors for Fusarium graminearum. Journal of Zhejiang University(Science Edition), 42 (2):166-171. (in Chinese)
	叶朝燕, 张阳, 汪冒君, 吴军, 陈卫良. 2015. 镰刀菌乙酰乳酸合成酶抑制剂的筛选及抗菌性分析. 浙江大学学报(理学版), 42 (2):166-171.
[25]	Zhang L. 2017. Construction of infectious full-length cDNA clones of apple viruses and plant viral vector development. Heidelberg:Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg.

序号 No.	候选蛋白 Putative protein	物种 Species	GenBank登录号 GenBank accession No.	总分值 Total score	覆盖率/% Query cover	E值 E-value	序列一致性/% Sequence identity
1	40S ribosomal protein	野生烟草Nicotiana attenuata	XM_019380942.1	314	74	0	95.40
2	Putative dual specificity protein	绒毛状烟草N. tomentosiformis	XM_009598741.3	1 831	94	0	95.79
3	Putative late blight resistance protein	美花烟草N. sylvestris	XM_009777746.1	1 419	82	0	94.17
4	Fructose-1,6-bisphosphatase	普通烟草N. tabacum	XM_016645668.1	1 962	93	0	97.16
5	Transmembrane protein	普通烟草N. tabacum	XM_016608120.1	1 541	94	0	93.88
6	DEAD-box ATP-dependent RNA helicase	普通烟草N. tabacum	XM_016614675.1	1 652	86	0	95.38
7	Acetolactate synthase	野生烟草N. attenuata	XM_019368947.1	1 552	92	0	97.99
8	Chlorophyll a-b binding protein	美花烟草N. sylvestris	XM_009797464.1	1 611	84	0	97.66
9	Lysine--tRNA ligase	野生烟草N. attenuata	XM_019388504.1	1 877	95	0	96.12
10	L-ascorbate peroxidase 2	绒毛状烟草N. tomentosiformis	XM_009608760.3	1 644	89	0	95.63
11	Photosystem I reaction center subunit III	普通烟草N. tabacum	XM_016584537.1	1 321	86	0	97.07
12	Ribulose bisphosphate carboxylase small chain	野生烟草N. attenuata	XM_019393743.1	1 206	86	0	94.63
13	60S ribosomal protein	普通烟草N. tabacum	XM_016621890.1	1 184	79	0	91.28
14	Probable ubiquitin-like-specific protease	野生烟草N. attenuata	XM_019370696.1	507	55	7e-139	95.33
15	Phototropin	普通烟草N. tabacum	XM_016615622.1	1 075	61	0	93.62
16	Ras-related protein	绒毛状烟草N. tomentosiformis	XM_009613476.3	1 544	90	0	94.64
17	Persulfide dioxygenase	野生烟草N. attenuata	XM_019394068.1	424	76	1e-113	78.43
18	Fructose-bisphosphate aldolase	野生烟草N. attenuata	XM_019380711.1	1 537	93	0	98.07
19	Peroxidase	野生烟草N. attenuata	XM_019378242.1	1 038	87	0	90.70
20	Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)	野生烟草N. attenuata	XM_019383154.1	1 557	93	0	98.53
21	Riboflavin synthase	普通烟草N. tabacum	XM_016652029.1	676	86	0	93.42
22	Cytosolic Fe-S cluster assembly factor	普通烟草N. tabacum	XM_016639050.1	1 526	94	0	95.68
23	Probable ribose-5-phosphate isomerase	普通烟草N. tabacum	XM_016602671.1	1 517	90	0	94.96
24	Proline synthase co-transcribed bacterial homolog protein	野生烟草N. attenuata	XM_019396117.1	715	86	0	95.14
25	L-ascorbate peroxidase 1	普通烟草N. tabacum	XR_001646090.1	780	59	0	96.41
26	Phosphoglycerate kinase	绒毛状烟草N. tomentosiformis	XM_009623760.3	817	71	0	96.73
27	DNA-directed RNA polymerase	野生烟草N. attenuata	XM_019380496.1	1 565	93	0	97.00
28	WD repeat-containing protein	美花烟草N. sylvestris	XM_009763517.1	1 291	79	0	97.13
29	Chloroplast PsbP2 precursor	本氏烟草N. benthamiana	JF897608.1	1 469	93	0	97.15
30	mRNA for aquaporin	普通烟草N. tabacum	AJ001416.1	1 718	92	0	94.29

序号 No.	候选蛋白 Putative protein	物种 Species	GenBank登录号 GenBank accession No.	总分值 Total score	覆盖率/% Query cover	E值 E-value	序列一致性/% Sequence identity
1	40S ribosomal protein	野生烟草Nicotiana attenuata	XM_019380942.1	314	74	0	95.40
2	Putative dual specificity protein	绒毛状烟草N. tomentosiformis	XM_009598741.3	1 831	94	0	95.79
3	Putative late blight resistance protein	美花烟草N. sylvestris	XM_009777746.1	1 419	82	0	94.17
4	Fructose-1,6-bisphosphatase	普通烟草N. tabacum	XM_016645668.1	1 962	93	0	97.16
5	Transmembrane protein	普通烟草N. tabacum	XM_016608120.1	1 541	94	0	93.88
6	DEAD-box ATP-dependent RNA helicase	普通烟草N. tabacum	XM_016614675.1	1 652	86	0	95.38
7	Acetolactate synthase	野生烟草N. attenuata	XM_019368947.1	1 552	92	0	97.99
8	Chlorophyll a-b binding protein	美花烟草N. sylvestris	XM_009797464.1	1 611	84	0	97.66
9	Lysine--tRNA ligase	野生烟草N. attenuata	XM_019388504.1	1 877	95	0	96.12
10	L-ascorbate peroxidase 2	绒毛状烟草N. tomentosiformis	XM_009608760.3	1 644	89	0	95.63
11	Photosystem I reaction center subunit III	普通烟草N. tabacum	XM_016584537.1	1 321	86	0	97.07
12	Ribulose bisphosphate carboxylase small chain	野生烟草N. attenuata	XM_019393743.1	1 206	86	0	94.63
13	60S ribosomal protein	普通烟草N. tabacum	XM_016621890.1	1 184	79	0	91.28
14	Probable ubiquitin-like-specific protease	野生烟草N. attenuata	XM_019370696.1	507	55	7e-139	95.33
15	Phototropin	普通烟草N. tabacum	XM_016615622.1	1 075	61	0	93.62
16	Ras-related protein	绒毛状烟草N. tomentosiformis	XM_009613476.3	1 544	90	0	94.64
17	Persulfide dioxygenase	野生烟草N. attenuata	XM_019394068.1	424	76	1e-113	78.43
18	Fructose-bisphosphate aldolase	野生烟草N. attenuata	XM_019380711.1	1 537	93	0	98.07
19	Peroxidase	野生烟草N. attenuata	XM_019378242.1	1 038	87	0	90.70
20	Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)	野生烟草N. attenuata	XM_019383154.1	1 557	93	0	98.53
21	Riboflavin synthase	普通烟草N. tabacum	XM_016652029.1	676	86	0	93.42
22	Cytosolic Fe-S cluster assembly factor	普通烟草N. tabacum	XM_016639050.1	1 526	94	0	95.68
23	Probable ribose-5-phosphate isomerase	普通烟草N. tabacum	XM_016602671.1	1 517	90	0	94.96
24	Proline synthase co-transcribed bacterial homolog protein	野生烟草N. attenuata	XM_019396117.1	715	86	0	95.14
25	L-ascorbate peroxidase 1	普通烟草N. tabacum	XR_001646090.1	780	59	0	96.41
26	Phosphoglycerate kinase	绒毛状烟草N. tomentosiformis	XM_009623760.3	817	71	0	96.73
27	DNA-directed RNA polymerase	野生烟草N. attenuata	XM_019380496.1	1 565	93	0	97.00
28	WD repeat-containing protein	美花烟草N. sylvestris	XM_009763517.1	1 291	79	0	97.13
29	Chloroplast PsbP2 precursor	本氏烟草N. benthamiana	JF897608.1	1 469	93	0	97.15
30	mRNA for aquaporin	普通烟草N. tabacum	AJ001416.1	1 718	92	0	94.29