中国葡萄卷叶相关病毒7检测及基因变异分析

doi:10.16420/j.issn.0513-353x.2020-0241

摘要/Abstract

摘要：

葡萄卷叶相关病毒7（grapevine leafroll-associated virus 7,GLRaV-7）是与葡萄卷叶病相关的重要病原物之一。为明确中国GLRaV-7分离物的基因序列变异,采用RT-PCR方法对采自中国15个省（市）、自治区的188份葡萄样品进行检测,检出率为24.5%（44/188）。对来源于42个GLRaV-7分离物的外壳蛋白（coat protein,CP）和15个分离物的热休克蛋白70（heat-shock protein 70,HSP70）基因进行克隆、测序。序列分析结果显示,来源于国内外GLRaV-7分离物的CP和HSP70基因的核苷酸和氨基酸序列同源性分别为89.12% ~ 100.00%和88.94% ~ 100.00%、85.33% ~ 100.00%和88.97% ~ 100.00%。基于CP和HSP70基因序列构建的进化树,分别将GLRaV-7分离物划分为6个和5个明显区分的组群。Gp3、Gp4和Gp5仅由中国分离物组成。本研究中初次分析国内外GLRaV-7分离物的CP和HSP70基因序列变异,可为进一步开展GLRaV-7分子检测的研发及不同变种的致病性研究提供依据。

Abstract:

Grapevine leafroll disease（GLD）is a virus disease widely distributed in the world and causes great damage on grapevine. Grapevine leafroll-associated virus 7（GLRaV-7） is one of its pathogens. To investigate the genetic variation of GLRaV-7 isolates,42 positive samples were screened out by reverse transcription-PCR（RT-PCR）from 188 grapevine samples which were collected from 15 Chinese provinces and regions. The detection rate of GLRaV-7 was 24.5%. The coat protein（CP）genes of 42 isolates and heat-shock protein 70（HSP70）genes of 15 isolates were sequenced and analyzed respectively,and the results showed identities ranging 89.12%-100.00% and 88.94%-100.00%,85.33%-100.00% and 88.97%-100.00% at the nucleotide and amino acid sequences respectively. Phylogenetic analysis of CP and HSP70 gene sequences showed that all of GLRaV-7 isolates can be divided into six and five groups,respectively. Gp3,Gp4 and Gp5 only included GLRaV-7 Chinese isolates obtained in this study. This is the first report on the genetic variation of CP and HSP70 genes of GLRaV-7 isolates in China,and this study will be helpful for pathogenicity studies and development of reliable diagnostic methods.

Key words: grapevine, grapevine leafroll-associated virus 7, RT-PCR detection, CP gene, HSP70 gene, phylogenetic analysis

中图分类号:

S663.1

范旭东, 董雅凤, 张尊平, 任芳, 胡国君, 张梦妍, 李晨. 中国葡萄卷叶相关病毒7检测及基因变异分析[J]. 园艺学报, 2021, 48(2): 347-354.

FAN Xudong, DONG Yafeng, ZHANG Zunping, REN Fang, HU Guojun, ZHANG Mengyan, LI Chen. Detection and Sequence Analyses of Grapevine Leafroll-associated Virus 7 Isolates in China[J]. Acta Horticulturae Sinica, 2021, 48(2): 347-354.

图/表 4

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扩增区域 Genomic region	引物 Primer	序列（5′-3′） Sequences	产物大小/bp Size
CP	L7CP	F：GGRAATAGGWCRCAAAGTGAA;R：CTCYTRTTYAATGTRCGTCC	844
CP	L7CPn	F：GAATTACACCGAGAAGTCTG;R：TGTTGCCGCAAGTTGAGAAGT	709
HSP70	L7HSP	F：GGGTTTGATTTTGGTACTACTTTTA;R：ACCACCTCCTTGTAAGCT	450
HSP70	L7HSPn	F：GAATTAAATATGAACGGATCAC;R：CCTTCAAGAGAGATTTCTGATA	409

扩增区域 Genomic region	引物 Primer	序列（5′-3′） Sequences	产物大小/bp Size
CP	L7CP	F：GGRAATAGGWCRCAAAGTGAA;R：CTCYTRTTYAATGTRCGTCC	844
CP	L7CPn	F：GAATTACACCGAGAAGTCTG;R：TGTTGCCGCAAGTTGAGAAGT	709
HSP70	L7HSP	F：GGGTTTGATTTTGGTACTACTTTTA;R：ACCACCTCCTTGTAAGCT	450
HSP70	L7HSPn	F：GAATTAAATATGAACGGATCAC;R：CCTTCAAGAGAGATTTCTGATA	409

来源 Location	样品 Sample	分离物编号 Isolate ID	扩增区域 Genomic region	GenBank登录号 GenBank accession number
北京Beijing	品丽珠Cabernet Franc	BJ-CaF	CP	KT306553
河北Heibei	巨峰Kyoho	HB- Kyoho1	CP	KT306560
		HB- Kyoho2	CP	KT306561
	马瑟兰Marselen	HB- MSL	CP	KT306562
		HB-MSL2	CP	KT306563
辽宁Liaoning	阿尔曼玫瑰Aerman Meigui	LN-AEMMG	CP,HSP70	KT306566,KT306603
	贝达Beda	LN-BD1	CP	KT306567
		LN-BD2	CP	KT306568
		LN-BD3	CP	KT306569
	白塞涅Baisenie	LN-BSN	CP	KT306570
	白瓦沙Baiwashaka	LN-BWSK	CP,HSP70	KT306571,KT306604
	瑰宝Guibao	LN-GB1	CP	KT306572
		LN-GB2	CP	KT306573
	红丹Hongdan	LN-HD	CP	KT306574
	琥珀Hupo	LN-HP	CP	KT306575
	李子香Lizixiang	LN-LZX	CP	KT306576
	牛心Niuxin	LN-NX	CP	KT306577
	巧保2号Qiaobao 2	LN-QB2	CP,HSP70	KT306578,KT306605
	秋蜜Qiumi	LN-QM	CP	KT306579
	秋无核Qiuwuhe	LN-QWH1	CP,HSP70	KT306580,KT306606
		LN-QWH2	CP,HSP70	KT306581,KT306607
	桑多哈尼Santos Hani	LN-SDHN1	CP,HSP70	KT306582,KT306608
来源 Location	样品 Sample	分离物编号 Isolate ID	扩增区域 Genomic region	GenBank登录号 GenBank accession number
		LN-SDHN2	CP,HSP70	KT306583 ~ 84,KT306609
	乌兹别克玫瑰Uzbekistan Rose	LN-WZBKMG	CP	KT306585
	紫峰Zifeng	LN-ZF	CP	KT306586 ~ 88
	着色香Zhaosexiang	LN-ZSX	CP,HSP70	KT306589,KT306611 ~ 12
	102459	LN-102495	CP,HSP70	KT306590,KT306613
	228	LN-228	CP,HSP70	KT306591,KT306610
山东Shandong	北冰红Beibinghong	SD-BBH	CP	KT306592
	贵人香Italian Riesling	SD-GRX5	CP	KT306593
		SD-GRX1	CP	KT306594
		SD-GRX2	CP	KT306595
		SD-GRX3	CP	KT306596
		SD-GRX4	CP	KT306597
天津Tianjin	赤霞珠Caberbet Sauvignon	TJ-CaS	CP	KT306565
	贵人香Italian Riesling	TJ-GRX	CP	KT306564
新疆Xinjiang	克瑞森无核Crimson Seedless	XJ-KRS	CP,HSP70	KT306559,KT306602
	无核白Thompson Seedless	XJ-WHB1	CP,HSP70	KT306558,KT306601
		XJ-WHB2	CP	KT306554
		XJ-WHB3	CP,HSP70	KT306555,KT306598
		XJ-WHB4	CP,HSP70	KT306556,KT306599
		XJ-WHB5	CP,HSP70	KT306557,KT306600

来源 Location	样品 Sample	分离物编号 Isolate ID	扩增区域 Genomic region	GenBank登录号 GenBank accession number
北京Beijing	品丽珠Cabernet Franc	BJ-CaF	CP	KT306553
河北Heibei	巨峰Kyoho	HB- Kyoho1	CP	KT306560
		HB- Kyoho2	CP	KT306561
	马瑟兰Marselen	HB- MSL	CP	KT306562
		HB-MSL2	CP	KT306563
辽宁Liaoning	阿尔曼玫瑰Aerman Meigui	LN-AEMMG	CP,HSP70	KT306566,KT306603
	贝达Beda	LN-BD1	CP	KT306567
		LN-BD2	CP	KT306568
		LN-BD3	CP	KT306569
	白塞涅Baisenie	LN-BSN	CP	KT306570
	白瓦沙Baiwashaka	LN-BWSK	CP,HSP70	KT306571,KT306604
	瑰宝Guibao	LN-GB1	CP	KT306572
		LN-GB2	CP	KT306573
	红丹Hongdan	LN-HD	CP	KT306574
	琥珀Hupo	LN-HP	CP	KT306575
	李子香Lizixiang	LN-LZX	CP	KT306576
	牛心Niuxin	LN-NX	CP	KT306577
	巧保2号Qiaobao 2	LN-QB2	CP,HSP70	KT306578,KT306605
	秋蜜Qiumi	LN-QM	CP	KT306579
	秋无核Qiuwuhe	LN-QWH1	CP,HSP70	KT306580,KT306606
		LN-QWH2	CP,HSP70	KT306581,KT306607
	桑多哈尼Santos Hani	LN-SDHN1	CP,HSP70	KT306582,KT306608
来源 Location	样品 Sample	分离物编号 Isolate ID	扩增区域 Genomic region	GenBank登录号 GenBank accession number
		LN-SDHN2	CP,HSP70	KT306583 ~ 84,KT306609
	乌兹别克玫瑰Uzbekistan Rose	LN-WZBKMG	CP	KT306585
	紫峰Zifeng	LN-ZF	CP	KT306586 ~ 88
	着色香Zhaosexiang	LN-ZSX	CP,HSP70	KT306589,KT306611 ~ 12
	102459	LN-102495	CP,HSP70	KT306590,KT306613
	228	LN-228	CP,HSP70	KT306591,KT306610
山东Shandong	北冰红Beibinghong	SD-BBH	CP	KT306592
	贵人香Italian Riesling	SD-GRX5	CP	KT306593
		SD-GRX1	CP	KT306594
		SD-GRX2	CP	KT306595
		SD-GRX3	CP	KT306596
		SD-GRX4	CP	KT306597
天津Tianjin	赤霞珠Caberbet Sauvignon	TJ-CaS	CP	KT306565
	贵人香Italian Riesling	TJ-GRX	CP	KT306564
新疆Xinjiang	克瑞森无核Crimson Seedless	XJ-KRS	CP,HSP70	KT306559,KT306602
	无核白Thompson Seedless	XJ-WHB1	CP,HSP70	KT306558,KT306601
		XJ-WHB2	CP	KT306554
		XJ-WHB3	CP,HSP70	KT306555,KT306598
		XJ-WHB4	CP,HSP70	KT306556,KT306599
		XJ-WHB5	CP,HSP70	KT306557,KT306600

重组序列 Recombinant Sequences	亲本序列（major × min） Parental sequences	重组区域 Breakpoints（begin-end）	重组分析方法 RDP-implemented method	P值 P value
LN-ZF clone 9	LN-228 × LN-QWH1	59 ~ 415	GeneConv	4.885 × 10^-03
			Bootscan	3.031 × 10^-07
			MaxChi	8.660 × 10^-08
			CHIMAERA	2.498 × 10^-07
			SiScan	1.422 × 10^-07
			3SEQ	1.471 × 10^-14