甘薯羽状斑驳病毒O株系和RC株系中国分离物全基因组 序列分析及其遗传特征

doi:10.3864/j.issn.0578-1752.2020.11.007

摘要/Abstract

摘要：

【目的】对甘薯羽状斑驳病毒（Sweet potato feathery mottle virus,SPFMV）O株系中国分离物（SPFMV-O-Ch1）和RC株系中国分离物（SPFMV-RC-Ch1）的基因组全序列进行克隆,明确SPFMV-O-Ch1和SPFMV-RC-Ch1的基因组结构特征及其遗传变异情况,为研究甘薯羽状斑驳病毒的致病机制打下基础。【方法】根据GenBank中登录的SPFMV基因组全序列设计2对简并引物和3对特异性引物,利用RT-PCR方法,从感染SPFMV的甘薯叶片中扩增SPFMV O株系和RC株系中国分离物的基因组全长序列,将目的片段分别克隆到pMD19-T载体上,经序列测定、分析和拼接,获得SPFMV-O-Ch1和SPFMV-RC-Ch1的全序列,利用DNAMAN和MEGA7对SPFMV基因组全序列及不同编码区序列进行遗传变异和系统进化树分析,利用RDP软件分析SPFMV基因组重组情况。【结果】经序列测定和拼接,结果表明SPFMV-O-Ch1和SPFMV-RC-Ch1基因组分别包含10 922和10 851 nt,均包含一个开放阅读框,分别由10 557和10 482 nt组成,编码一个多聚蛋白,分别由3 518和3 493个氨基酸残基组成。两个分离物均在P1蛋白内编码一个P1N-PISPO蛋白,在P3蛋白内编码一个P3N-PIPO蛋白。基因组全序列核苷酸一致性分析表明,SPFMV-O-Ch1与SPFMV-RC-Ch1的一致性为87.3%,与GenBank登录的其他分离物基因组全序列一致性为86.0%—95.8%,与Ruk73分离物的一致性最高,为95.8%,与11-1分离物的一致性最低,为86.0%。SPFMV-RC-Ch1与GenBank登录的其他分离物基因组全序列一致性为85.9%—98.7%,与IS90分离物的一致性最高,为98.7%,与Aus1-2B分离物的一致性最低,为85.9%。基于多聚蛋白基因核苷酸序列的遗传进化树分析表明,SPFMV-O-Ch1与Ordinary、10-O和17-O等O株系的分离物形成一个分支,SPFMV-RC-Ch1与S、IS90和CW137等RC株系的分离物形成一个分支。重组分析结果表明,O-Ch1分离物中发现3个重组事件,分别发生在7 731—9 710、135—10 012和4 825—6 948 nt,RC-Ch1没有发现重组事件。【结论】我国的SPFMV-O-Ch1和SPFMV-RC-Ch1分离物的基因组结构与其他分离物相同,O-Ch1与O株系分离物一致性较高,RC-Ch1与RC株系分离物一致性较高,O-Ch1分离物检测到3个重组事件,RC-Ch1未发现重组事件。

关键词: 甘薯羽状斑驳病毒, 株系, 全基因组序列, 遗传变异, 重组分析

Abstract:

【Objective】The objective of this study is to clone the complete nucleotide sequence of Chinese isolate of Sweet potato feathery mottle virus (SPFMV) O and RC strains, elucidate the genomic structural characterization and variation of SPFMV-O-Ch1 and SPFMV-RC-Ch1, and to lay a foundation for the study of pathogenic mechanism of SPFMV. 【Method】According to the SPFMV genome sequences available in GenBank database, 2 pairs of degenerate primers and 3 pairs of specific primers were designed, the whole genome of SPFMV O and RC strains isolated from China was amplified by RT-PCR from sweet potato leaves infection with SPFMV and subsequently cloned into vector pMD19-T and sequenced. The complete genome sequences of SPFMV-O-Ch1 and SPFMV-RC-Ch1 isolates were assembled by using DNAMAN. Genetic variation analyses of complete genomic sequences, polyproteins, and individual protein sequences were performed using DNAMAN. Neighbor-joining phylogenetic tree of SPFMV-O-Ch1 and SPFMV-RC-Ch1 isolates with other isolates was constructed using MEGA7.0 software. Recombination analyses were carried out using RDP software. 【Result】The amplification and sequencing revealed that the complete nucleotide sequence of SPFMV-O-Ch1 and SPFMV-RC-Ch1 isolates was 10 992 nucleotides (nt) and 10 851 nt in length, respectively. The viral genome of SPFMV-O-Ch1 isolate contained a single open reading frame (ORF) of 10 557 nt encoding a polyprotein of 3 518 aa. SPFMV-RC-Ch1 isolate polyprotein consisted of 10 482 nt and encoded 3 493 aa. Two small ORFs, P1N-PISPO and P3N-PIPO were identified in the P1 and P3 proteins of these two isolates. Pairwise comparisons of the complete genome nucleotide sequence showed that O-Ch1 had 87.3% identity with RC-Ch1 isolate and shared 86.0%-95.8% sequence identity with other SPFMV isolates. It was most closely related to the isolate Ruk73 with 95.8% nt identity and lowest nt identity with 11-1 isolate (86.0%). RC-Ch1 and other SPFMV isolates shared 85.9%-98.7% sequence identity at the complete genome nucleotide sequence level. It had the highest nt identity with IS90 isolate (98.7%), and had lowest nt identity with Aus1-2B isolate (85.9%). Phylogenetic tree analysis based on polyprotein gene indicated that SPFMV-O-Ch1 formed a branch with the isolates of O strain containing Ordinary, 10-O and 17-O, and SPFMV-RC-Ch1 formed a branch with the isolates of RC strain containing S, IS90 and CW137. Recombination analysis showed that there were three potential significant recombination events occurred in 7 731- 9 710, 135-10 012 and 4 825-6 948 nt of O-Ch1 isolate genome, respectively. No recombination event was detected in the complete genome of RC-Ch1 isolate. 【Conclusion】The genomic organizations of SPFMV-O-Ch1 and SPFMV-RC-Ch1 isolates were same to other isolates. O-Ch1 isolate was closely related to the isolates of O strain and RC-Ch1 isolate was closely related to those isolates of RC strain. Three recombination events were detected in O-Ch1 isolate, but no recombination event was detected in RC-Ch1 isolate.

Key words: Sweet potato feathery mottle virus (SPFMV), strain, complete nucleotide sequence, genetic variation, recombination analysis

秦艳红,王永江,王爽,乔奇,田雨婷,张德胜,张振臣. 甘薯羽状斑驳病毒O株系和RC株系中国分离物全基因组序列分析及其遗传特征[J]. 中国农业科学, 2020, 53(11): 2207-2218.

QIN YanHong,WANG YongJiang,WANG Shuang,QIAO Qi,TIAN YuTing,ZHANG DeSheng,ZHANG ZhenChen. Complete Nucleotide Sequence Analysis and Genetic Characterization of the Sweet potato feathery mottle virus O and RC Strains Isolated from China[J]. Scientia Agricultura Sinica, 2020, 53(11): 2207-2218.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2020.11.007

https://www.chinaagrisci.com/CN/Y2020/V53/I11/2207

图/表 7

表1

本研究所用分离物的名称、株系、来源和登录号信息"

序号Number	分离物名称Isolate name	株系Strain	地理来源Geographic origin	登录号Accession number
1	RC-Ch1	RC	中国China	KY296451
2	O-Ch1	O	中国China	KY296450
3	CW137	RC	韩国South Korea	KP115608
4	IS90	RC	韩国South Korea	KP115610
5	GJ122	O	韩国South Korea	KP115609
6	S	RC	日本Japan	D86371
7	10-O	O	日本Japan	AB439206
8	Ordinary	O	日本Japan	AB465608
9	17-O	O	日本Japan	AB509454
10	11-8	RC	美国USA	MH782223
11	95-204R	RC	美国USA	MH782224
12	11-1	RC	美国USA	MH778648
13	TFSW-1J	RC	美国USA	MH782227
14	95-2T	O	美国USA	MH782225
15	Aus7D	RC	澳大利亚Australia	MF572046
16	Aus12D	RC	澳大利亚Australia	MF572049
17	Aus8-8CA	RC	澳大利亚Australia	MG656422
18	Aus11D	RC	澳大利亚Australia	MF572048
19	Aus15-21CA	RC	澳大利亚Australia	MG656429
20	Aus11-13CA	RC	澳大利亚Australia	MG656425
21	Aus6-3B	RC	澳大利亚Australia	MG656420
22	Aus9-9B-2	RC	澳大利亚Australia	MG656423
23	Aus10-10D	RC	澳大利亚Australia	MG656424
24	Aus13-18B-1	RC	澳大利亚Australia	MG656427
25	Aus4D	RC	澳大利亚Australia	MF572047
26	Aus3D	RC	澳大利亚Australia	MF572052
27	Aus7-7B	RC	澳大利亚Australia	MG656421
28	Aus2D	RC	澳大利亚Australia	MF572051
29	Aus4-3A	RC	澳大利亚Australia	MG656418
30	Aus9D	RC	澳大利亚Australia	MF572054
31	Aus14-20CA	RC	澳大利亚Australia	MG656428
32	Aus12-16B	RC	澳大利亚Australia	MG656426
33	Aus16-24A	RC	澳大利亚Australia	MG656430
34	Aus3-2BC	RC	澳大利亚Australia	MG656417
35	Aus17-11D	O	澳大利亚Australia	MG656431
36	Aus5-3B	O	澳大利亚Australia	MG656419
37	Aus13B	O	澳大利亚Australia	MF572050
38	Aus1-2B	O	澳大利亚Australia	MG656415
39	Aus2-2BC	O	澳大利亚Australia	MG656416
40	UNB-01	RC	巴西Brazil	MF185715
41	TM33C	O	东帝汶East Timor	MF572055
42	TM64B	O	东帝汶East Timor	MF572053
43	TM66B	O	东帝汶East Timor	MF572056
44	Ruk73	O	乌干达Uganda	KP729265
45	RC-Arg	RC	阿根廷Argentina	KF386014
46	O-Arg	O	阿根廷Argentina	KF386013
47	SRF109a	O	肯尼亚Kenya	MH264535
48	ZA-O	O	南非South Africa	KT069222
49	Piu3	EA	秘鲁Peru	FJ155666
50	AM-MB2		西班牙Spain	KU511268

表1

表2

图1

表3

图2

图3

表4

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扩增片段 Amplified fragment	引物名称 Primer name	引物序列 Primer sequence (5′→3′)	目的片段大小 Expected size (bp)
O-1/RC-1	1-F	AATAACACAACWCAAYACAACAYAASAAAACT	4784/4711
O-1/RC-1	4711-R	GTGATATCATCTAARCTYTGATG	4784/4711
O-2	4689-F	CATCARAGYTTAGATGATATCAC	5692
O-2	10380-R	CATATCGCGCAAGACTCATATC	5692
O-3	SPFMV-O-4160-F	AAGCGACAGAGCAACAGACTTATGTACTA	1124
O-3	SPFMV-O-5284-R	GATATTGCGTTGTAAATTCAACCTCACGTC	1124
RC-2	SPFMV-RC-4260-F	AAGTTTGTGAATATGCTACTAGTCATCA	3814
RC-2	SPFMV-RC-8220-R	TACCTTCTAGGTCTGTGATCAGTTTAGAC	3814
RC-3	SPFMV-RC-7580-F	CCGATAGCTAGCGTCATATGCCATCTCG	3426
RC-3	SPFMV-11000-R	TTGGCTCGATCACGAACCAAAAAGGCT	3426

基因组区域 Genome region	O-Ch1蛋白大小及与48个分离物比对 Protein size of O-Ch1 and comparison with 48 isolates			RC-Ch1蛋白大小及与48个分离物比对 Protein size of RC-Ch1 and comparison with 48 isolates			O-Ch1与RC-Ch1比对 Comparison of O-Ch1 and RC-Ch1
基因组区域 Genome region	蛋白大小（氨基酸） Protein size (aa)	核苷酸一致性 nt identity (%)	氨基酸一致性 aa identity (%)	蛋白大小（氨基酸） Protein size (aa)	核苷酸一致性 nt identity (%)	氨基酸一致性 aa identity (%)	核苷酸（氨基酸）一致性 nt (aa) identity (%)
全长Complete sequence	-	86.0-95.8	-	-	85.9-98.7	-	87.3 (-)
多聚蛋白Polyprotein	3518	87.0-95.8	92.1-97.6	3493	87.0-98.8	92.8-99.3	88.2 (94.0)
P1	689	83.4-97.8	81.2-96.4	664	82.8-99.0	80.6-99.2	85.4 (83.9)
HC	458	83.3-98.2	91.9-98.9	458	83.4-99.0	92.1-100.0	84.0 (93.2)
P3	352	93.2-98.5	95.5-98.6	352	92.8-99.3	94.3-99.4	93.8 (96.0)
6K1	52	92.9-100.0	92.3-100.0	52	91.7-98.7	94.2-100.0	94.2 (98.1)
CI	643	89.0-98.0	97.0-99.7	643	89.7-98.9	97.5-99.8	89.9 (98.0)
6K2	53	91.2-98.7	94.3-100.0	53	89.3-98.7	96.2-100.0	91.8 (98.1)
NIa-VPg	192	78.1-97.7	90.6-100.0	192	83.9-99.3	89.6-100.0	84.4 (97.4)
NIa-Pro	243	83.4-96.3	93.4-98.8	243	79.0-98.6	92.6-99.6	85.9 (96.3)
NIb	521	86.3-97.3	93.1-98.5	521	85.3-98.7	92.9-99.8	89.5 (96.2)
CP	315	76.7-96.6	94.0-99.7	315	79.9-98.9	95.6-99.7	92.5 (96.8)

序号 Number	主要亲本 Major parent	次要亲本 Minor parent	位置 Position (nt)	P值P-Value
序号 Number	主要亲本 Major parent	次要亲本 Minor parent	位置 Position (nt)	RDP	GENECONV	BootScan	MaxChi	Chimaera	SiScan	3Seq
1	Ordinary- AB465608	TM33C- MF572055	7731- 9710	5.583×10^-33	8.317×10^-17	7.407×10^-31	3.758×10^-20	3.466×10^-22	2.430×10^-25	1.095×10^-44
2	EA-Piu3- FJ155666	Aus5-3B- MG656419	135- 10012	-	-	-	8.783×10^-4	7.437×10^-3	1.803×10^-20	1.561×10^-56
3	Aus13B- MF572050	EA-Piu3- FJ155666	4825- 6948	-	-	-	1.104×10^-3	1.071×10^-2	-	1.256×10^-2