中国农业科学 ›› 2020, Vol. 53 ›› Issue (11): 2207-2218.doi: 10.3864/j.issn.0578-1752.2020.11.007
收稿日期:
2019-11-07
接受日期:
2019-11-28
出版日期:
2020-06-01
发布日期:
2020-06-09
通讯作者:
张振臣
作者简介:
秦艳红,E-mail: qinyanhong6040@163.com。
基金资助:
QIN YanHong,WANG YongJiang,WANG Shuang,QIAO Qi,TIAN YuTing,ZHANG DeSheng,ZHANG ZhenChen()
Received:
2019-11-07
Accepted:
2019-11-28
Online:
2020-06-01
Published:
2020-06-09
Contact:
ZhenChen ZHANG
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。
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.
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2
引物序列及目的片段大小"
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 |
4711-R | GTGATATCATCTAARCTYTGATG | ||
O-2 | 4689-F | CATCARAGYTTAGATGATATCAC | 5692 |
10380-R | CATATCGCGCAAGACTCATATC | ||
O-3 | SPFMV-O-4160-F | AAGCGACAGAGCAACAGACTTATGTACTA | 1124 |
SPFMV-O-5284-R | GATATTGCGTTGTAAATTCAACCTCACGTC | ||
RC-2 | SPFMV-RC-4260-F | AAGTTTGTGAATATGCTACTAGTCATCA | 3814 |
SPFMV-RC-8220-R | TACCTTCTAGGTCTGTGATCAGTTTAGAC | ||
RC-3 | SPFMV-RC-7580-F | CCGATAGCTAGCGTCATATGCCATCTCG | 3426 |
SPFMV-11000-R | TTGGCTCGATCACGAACCAAAAAGGCT |
表3
O-Ch1、RC-Ch1与其他分离物核苷酸和氨基酸序列一致性"
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 | ||||
---|---|---|---|---|---|---|---|
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) |
表4
O-Ch1分离物的重组分析"
序号 Number | 主要亲本 Major parent | 次要亲本 Minor parent | 位置 Position (nt) | P值P-Value | ||||||
---|---|---|---|---|---|---|---|---|---|---|
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 |
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