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侵染地黄的瓜类褪绿黄化病毒基因组序列克隆及系统进化分析
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引用本文:秦艳红,文艺,刘玉霞,高素霞,王飞,鲁传涛.侵染地黄的瓜类褪绿黄化病毒基因组序列克隆及系统进化分析.植物保护学报,2023,50(2):359-369
DOI:10.13802/j.cnki.zwbhxb.2023.2021165
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作者单位E-mail
秦艳红 河南省农业科学院植物保护研究所, 河南省农作物病虫害防治重点实验室, 农业农村部华北南部作物有害生物综合治理重点实验室, 郑州 450002  
文艺 河南省农业科学院植物保护研究所, 河南省农作物病虫害防治重点实验室, 农业农村部华北南部作物有害生物综合治理重点实验室, 郑州 450002  
刘玉霞 河南省农业科学院植物保护研究所, 河南省农作物病虫害防治重点实验室, 农业农村部华北南部作物有害生物综合治理重点实验室, 郑州 450002  
高素霞 河南省农业科学院植物保护研究所, 河南省农作物病虫害防治重点实验室, 农业农村部华北南部作物有害生物综合治理重点实验室, 郑州 450002  
王飞 河南省农业科学院植物保护研究所, 河南省农作物病虫害防治重点实验室, 农业农村部华北南部作物有害生物综合治理重点实验室, 郑州 450002 yunfeiren@163.com 
鲁传涛 河南省农业科学院植物保护研究所, 河南省农作物病虫害防治重点实验室, 农业农村部华北南部作物有害生物综合治理重点实验室, 郑州 450002 chuantaolu@qq.com 
中文摘要:为地黄脱毒及防控瓜类褪绿黄化病毒(cucurbit chlorotic yellows virus,CCYV),从河南省温县、武陟县和禹州市3个主产区采集60份具有花叶、黄化和褪绿等典型症状的地黄病叶样品,利用高通量测序技术对混合病样进行测定,利用CCYV-CPF/CPR和CCYV-P22F/P22R引物对60份样品进行反转录聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)检测,并利用9对引物对侵染地黄的CCYV全基因组序列进行扩增和克隆,用DNAMAN 6.0软件对CCYV的分子变异进行分析,利用MEGA X 10.0软件构建系统发育树。结果显示,60份地黄病叶样品中检测出了蚕豆萎蔫病毒2(broad bean wilt virus 2,BBWV2)、地黄花叶病毒(Rehmannia mosaic virus,ReMV)、油菜花叶病毒(youcai mosaic virus,YoMV)和CCYV等病毒;在60份样品中,有22份样品中检测到CCYV,检出率为36.7%;21个地黄分离物之间的CP基因核苷酸和氨基酸序列一致率分别为99.3%~100.0%和98.4%~100.0%,与其他分离物之间的CP基因核苷酸和氨基酸序列一致率分别为94.6%~100.0%和96.8%~100.0%,17个地黄分离物之间的P22基因核苷酸和氨基酸序列一致率分别为98.4%~100.0%和96.3%~100.0%,与其他分离物之间的P22基因核苷酸和氨基酸序列一致率分别为96.1%~100.0%和94.1%~100.0%;基于CPP22基因核苷酸序列的系统发育树结果显示,CCYV不同分离物形成2个分支。CCYV地黄分离物的基因组包含RNA1和RNA2两个组分,全长分别为8 607 nt和8 041 nt,共编码12个蛋白;CCYV地黄分离物基因组高度保守,与其他分离物基因组RNA1和RNA2的核苷酸一致率均为99.7%~100.0%,基于基因组的系统发育树结果显示,CCYV地黄分离物的核苷酸序列与其他寄主上CCYV分离物的核苷酸序列聚为一个分支,而与毛形病毒属Crinivirus的其他病毒分开。
中文关键词:地黄  瓜类褪绿黄化病毒  全基因组序列  系统进化分析  外壳蛋白
 
Complete nucleotide sequence and phylogenetic analysis of the cucurbit chlorotic yellows virus infecting Chinese foxglove Rehmannia glutinosa
Author NameAffiliationE-mail
Qin Yanhong Key Laboratory in Southern part of North China for Ministry of Agriculture and Rural Affairs, Henan Key Laboratory of Crop Pest Control Integrated Pest Management, Institute of Plant Protection, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan Province, China  
Wen Yi Key Laboratory in Southern part of North China for Ministry of Agriculture and Rural Affairs, Henan Key Laboratory of Crop Pest Control Integrated Pest Management, Institute of Plant Protection, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan Province, China  
Liu Yuxia Key Laboratory in Southern part of North China for Ministry of Agriculture and Rural Affairs, Henan Key Laboratory of Crop Pest Control Integrated Pest Management, Institute of Plant Protection, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan Province, China  
Gao Suxia Key Laboratory in Southern part of North China for Ministry of Agriculture and Rural Affairs, Henan Key Laboratory of Crop Pest Control Integrated Pest Management, Institute of Plant Protection, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan Province, China  
Wang Fei Key Laboratory in Southern part of North China for Ministry of Agriculture and Rural Affairs, Henan Key Laboratory of Crop Pest Control Integrated Pest Management, Institute of Plant Protection, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan Province, China yunfeiren@163.com 
Lu Chuantao Key Laboratory in Southern part of North China for Ministry of Agriculture and Rural Affairs, Henan Key Laboratory of Crop Pest Control Integrated Pest Management, Institute of Plant Protection, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan Province, China chuantaolu@qq.com 
Abstract:To develop virus-free Chinese foxglove Rehmannia glutinosa and control the cucurbit chlorotic yellows virus, 60 leaf samples with typical symptoms of mosaic, yellowing and chlorotic were collected from the main production region including Wenxian, Wuzhi and Yuzhou City of Henan Province. Mixed samples were further analyzed with high-throughput sequencing (HTS). Sixty samples were detected by using RT-PCR with primer pairs CCYV-CPF/CPR and CCYV-P22F/P22R. The genomic nucleotide sequences of CCYV infecting R. glutinosa were amplified and cloned by using nine primer pairs. Molecular variability was analyzed by using DNAMAN 6.0 software and phylogenetic trees were constructed using MEGA X 10.0. The result of HTS showed that BBWV2, ReMV, YoMV and CCYV were detected from the 60 leaf samples of R. glutinosa from Henan Province. CCYV was detected from 22 of the 60 samples with a detection rate of 36.7%. The nucleotide and amino acid identities of CP gene were respectively 99.3%-100.0% and 98.4%-100.0% in 21 CCYV-Rg isolates, 94.6%-100.0% and 96.8%-100.0% between CCYV-Rg isolates and other isolates. The nucleotide and amino acid identities of P22 gene were respectively 98.4%-100.0% and 96.3%-100.0% in 17 CCYV-Rg isolates, 96.1%-100.0% and 94.1%-100.0% between CCYV-Rg isolates and other isolates. Phylogenetic analysis based on nucleotide sequences of CP and P22 genes demonstrated that these isolates were grouped into two clades. The genome of CCYV R. glutinosa isolate (CCYV-Rg) consist of two segments and the length of RNA1 and RNA2 segments are 8 607 and 8 041 nt, respectively. The genome encode 12 proteins and their nucleotide sequences are highly conserved. The CCYV-Rg isolate had 99.7%-100.0% (RNA1 and RNA2 segments) nucleotide identities with other isolates deposited in GenBank. Phylogenetic analysis based on genome showed that the nucleotide sequences of CCYV-Rg isolate was grouped into one cluster together with CCYV isolates from other hosts but not with other virus species in crinivirus.
keywords:Rehmannia glutinosa  cucurbit chlorotic yellows virus  complete genomic sequence  phylogenetic analysis  coat protein
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