我国甘薯E病毒全基因组序列特征及荧光定量检测技术的建立

doi:10.3864/j.issn.0578-1752.2023.20.007

摘要/Abstract

摘要：

【目的】甘薯E病毒（sweet potato virus E，SPVE）是甘薯上新发现的一种病毒。本研究对我国甘薯E病毒江苏徐州分离物（SPVE-XZ）的全基因组序列进行测定，分析该病毒基因组序列特征，并建立针对SPVE的荧光定量（quantitative PCR，qPCR）检测技术，为监测SPVE发生分布、检测种薯种苗中SPVE带毒情况并及时防控该病毒提供理论依据和技术支持。【方法】采用small RNA深度测序，结合RT-PCR和RACE技术，获得SPVE-XZ的全基因组序列，利用MegAlign、MEGA11等软件对获得的全基因组序列进行序列比对、系统发育关系分析。设计SPVE荧光定量检测引物，并通过对退火温度及引物浓度的优化，建立针对SPVE的qPCR检测方法，测定其特异性和灵敏度，应用于江苏省和山东省田间甘薯样品的检测。【结果】除ploy(A)外，所获得的SPVE-XZ全基因组序列全长为10 919 nt，包含1个长为10 560 nt的开放阅读框（open reading frame，ORF），编码3 519 aa的多聚蛋白。5′非翻译区（5′ untranslated region，5′UTR）和3′非翻译区（3′UTR）分别为129和230 nt。在P1和P3蛋白内分别包含由移码产生的PISPO和PIPO蛋白。全基因组序列分析表明，SPVE-XZ与韩国SPVE GS分离物（SPVE-GS）全基因组核苷酸一致率最高，为98.6%，多聚蛋白氨基酸一致率为98.5%。利用邻接法基于cp基因核苷酸序列和多聚蛋白氨基酸序列构建的系统进化树发现，SPVE-XZ与SPVE-GS均聚类在一起。建立的SPVE荧光定量检测体系可检测到SPVE，而甘薯病毒2（sweet potato virus 2，SPV2）、甘薯C病毒（sweet potato virus C，SPVC）、甘薯G病毒（sweet potato virus G，SPVG）、甘薯羽状斑驳病毒（sweet potato feathery mottle virus，SPFMV）、甘薯褪绿矮化病毒（sweet potato chlorotic stunt virus，SPCSV）、甘薯潜隐病毒（sweet potato latent virus，SPLV）、甘薯褪绿斑病毒（sweet potato chlorotic fleck virus，SPCFV）、黄瓜花叶病毒（cucumber mosaic virus，CMV）均未能检测到;灵敏度可达3.12×10²copies/μL，是常规PCR 100倍。应用建立的qPCR检测方法，在山东省采集的6个样品和江苏省采集的52个样品中，分别在1个样品和13个样品中检测到SPVE。【结论】SPVE-XZ的全基因组大小为10 919 nt，与韩国SPVE-GS基因组结构一致;建立的qPCR检测体系对SPVE灵敏度高、特异性强，可用于对SPVE的快速检测。

关键词: 甘薯, 甘薯E病毒, 全基因组, 系统进化分析, qPCR检测

Abstract:

【Objective】Sweet potato virus E (SPVE) is a novel virus infecting Ipomoea batatas. The objectives of this study are to determine the complete genomic sequence of SPVE Xuzhou isolate (SPVE-XZ) in China, analyze the genomic sequence characteristics of SPVE-XZ, and develop the quantitative PCR (qPCR) assay for the specific detection of SPVE. This study will provide theoretical basis and technical support for the detection, monitoring and controlling SPVE in China. 【Method】The complete genomic sequence of SPVE-XZ was obtained by using small RNA deep sequencing, combined with RT-PCR and RACE technologies. The sequence alignment and phylogenetic relationship analysis of SPVE-XZ were performed by using software MegAlign and MEGA11. The primers of qPCR for rapid detection of SPVE were designed, and the qPCR detection method for SPVE was established by optimizing the annealing temperature and primer concentration. The specificity and sensitivity of the qPCR were determined. The developed qPCR technology was used to detect sweet potato samples collected from Jiangsu Province and Shandong Province. 【Result】The complete genomic sequence of SPVE-XZ was 10 919 nt, excluding the 3′-terminal poly (A) tail, containing the typical open reading frame of 10 560 nt in length and encoding a putative large polyprotein of 3 519 amino acids. The 5′UTR and 3′UTR of SPVE-XZ were 129 and 230 nt, respectively. PISPO and PIPO were produced with the frameshift in P1 and P3, respectively. Genomic sequence analysis showed that the genomic nucleotide sequence identity between SPVE-XZ and Korean SPVE-GS isolate was 98.6%, and the amino acid sequence identity of polyprotein between them was 98.5%. Phylogenetic trees were constructed based on the coat protein gene sequences and polyprotein amino acid sequence using the neighbor-joining method. The result showed that SPVE-XZ was clustered with SPVE-GS in these phylogenetic analyses. The established qPCR method could detect SPVE specifically, however sweet potato virus 2 (SPV2), sweet potato virus C (SPVC), sweet potato virus G (SPVG), sweet potato feathery mottle virus (SPFMV), sweet potato chlorotic stunt virus (SPCSV), sweet potato latent virus (SPLV), sweet potato chlorotic fleck virus (SPCFV), cucumber mosaic virus (CMV) could not be detected in this qPCR system. The lowest sensitivity of the qPCR was 3.12×10²copies/μL and it was 100 times that of conventional RT-PCR. SPVE was detected in 1 sample of 6 samples collected from Shandong Province and 13 samples of 52 samples collected from Jiangsu Province by using the established qPCR detection method. 【Conclusion】The complete genome sequence of SPVE-XZ is 10 919 nt. The genome structure of SPVE-XZ is consistent with SPVE-GS reported in Korean. The established qPCR detection system for SPVE is specific and highly sensitive, which can be used for the rapid detection of SPVE.

Key words: Ipomoea batatas, sweet potato virus E (SPVE), complete genomic sequence, phylogenetic analysis, qPCR detection

唐伟, 张成玲, 杨冬静, 马居奎, 陈晶伟, 高方园, 谢逸萍, 孙厚俊. 我国甘薯E病毒全基因组序列特征及荧光定量检测技术的建立[J]. 中国农业科学, 2023, 56(20): 4010-4020.

TANG Wei, ZHANG ChengLing, YANG DongJing, MA JuKui, CHEN JingWei, GAO FangYuan, XIE YiPing, SUN HouJun. Complete Genomic Sequence Characteristics and Establishment of qPCR Detection Technique of Sweet Potato Virus E in China[J]. Scientia Agricultura Sinica, 2023, 56(20): 4010-4020.

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引物名称 Primer name	序列 Sequence (5′-3′)	片段大小 Length (bp)	退火温度 TM (℃)	备注 Note
SPVE-F1	CAATACAACACAACAAAAATCAAAG	1708	58	基因组扩增Genome amplification
SPVE-R1	GAAATCCTCCCACTCTCCATA	1708	58	基因组扩增Genome amplification
SPVE-F2	ATTCAAAAGGTGGAATCTGA	1803	55	基因组扩增Genome amplification
SPVE-R2	CACATAGCAATACCCATCTTTC	1803	56	基因组扩增Genome amplification
SPVE-F3	TTTGCCAATTACTTCGATATT	2745	54	基因组扩增Genome amplification
SPVE-R3	CCCTGCATCAATTAAGAGG	2745	50	基因组扩增Genome amplification
SPVE-F4	AACTGCTGACAATATTCTCGTATAC	2776	52	基因组扩增Genome amplification
SPVE-R4	AAGGTCAAATAACATGCATTTC	2776	50	基因组扩增Genome amplification
SPVE-F5	TAGTGAAAAATGGGTCTTAGATAGA	2529	50	基因组扩增Genome amplification
SPVE-R5	GACAACATAAGTAGATTTTAAAGGC	2529	55	基因组扩增Genome amplification
SPVE-F6	CAATTCGACAACACCCGCT	~400	56	3′RACE
M4T	GTTTTCCCAGTCACGACTTTTTTTTTTTTTTTT	~400		3′RACE
SPVE-InR	ATGGAGTTGTGCGAGCATAAC	~400	55	5′RACE
SPVE-OutR	GTCTCTTCAGCCTCTTCCC		55	5′RACE
Q_T	CCAGTGAGCAGAGTGACGAGGA CTCGAGCTCAAGCTTTTTTTTTTTTTTTTT			5′RACE
Q_O	CCAGTGAGCAGAGTGACG			5′RACE