利用小RNA深度测序技术检测灰飞虱病毒种类

doi:10.13560/j.cnki.biotech.bull.1985.2021-0461

摘要/Abstract

摘要：

灰飞虱是一种重要农业害虫,作为病毒介体,可以传播多种植物病毒引起水稻、小麦和玉米等粮食作物病毒病害。目前对于灰飞虱体内昆虫病毒种类尚缺乏系统认识,难以开展利用昆虫病毒防治灰飞虱相关研究工作。为挖掘灰飞虱体内昆虫病毒资源,本文通过小RNA深度测序技术对灰飞虱所携带的病毒种类进行分析鉴定。结果显示,测序数据比对得到13种病毒,涉及8个病毒科和2种未分类病毒。除占据优势的水稻条纹病毒外,其余均为专性寄生的昆虫病毒,包括5种正链RNA病毒、2种单链DNA病毒和5种双链DNA病毒。在这些病毒中,发现了一种与果蝇A病毒较相似的新病毒,克隆出其依赖RNA的RNA聚合酶（RdRP）基因1-1 932位核酸序列,经Blast比对和系统进化分析,在氨基酸序列中发现RdRP掌型亚结构域保守区呈现复制酶置换四体病毒科（Permutotetraviridae）病毒所具有的“C-A-B”排列样式,确定该病毒是一种新的类复制酶置换四体病毒,暂命名为Laodelphax striatellus permutotetra-like virus（LsPLV）。这是首次在半翅目昆虫中发现类复制酶置换四体病毒。本研究表明灰飞虱体内昆虫病毒种类较为丰富,为今后利用昆虫病毒生物防治灰飞虱奠定了基础。

关键词: 灰飞虱, 昆虫病毒, 小RNA深度测序, 类复制酶置换四体病毒

Abstract:

Small brown planthopper（SBPH）（Laodelphax striatellus Fallén）is an important agricultural pest. As a virus vector,it can transmit some plant viruses,which cause viral diseases of grain crops,including rice,wheat and maize. At present,there is no comprehensive understanding of insect virus species in SBPH,thus it is difficult to conduct relevant research on SBPH management by exploiting insect viruses. In this paper,in order to excavate insect virus resources,the virus species in SBPH were identified by small RNA deep sequencing technology. The results showed that 13 viruses were found via sequencing data alignment,involving in 8 families and 2 unclassified viruses. Besides the dominant rice stripe virus,the rest were specialized parasitic insect viruses,including 5 positive-stranded RNA viruses,2 single-stranded DNA viruses and 5 double-stranded DNA viruses. Among them,a new virus similar to Drosophlia A virus was identified,and the 1-1 932 nt nucleotides of viral RNA-dependent RNA polymerase（RdRp）gene was obtained. After Blast and phylogenetic analysis,the unusual “C-A-B” permutation was found in the palm subdomain conserved region of RdRp,which was a unique arrangement pattern of Permutotetraviridae viruses,thus it was considered that the virus was a novel permutotetra-like virus,temporarily named Laodelphax striatellus permutotetra-like virus（LsPLV）. A permutotetra-like virus was first identified in Hemiptera insect. The study suggests that insect virus species are abundant in SBPH,which lays the foundation for biological control of SBPH by exploiting insect viruses in the future.

Key words: small brown planthopper, insect virus, small RNA deep sequencing, Laodelphax striatellus permutotetra-like virus

朴君, 张璐婕, 朴敬爱, 周益军, 李硕. 利用小RNA深度测序技术检测灰飞虱病毒种类[J]. 生物技术通报, 2022, 38(2): 281-288.

PIAO Jun, ZHANG Lu-Jie, PIAO Jing-Ai, ZHOU Yi-Jun, LI Shuo. Discovery of Viruses from Small Brown Planthopper by Small RNA Deep Sequencing[J]. Biotechnology Bulletin, 2022, 38(2): 281-288.

图/表 4

表1 灰飞虱中经测序比对鉴定的病毒

Table 1 Identified viruses in Laodelphax striatellus via alignment

同源病毒名称 Name of virus		病毒核酸类型 Nucleic acid	Contigs数 Nos. of contigs	Blast算法 Blast algorithm	E值 E-value	参考序列 Reference	属 Genus	科 Family
Rice stripe virus	RNA1	分节段负链RNA segmented -sRNA	92	Blastn	1.3E-16-2.0E-54	NC_003755.1	Tenuivirus	白纤病毒科 Phenuiviridae
	RNA2		32	Blastn	1.2E-17-1.1E-56	NC_003754.1
	RNA3		69	Blastn	9.7E-18-1.2E-34	NC_003776.1
	RNA4		38	Blastn	3.4E-17-9.0E-58	NC_003753.1
Laodelphax striatellus honeydew virus 1		正链RNA +ssRNA	20	Blastn	1.2E-12-8.6E-43	KF934491.1	unclassified	传染性软腐病病毒科 Iflaviridae
Laodelphax striatellus iflavirus 1			7	Blastn	9.1E-13-6.0E-70	MG815140.1	unclassified	传染性软腐病病毒科 Iflaviridae
Himetobi P virus			14	Blastn	1.1E-06-7.3E-74	NC_003782.1	Triatovirus	双顺反子病毒科 Dicistroviridae
Nilaparvata lugens commensal X virus			1	BlastX	1.7E-10	AB183424.1	未分类的正链RNA病毒 Unclassified positive-strand ssRNA viruses
Drosophila A virus			4	BlastX	2.8E-06-1.5E-13	YP_003038595.1	未分类的正链RNA病毒 Unclassified positive-strand ssRNA viruses
Periplaneta fuliginosa densovirus		单链DNA ssDNA	3	BlastX	4.5E-05-2.4E-12	NP_051016.1	Pefudensovirus	浓核症病毒亚科 Densovirinae
Dendrolimus punctatus densovirus		单链DNA ssDNA	1	BlastX	7.0E-08	YP_164339.1	Iteradensovirus	浓核症病毒亚科 Densovirinae
Amsacta moorei entomopo-xvirus		双链DNA dsDNA	4	BlastX	1.9E-05-1.2E-12	NP_064883.1	Betaentomopoxvirus	昆虫痘病毒亚科 Entomopoxvirinae
Invertebrate iridescent virus 6			3	Blastn	2.3E-09-2.3E-21	NC_003038.1	Iridovirus	虹彩病毒科 Iridoviridae
Cotesia congregata bracovirus			3	BlastX	8.9E-09-2.8E-15	NC_006645.1	Bracovirus	多分体DNA病毒科 Polydnaviridae
Choristoneura occidentalis granulovirus			4	Blastn	2.4E-10-4.3E-16	NC_008168.1	Betabaculovirus	杆状病毒科 Baculoviridae
Neodiprion abietis nucleopolyhedrovirus			1	BlastX	6.0E-05	YP_667900.1	Gammabaculovirus	杆状病毒科 Baculoviridae

表1 灰飞虱中经测序比对鉴定的病毒

Table 1 Identified viruses in Laodelphax striatellus via alignment

同源病毒名称 Name of virus		病毒核酸类型 Nucleic acid	Contigs数 Nos. of contigs	Blast算法 Blast algorithm	E值 E-value	参考序列 Reference	属 Genus	科 Family
Rice stripe virus	RNA1	分节段负链RNA segmented -sRNA	92	Blastn	1.3E-16-2.0E-54	NC_003755.1	Tenuivirus	白纤病毒科 Phenuiviridae
	RNA2		32	Blastn	1.2E-17-1.1E-56	NC_003754.1
	RNA3		69	Blastn	9.7E-18-1.2E-34	NC_003776.1
	RNA4		38	Blastn	3.4E-17-9.0E-58	NC_003753.1
Laodelphax striatellus honeydew virus 1		正链RNA +ssRNA	20	Blastn	1.2E-12-8.6E-43	KF934491.1	unclassified	传染性软腐病病毒科 Iflaviridae
Laodelphax striatellus iflavirus 1			7	Blastn	9.1E-13-6.0E-70	MG815140.1	unclassified	传染性软腐病病毒科 Iflaviridae
Himetobi P virus			14	Blastn	1.1E-06-7.3E-74	NC_003782.1	Triatovirus	双顺反子病毒科 Dicistroviridae
Nilaparvata lugens commensal X virus			1	BlastX	1.7E-10	AB183424.1	未分类的正链RNA病毒 Unclassified positive-strand ssRNA viruses
Drosophila A virus			4	BlastX	2.8E-06-1.5E-13	YP_003038595.1	未分类的正链RNA病毒 Unclassified positive-strand ssRNA viruses
Periplaneta fuliginosa densovirus		单链DNA ssDNA	3	BlastX	4.5E-05-2.4E-12	NP_051016.1	Pefudensovirus	浓核症病毒亚科 Densovirinae
Dendrolimus punctatus densovirus		单链DNA ssDNA	1	BlastX	7.0E-08	YP_164339.1	Iteradensovirus	浓核症病毒亚科 Densovirinae
Amsacta moorei entomopo-xvirus		双链DNA dsDNA	4	BlastX	1.9E-05-1.2E-12	NP_064883.1	Betaentomopoxvirus	昆虫痘病毒亚科 Entomopoxvirinae
Invertebrate iridescent virus 6			3	Blastn	2.3E-09-2.3E-21	NC_003038.1	Iridovirus	虹彩病毒科 Iridoviridae
Cotesia congregata bracovirus			3	BlastX	8.9E-09-2.8E-15	NC_006645.1	Bracovirus	多分体DNA病毒科 Polydnaviridae
Choristoneura occidentalis granulovirus			4	Blastn	2.4E-10-4.3E-16	NC_008168.1	Betabaculovirus	杆状病毒科 Baculoviridae
Neodiprion abietis nucleopolyhedrovirus			1	BlastX	6.0E-05	YP_667900.1	Gammabaculovirus	杆状病毒科 Baculoviridae

图1 LsPLV RdRP基因片段的扩增 M：DNA标准分子量5000;1：RdRP基因片段1扩增产物;2：RdRP基因片段2扩增产物

Fig.1 PCR amplification of RdRP gene segment M：DNA Marker 5000. 1：PCR products of RdRP gene segment 1. 2：PCR products of RdRP gene segment 2

图2 类复制酶置换四体病毒RdRP氨基酸序列比对显示“C-A-B”motif的排列 A、B和C motif用单划线标出,GDD box用双划线标出。比对所涉及病毒序列信息：LsPLV为本研究所获序列,A_nodav：A Nodaviridae virus（GenBank登录号：QIJ25871.1）,CDLV：Culex Daeseongdong-like virus（AXQ04818.1）,DAV：Drosophila A virus（AKH67411.1）,HPLV11：Hubei permutotetra-like virus 11（YP_009337276.1）,SmPLV：Smithfield permutotetra-like virus（QIJ25871.1）,VvPLV2：Vespa velutina associated permutotetra-like virus 2（QGL51736.1）

Fig.2 Amino acid alignment of permutotetra-like virus RdRP showing C-A-B motif arrangement A,B and C motif are marked with a single line,and GDD box is double underlined. Viral sequence information used in alignment is as follows：LsPLV is obtained in this study,A_nodav：A Nodaviridae virus（GenBank No. QIJ25871.1）,CDLV：Culex Daeseongdong-like virus（AXQ04818.1）,DAV：Drosophila A virus（AKH67411.1）,HPLV11：Hubei permutotetra-like virus 11（YP_009337276.1）,SmPLV：Smithfield permutotetra-like virus（QIJ25871.1）,VvPLV2：Vespa velutina associated permutotetra-like virus 2（QGL51736.1）

图3 根据不同类复制酶置换四体病毒RdRP序列构建的系统进化树 A：核苷酸序列系统进化树;B：氨基酸序列系统进化树。系统树所用病毒序列信息中LsPLV为本研究所获序列

Fig.3 Phylogenetic tree based on RdRP sequence consistency of different permutotetra-like viruses A：Phylogenetic tree based on nucleotide sequence,B：Phylogenetic tree based on amino acid sequence. Viral sequence information used in phylogenetic tree is as follows：LsPLV is obtained in this study,CDLV（GenBank No. MH188027.1）,DdV2（NC_028489.1）,SmPLV（MN784079.1）, A Nodaviridae virus（MT138111.1）, APLV1（MT896207.1）,DAV（KP969947.1）, VvPLV2（MN565052.1）, HPLV6（NC_033114.1）, HPLV11（NC_033097.1）, VpPLV（MN832441.1）, SWSV19（NC_033196.1）, EeV（NC_003412.1）, TaV（NC_043232.1）, ShPLV1（KX883439.1）, WHCV9（NC_033470.1）

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