胡逸超 卢晓静 李璞 廖咏梅 何新华 邹承武 陈琦

摘要：【目的】探究侵染掌叶半夏（Pinellia pedatisecta）的大豆花叶病毒（Soybean mosaic virus，SMV）衍生的干扰小RNA（virus-derived small interfering RNA，vsiRNA）序列特征，为掌叶半夏抵御病毒的作用机制研究提供参考。【方法】以一株来自广西的具有典型病毒病症状的掌叶半夏为材料，取其褪绿斑驳的叶片采用TRIzol法提取总RNA，并进行小RNA深度测序（Small RNA Deep Sequencing）;利用快速扩增cDNA末端（Rapid Amplification of cDNA Ends，RACE）和分段克隆技术获得病毒的全长基因组序列，利用MEGA 7.0将其与有代表性的病毒序列构建系统发育进化树并分析亲缘关系;以克隆测序获得的基因组序列作为参考进行vsiRNA特征分析。【结果】小RNA深度测序共获得4851249条高质量的读段（reads），长度为21、22和24 nt的reads具有较高的丰度，占比分别为33.4%、13.7%和11.3%。该病毒分离物的基因组全长为9735 nt，编码3105个氨基酸，其基因组全序列与SMV HZ1分离物核苷酸序列相似性为86.93%，暂命名为SMV NN;系统发育进化树显示，SMV NN与分离自半夏的SMV HZ1分離物的亲缘关系最近。将vsiRNA定位到克隆测序后获得的SMV NN的基因组上，分析该病毒的vsiRNA特征，结果发现长度为21和22 nt的vsiRNA具有较高的丰度，病毒全基因组的正链和负链均能被vsiRNA覆盖，且分别在HC-Pro和P3蛋白编码区具有最强热点。【结论】掌叶半夏主要通过dicer样核糖核酸酶4（dicer-like ribonuclease 4，DCL4）和Argonaute蛋白1（Argonaute1，AGO1）对SMV的HC-Pro和P3蛋白编码区进行剪切，主要产生长度为21和22 nt的vsiRNA，从而抑制SMV在植株体内的复制。

关键词： 掌叶半夏;病毒基因组;大豆花叶病毒;小RNA深度测序;vsiRNA

中图分类号： S432                               文献标志码： A 文章编号：2095-1191（2021）12-3392-08

Characterization of virus-derived small interfering RNAs and complete sequence of a strain of Soybean mosaic virus

infected Pinellia pedatisecta

HU Yi-chao1， LU Xiao-jing2，3， LI Pu4， LIAO Yong-mei2， HE Xin-hua2，

ZOU Cheng-wu2*， CHEN Qi4*

（1Technical Center，China Tobacco Guangxi Industrial Co.， Ltd.， Nanning  530001， China; 2 National Demonstration Center for Experimental Plant Science Education， College of Agriculture， Guangxi University， Nanning  530004， China; 3Nanning Center for Disease Control and Prevention， Nanning  530023， China; 4Nanning Gold-tech

Bio-science Co.， Ltd.， Nanning  530001， China）

Abstract：【Objective】To illuminate the virus silencing mechanism in Pinellia pedatisecta， performed an analysis of the sequence characteristics of virus-derived small interfering RNA （vsiRNA） in a Soybean mosaic virus （SMV） infec-ting P. pedatisecta. 【Method】In this study， a plant of P. pedatisecta， which displayed typical virus infection symptoms， was found in Guangxi. The total RNA of the chlorotic and mottled leaves was extracted using the TRIzol method. A small RNA deep sequencing was employed to identify the vsiRNA. The full-length genome sequence of the virus was obtained by rapid amplification of cDNA ends （RACE） and segmented cloning. The phylogenetic relationships of the assembled virus sequence was analyzed using MEGA 7.0  software. The characterization of vsiRNA was performed using the cloned genome as a reference sequence. 【Result】In total， 4851249 high-quality reads， in which the relatively rich reads were 21， 22， and 24 nt with the percentage of 33.4%， 13.7% and 11.3%， respectively， were obtained by small RNA deep sequencing. According to the small RNA sequencing results， the full-length genome sequence cloning of the virus was carried out. The virus genome was found to be 9735 nt in length， encoding a polyprotein that contained 3105 amino acids. Sequence analysis showed that the nucleotide sequence of the virus isolate genome shared 86.93% identical identity with the SMV HZ1 isolate. This virus isolate was named SMV NN tentatively. The phylogenetic relationship anlysis revealed that the SMV NN shared the closest genetic relation with the SMV HZ1 isolated from P. ternate. The small RNAs of the virus were mapped to the genome of SMV NN for vsiRNA characterization. The results showed that the vsiRNA with lengths of 21 nt and 22 nt was in high abundance; both positive and negative virus vsiRNA could cover the entire virus genome and had the strongest hot spots in the HC-Pro and P3 protein-coding regions， respectively. 【Conclusion】P. pedatisecta mainly uses dicer like ribonuclease 4 （DCL4） and Argonaute1 （AGO1） to cleave the HC-Pro and P3 domain of SMV， and mainly produces vsiRNA with the lengths of 21 nt and 22 nt， resulting in the inhibition of SMV replication in plants.

Key words： Pinellia pedatisecta; rirus genome; Soybean mosaic virus; small RNA deep sequencing; vsiRNA

Foundation item： Guangxi Innovation Driven Development Special Project （Major Science and Technology Proje-cts） （Guike AA17204054）; Project of China Tobacco Guangxi Industrial Company （Keji 20190029）

0 引言

【研究意义】掌叶半夏（Pinellia pedatisecta）是我国特有的天南星科（Araceae）植物，其药材名为虎掌，具有镇痛、抗惊厥和治疗心血管疾病等多种功效（Lin et al.，2007;刘寨东等，2009;Wang et al.，2019）。……