侵染小构树的双生病毒及其缺陷DNA形式的检测和特征分析

Detection and Characteristic Analysis of Geminivirus and Its Defective DNA Forms Infecting Broussonetia kazinoki

为明确侵染小构树(Broussonetia kazinoki)并引起叶片黄化卷曲症状的病毒种类及其基因组特征,本研究利用转录组高通量测序技术获得病毒基因组,利用PCR扩增和基因克隆技术对病毒基因组及其缺陷DNA进行鉴定。结果表明,通过转录组高通量测序和无参组装,获得引起小构树叶片黄化卷曲的双生病毒,命名为小构树卷叶病毒2(Kozo leaf curl virus 2,KozoLCV-2)。进一步克隆测序获得的KozoLCV-2基因组全长为3756~3758 bp,其与构树卷叶病毒(Paper mulberry leaf curl virus 2,PMLCV-2)具有相同的基因组结构和较高的序列一致性;病毒正义链编码4个ORF(V1、V2、V3和V4),互补链编码2个(C1和C1:C2),互补链存在选择性剪接。PCR扩增和克隆测序获得了10种KozoLCV-2的缺陷DNA(defective DNA,D-DNA)形式,其中9种完全或部分缺失复制酶区域,4种形式的D-DNAs与来自PMLCV-2-TL RepA第二外显子末端的序列发生了重组。

In order to clarify the virus causing leaf yellowing and curling symptoms of Broussonetia kazi⁃noki and its genome characteristics,the viral genome was obtained by high throughput sequencing of transcrip⁃tome,and the genome and defective DNA(D⁃DNA)form were identified by PCR amplification and gene clo⁃ning technology.The results showed that a geminivirus that caused leaf yellowing and curling in B.kazinoki was obtained through high⁃throughput sequencing and non parametric assembly of the transcriptome,which was named Kozo leaf curl virus 2(KozoLCV⁃2).The genome of KozoLCV⁃2 had a total length of 3756~3758 bp,which had the same genomic structure and high sequence identity with Paper mulberry leaf curl virus 2(PMLCV⁃2).The genome structure of the two geminiviruses were the same,both had four ORFs(V1,V2,V3 and V4)in the sense strand and two ORFs(C1 and C1:C2)in the complementary strand,and splicing form was found in the com⁃plementary⁃strand.A total of ten defective DNA(D⁃DNA)forms of KozoLCV⁃2 were obtained by PCR amplification and clone,nine of which were completely or partially lost the replicase region.Four forms of D⁃DNAs were recom⁃bined with the sequence from the end of the second exon of PMLCV⁃2⁃TL RepA.