Cloning Of The Full Length CDNA Of CmRV And Transformation Of Virus-free Strain ZS-1 Of Coniothyrium Minitans

Coniothyrium minitans, an important sclerotial mycoparasite of Sclerotiniasclerotiorum with worldwide distribution, has great potential for biological control ofplant diseases caused by S. sclerotiorum and other Sclerotinia spp., C. minitans RNAvirus (CmRV) is one type of double-stranded RNA (dsRNA) mycovirus belong toTotiviridae infecting C. minitans strain Chy-1, its genomic RNA has been completedsequencing analysed. Previously, biological observation showed that CmRV haslittle negative influence on its host. In this paper, reverse genetics technology wasused to analyse the potential effect of CmRV on its host, C. minitans.To clone the full length cDNA of virus, RT-PCR technology was used. Firstly,two pairs of primers were designed based on the genomic dsRNA sequence of CmRVto obtain two overlapped cDNA fragments; secondly, the two fagments were digestedwith PstI, and ligated onto pMD18-TCmRV.The CmRV genome that sequenced in 2003 was noted as 4975 nt in length, whilewe found that this sequence was not correct, the 21 nucleic tides located at theextreme terminus was not exist actually, it was the oligo nt artificially ligated on thegenomic dsRNA during cloning. Thus, the actual length of viral dsRNA is 4954 bp.As these 21 base pairs located at the 3 extremely terminus which is belong to thenon-translated region, it dose not affect the classification of CmRV.To obtain the expression vector of the full-length viral cDNA, the cDNA wasdigested with XbaI, and then was ligated onto the same enzyme digested vectorp1301V which was constructed based on the backbone of pCAMBIAN1301 fortransformation of Sclerotinia sclerotiorum debilitation associated RNA virus (SsDRV).The constructed vector was named as p1301-CmRV, since there were two directionfor the ligation of XbaI digested viral cDNA, two types of vector were obtained,namely pCmRV-cDNA (+) in which the two viral ORFs were under the control ofPtrpC, and pCmRV-cDNA(-), the reverse insertion of the viral cDNA. The twovectors were transformated into Agrobacterium tumefaciens EHA 105, and were usedfor transformation of virus-free strain ZS-1 of C. minitans.Candidates which could grow on PDA containing hygromycin were identifiedwith PCR amplification, and results showed that all tested candidates carried thehygromycin resistance gene (hph). To probe the expression of viral gene in transformants, RT-PCR technique was used with specific primers designed from theviral genome, and resluts showed that viral gene had expressed in tmasformants.However, no viral dsRNA could be detected in all tested transformants.Transformants with direct or reverse insertion of viral cDNA showed littledifferent from original strain ZS-1 in colony morphology, conidial production andparasitical ability with two exceptions. Thus, the data showed that CmRV eDNAmight not effect on its host significantly.