The General And Molecular Biology Study Of Yam Chlorotic Necrotic Mosaic Virus

Yam chlorotic necrotic mosaic virus is a tentative member of genus Macluravirus in family Potyviridae.It was firstly isolated from Dioscorea parviflora C.T.Ting in Yunnan province and named by our group in Yongsheng county,Yunnan province,2009.However,some more key informations are still not available due to only four isolates of YCNMV were sequenced to acquire the 3’-end(including partial complete cp gene and 3’-UTR)at that time.To ensure the taxonomic status of YCNMV and enhancing our cognition to YCNMV,we carried out a series of general biology and molecular biology researches on YCNMV,such as determining the complete genome sequence,investigating the general biology properties,analysising the molecular genetic diversity in CP region.Moreover,a set of the serology and molecular detection system were established to help further research,and the infectious clone of YCNMV under the control of T7 promoter was constructed.We have also localized partial of the presumed movement proteins(MPs)of YCNMV on the sub-cellular level.The details of our works are as follows:(1)The general biology property and genomic structure of YCNMV were investigated.The virus particles observed under the electron microscope(EM)are flexuous and filamentous particles which measured 600-700x13 nm.The typical "pinwheel" structure of family Potyviridae is also observed under the EM.The genomic structure analysis showed that YCNMV has the smallest genome in the family Potyviridae as the complete nucleotide sequence of YCNMV genomic RNA is only 8 208 nt,excluding the poly(A)tail.There is a large polyprotein reading frame existed in the genome which encoded nine function protein,and a PIPO ORF is also observed in the P3 region.As well as the P1 region,the conserved motifs of DAG,KITC and PTK,which are conserved in most members of family Potyviridae,are absent from the genomic sequence of YCNMV,and these motifs are well known as "aphid transmission" related.The complete genome nucleotide of YCNMV shared identity of 55.22-72.91%within isolates of CYNMV and ArLV(also members of genus Macluravirus).This identity of nucleotide and amino acide in ORF is 56.08-73.09%and 49.40-80.66%,and 51.40-70.57%when comparing cp nucleotide,40.74-70.24%when comparing CP amino acide,respectively.According to the criteria of ICTV,YCNMV should be a new member of family Potyviridae due to the homology of nucleotide in cp region and complete genome are lower than 76%,and the homology of amino acide in CP protein is lower than 80%when comparing with members of genus Macluravirus.Moreover,the phylogenetic tree based on the ORF amino acid sequence grouped the CYNMV and YCNMV into a same cluster,but within obviously different branch.These results support the conclusion that YCNMV is a member different to CYNMV in genus Macluravirus.The inoculation experiment conducted on commonly used host plants with different methods(including mechanical and infiltration inoculation)showed negative results.The aphid-transmission assay was conducted on both D.batata and N.benthamiana also failed to inoculate YCNMV.These results implied that the host range of YCNMV is relatively narrow,or possibly transmitted in another way.The test results of serological relationship between YCNMV and partial members of family Potyviridae showed negtive results or only slightly positive results,which indicate that the serological relationship of YCNMV distantly related with the tested members.(2)A set of detection system based on the serological and molecular system were established to detect YCNMV.The recombinant CP protein of YCNMV was obtained by constructing the prokaryotic expression vector of cp gene.The rabbit was immued with purified CP recombinant protein for preparing antigen with high titer.Some serological test systems,such as ID-ELISA,Dot-ELISA and Western blot were established based on the prepared antigen.YCNMV-specific molecular detection system,including RT-PCR and qRT-PCR were also established based on the designed degenerate primer,specific primers and specific qPCR primers.One-hundred and seventy-one yam samples collected from Yunnan province which showed symptoms were tested with the established detection method mentioned above,and twenty samples showed positive results.The 3’-end(including partial nib,complete cp and 3’-UTR sequence)of YCNMV isolates were acquired by using RT-PCR method.The successful application of established detection system proved that they are highly efficient,reliable and specific,and can be used in the further detection process of YCNMV.(3)The molecular genetic diversity analysis of YCNMV isolates were conducted.Combined with the downloaded sequence information(from NCBI)of partial members in family Potyviridae,the acquired sequence of YCNMV isolates were then subjected to the molecular genetic diversity analysis.The information biology analysis based on the cp gene,CP amino acid and 3’-UTR sequence showed that the isolates of YCNMV exhibited obviously higher level of genetic diversity than CYNMV.Further analysis showed that the recombination phenomenon in YCNMV is unremarkable,whereas the YCNMV group possess relatively high value in both nucleotide diversity(Pi)(0.16114±0.01333)and haplotype diversity(Hd)(0.996±0.013).This implies the population diversity of YCNMV is in a relatively high level.The selection analysis indicate that the YCNMV is generally experiencing a strong negative selection process with a value of 0.1257(dN/dS),and the gene of YCNMV is relatively conserved during the evolution.However,four sites are showed to have the trace of episodic diversifying selection(EDS)in cp gene of YCNMV isolates,especially for Codon273 which have ap-value≤0.05.(4)The infectious clone of YCNMV under the T7 promoter control was constructed by applying the infusion technology.The infectious clone of YCNMV(pSKycnmv)is constructed and the sequencing results showed that the reading frame is correct,and no insertion,deletion or recombination mutant was observed in the complete sequence.However,the inoculation experiment conducted by appling mechanical inoculation method with the transcripts on N.benthamiana and N.glutinosa showed negative results after 7 dpi and 22 dpi,respectively,thus the infectious of pSKycnmv need to be ensured in the further study.(5)The subcellular localization of four presumed movement proteins(MPs)of YCNMV were observed.Four presumed MPs(HC-Pro,VPg,CI,CP)of YCNMV were co-expressed with GFP in N.benthamiana seperately.The subcellular localization of these proteins were then observed with confocal laser microscopy.The results showed that HC-Pro,VPg and CP were mainly located in the cell nucleus and cytoplasm,no special tendency of distribution was observed,whereas Cl was located on the cytoderm/plasma membrane and formed stippling shaped.According to this result,we presume that the CI is located on the plasmodesma(PD)and may play an important role in the movement of YCNMV.In conclution,the study metioned above could provide evidences for the classification of YCNMV,and help us to further comprehend the population genetic structure,as well as the infection mechanism of YCNMV.