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Analyses of the genomic sequence of the mild strain HA 5-1 of Papaya ringspot virus
|關鍵字:||PRSV;木瓜輪點病毒;mild strain;HA 5-1;genomic sequence;nucleotide identities;amino acid identities;輕症突變株;基因體序列;核苷;酸相同度;胺基酸相同度||出版社:||植物病理學系||摘要:||
木瓜輪點病毒(Papaya Ringspot Virus, PRSV)屬於馬鈴薯Y病毒屬(The genus Potyvirus), 含有一條正極性(positive polarity)的單鏈狀核醣核酸(ssRNA)。之前以亞硝酸處理PRSV 夏威夷強病徵品系HA, 得到輕症突變株HA5-1, 經由溫室及田間試驗發現其保護效果良好, 但是發現在使用HA 5-1作為交互保護時, 會產生系統專一性保護(strain-specific protection)的現象, 使保護效果容易被破壞。因此為了找出更有效的防治策略, 本研究首先將HA 5-1全長度基因體序列解序完成, 與HA比對發現其中有67個核苷酸變異, 相同度為99.35 % , 胺基酸有29個變異, 相同度為99.16 %。進一步分析各基因的差異時發現P1基因有11個核苷酸差異, HC-Pro基因有7個, P3基因有15個, CIP基因有13個, P5基因有1個, NIa基因有6個, NIb基因有11個, CP基因有3個, 而比較兩者胺基酸的差異時則發現P1基因有8個胺基酸差異, HC-Pro基因有4個, P3基因有7個, CIP基因有2個, P5基因沒有差異, NIa基因有2個, NIb基因有3個, CP基因有3個。所有基因的核苷酸相同度在98.74 %到100 % 之間, 胺基酸相同度則在98.24 %到100 %之間。其中胺基酸序列差異最大的區域是在P1、HC-Pro、及P3 基因, 其胺基酸相同度為98.24 % 到 99.12 %之間。可以歸納出P3基因為核苷酸及胺基酸相似度變異最大的基因, 而5' 端非轉譯區(5'-NTR)及3' 端非轉譯區(3' —NTR)則是核苷酸相同度最高度保留的區域, 而6 K基因則是胺基酸相同度最高度保留的區域, 因此我們推論在 P1、HC-Pro、及P3基因的改變可能使病毒的病徵表現受到影響。而比較基因體其中的保留區域如與病徵表現相關的FRNK區域及和蚜蟲傳播相關的DAG、PTK、KITC區域等並無發生變化。在比較核苷酸與胺基酸變異分布情形發現, 共有38個核苷酸的變異是在遺傳密碼浮動鹼基(wobble base)的位置, 推測對其轉譯出的胺基酸可能相對較無影響。而在14個結構發生改變的胺基酸中, 就有8個集中分布於 P1、HC-Pro、及P3 基因中, 因此推論在P1、HC-Pro、及P3 基因中發生的改變, 對於HA 5-1的病原性或其功能改變有重要的影響。而在了解HA 5-1基因體序列之後, 可針對特定的PRSV強病徵型病毒作適當的突變以獲得輕症突變株, 用來保護當地的經濟栽培作物,避免因系統專一性保護現象而失去交互保護的效用。
The PRSV virus is a member of the genus Potyvirus of the family Potyviridae, with a single-stranded RNA (ssRNA) of positive polarity. The PRSV P HA 5-1 induced from nitrous acid treatment of a severe Hawaii strain PRSV P HA provides high degrees of protection for papaya against the infection by HA. However, a phenomenon of strain-specific protection occurs when HA 5-1 was practically used to control PRSV by cross protection. In this study, the complete genomic sequence of the HA 5-1 was determined and compared with that of HA. Sequence analysis showed that that they share 99.35 % nucleotide identity with 67 nucleotide changes and 99.16 % amino acid identity with 29 amino acid changes. Comparison of nucleotide difference of each genes between the HA 5-1 and HA genomes revealed that there are 11 nucleotides changes in the P1 gene, 7 in the HC- Pro gene, 15 in the P3 gene, 13 in the CIP gene, 1 in the 6 K gene, 6 in the NIa gene, 11 in the NIb gene, and 3 in the CP gene. Also, there are 8 amino acids changes in the P1 protein, 4 in the HC- Pro protein, 7 in the P3 protein, 2 in the CI protein, none in the P5 protein, 2 in the NIa protein, 3 in the NIb protein, and 3 in the CP protein. All the genes shared 98.74 % to 100 % nucleotide identities and 98.24 % to 100 % amino acid identities, and the most different regions in amino acid sequence were located in the P1, HC-Pro, and P3 genes, with identities of 98.24 % to 99.12 %. The results indicated that the P3 gene has the lowest degree of nucleotide and amino acid identity, and is more variable than the other proteins between HA 5-1 and HA. Both the 5' -NTR and 3' -NTR are the most conserved regions between HA 5-1 and HA, with 100 % sequence identity. The 6 K protein is the most conserved protein than the other proteins in between HA 5-1 and HA. It was suggested that the changes in the P1, HC-Pro, and P3 genes may affect the virus symptom expression in plants. Comparison of the each gene revealed that the conserved motifs such as the FRNK motif associated with the symptom expression, and the DAG, PTK, and KITC motifs associated with aphid transmissibility are not changed. Comparison of the distribution of the nucleotide and amino acid changes in the position of genetic codes of each gene revealed that 38 nucleotide changes are in the wobble bases of genetic codes and mostly did not affect the encoded amino acids. There are 14 amino acids changes were found structurally different, and the regions of the P1, HC-Pro, and P3 genes had a total of 8 amino acid changes, more than half of the total significant amino acid changes. It is suggested that the mutations in the regions of P1, HC-Pro, and P3 genes are more important for the change in pathogenicity of HA 5-1. After analyses of the genomic sequence of HA 5-1, it is possible to mutate a severe PRSV strain accordingly to generate a specific mild strain to prevent the breakdown of cross protection due to the phenomenon of the strain-specific protection.
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