木瓜畸葉嵌紋病毒的基因體分析及其與木瓜輪點病毒在木瓜上的交互作用關係研究

Abstract

木瓜是台灣重要熱帶經濟果樹之一，除了木瓜輪點病毒 (Papaya ringspot virus，PRSV) 會造成木瓜極嚴重的病害之外，近年來台灣也發現另一個新浮現的木瓜病毒－木瓜畸葉嵌紋病毒 (Papaya leaf distortion mosaic virus，PLDMV)，開始危害田間的木瓜。有關 PLDMV 的研究目前仍在少數，病毒之特性及其發病生態情況仍有待釐清。為此，本論文進行 PLDMV 之基因體全長解序並做比較分析，研發出 PLDMV 的 RT-PCR (反轉錄聚合酶連鎖反應) 與 real-time RT-PCR (即時定量反轉錄聚合酶連鎖反應) 分子檢測方法，且進一步應用於 PLDMV 的接種試驗以探討其致病性。將採集自屏東縣高樹鄉的 PLDMV 分離株 (PLDMV-KS) 進行全長解序，共計 10153 個核苷酸，已上傳至 NCBI GeneBank (Accession number：EU233272)。和日本 J56P 分離株進行比較，全長核苷酸序列相似度為 94.7%，胺基酸序列全長相似度則為 95.5%。就個別基因來做比較，核苷酸序列相似度以 3’UTR 為最高 (96.7%)，其次為 CI 以及 CP 基因 (95.8%)，而 5’UTR 最低 (86.6%)，其次為 P1 基因 (92.2%)；胺基酸序列相似度則以 NIa-Pro 最高 (98.4%)、CI 基因為第二 (98.3%)、其次為 6K1 基因 (98.1%，但只有一個胺基酸不同)，而 P1 基因相似度則最低 (87.9%)。PLDMV 在木瓜上造成的病徵會依木瓜品種 (系) 的不同而有些微的差異，接種試驗結果發現於台農二號 (TN2)、台大一號 (NTU1) 以及抗輪點病基因轉殖木瓜 (GM) 上會產生葉脈透化或黃化、葉畸形，有時會有絲狀葉的產生，於紅妃品種 (RL) 則不會觀察到絲狀葉的產生。利用 RT-PCR 及 real-time RT-PCR 追蹤病毒的增殖情況，在接種後 12 天 TN2、NTU1 及 GM 木瓜可偵測到 PLDMV 存在，約一個月後病毒攀升到最高增殖量；RL 則是在第 20 天才開始被偵測到，約兩個月後病毒才攀升到最高增殖量，顯示其對 PLDMV 耐病性較高。相對於 PLDMV，PRSV (畸形系統) 的接種試驗顯示於 TN2、NTU1 及 RL 可較早偵測到 PRSV，在第 14~16 天就能攀升到最高增殖量；而 GM 木瓜則對其具抗性。若木瓜同時混合接種 PLDMV 及 PRSV 時，會改變原本二病毒的增殖情形。由 real-time RT-PCR 的追蹤結果發現在混合接種的情況下，PRSV 在木瓜體內的含量會比單獨接種時的含量高，而且會延遲 PLDMV 達到飽和所需的時間。PLDMV 似乎有增強 PRSV 在木瓜體內的繁殖能力；且當兩病毒同時感染時，病徵也呈現協力作用而較為嚴重，因此在進行木瓜病毒病害防治時，PLDMV 不容忽視。

Papaya is one of the tropical fruits with economic importance in Taiwan. Another conspicuous pest, Papaya leaf distortion mosaic virus (PLDMV), recently occurred in the papaya orchards in Taiwan besides Papaya ringspot virus (PRSV). The pathological and molecular characters associated with PLDMV are still unclear because of rare studies on it. This thesis was dedicated to study the genomic nature and investigate the pathogenicity of PLDMV. Several important data such as the determination of full-length genomic sequence of PLDMV, development of RT-PCR (reverse- transcription polymerase chain reaction) and real-time RT-PCR (real-time reverse-transcription polymerase chain reaction) for efficiently qualitative and quantitative detection of PLDMV, and inoculation tests for comparative pathogenicity of PLDMV on different papaya cultivars (lines) were completely presented in this thesis. The full-length genomic sequence of PLDMV (Taiwan-KS isolate) was determined (total 10,153 nucleotides) and published in GenBank (Accession number: EU233272). Based on the results of alignment, the full-length nucleotide sequences were 94.7% homologous between KS and Japanese isolate (J56P). The nucleotide alignment of individual genes demonstrated that the 3’UTR, CI and CP genes have higher homology (96.7%, 95.8% and 95.8% respectively) whereas 5’UTR and P1 genes have lower homology (86.6% and 92.2% respectively) between them. The amino acid alignment of individual genes demonstrated that the NIa-Pro and CI have higher homology (98.4% and 98.3% respectively) whereas P1 has lower homology (87.9%). Symptoms induced by PLDMV were various on different papaya cultivars (lines) such as Tainung No.2 (TN2), National Taiwan University Hybrid No.1 (NTU1), Red Lady (RL) and the genetically modified papaya against PRSV (GM). PLDMV induced symptoms including vein-clearing, leaf-distortion and fern-leaf on TN2, NTU1 and GM. On RL, PLDMV induced vein-clearing and mild leaf-distortion without fern-leaf symptoms. The multiplicative fluctuation of PLDMV on different papaya cultivars (lines) was qualitatively and quantitatively monitored by RT-PCR and real-time RT-PCR. The results showed that PLDMV could be detected 12 days post-inoculation (dpi) on TN2, NTU1 and GM papaya, and it replicated to the maximum approximately 30 dpi. PLDMV was detected 20 dpi on RL, and it replicated to the maximum approximately 60 dpi. Compared to PLDMV-infection, PRSV seemed to show better susceptibility in papayas. PRSV could be earlierly detected than PLDMV after inoculation, and it rapidly replicated to the maximum approximately 14~16 dpi. On the other hand, the results of simultaneous inoculation with PLDMV and PRSV indicated that the amount of PRSV in papayas was increased more than individual infection of PRSV, and PRSV slightly delay the multiplication of PLDMV. The results also showed that papayas co-infected with PLDMV and PRSV produced more serious symptoms than those infected individual PLDMV or PRSV. PLDMV should be not neglected in the control of virus diseases of papaya.