Development and Application of Highly Efficient Methods for Detection of Transgenic Papayas Resistant to Papaya ringspot virus
Date Issued
2007
Date
2007
Author(s)
Wu, Hsuan-Yi
DOI
zh-TW
Abstract
This thesis was dedicated to develop various efficient methods with the DNA-based techniques for the identification of transgenic papayas resistant to PRSV. These developed methods contain (1) PCR detection：This was developed for detecting 35S promoter, PRSV coat protein gene, Nos terminator in transgenic papayas using our devised 35S-726, CP-739 and Nos-220 primer pairs. Nested-PCR assays were conducted using the 35S-348 and CP-372 primers to increase sensitivity. Both 35S-726 and Nos-220 primer pairs were used to perform multiplex PCR assays. Primer SC-670 could amplify the fragments between 35S promoter and PRSV CP gene；Primer CN-716 could amplify the fragments between PRSV CP gene and Nos terminator. Both SC-670 and CN-716 provide a more accurate and specific detection for the transgenic papayas. Primer RBNP-308 and NPTⅡ-718 designed to detect the right boarder region; Nos terminator and NPTⅡ gene were potential to be applied in the detection of other transgenic crops. All samples collected from young leaves, mature leaves, petioles, roots, flowers and fruits could be used in the detection of transgenic papayas. (2) Dot blot hybridization method: Dot blot hybridization provides a more accurate detection especially when gene-variation occurs in the inserted genes of transgenic papayas. AFLP techniques and fluoremetrical detection were used to increase sensitivity for dot blot hybridization in this study. (3) Real-time PCR method: This was applied for rapid detection and quantification of target gene for transgenic papayas. Our devised methods were directly applied in field survey for papaya plants. Many transgenic papaya plants were detected in the southern and eastern Taiwan. Interestingly, those transgenic papayas were also infected by PRSV in addition to PLDMV (Papaya leaf-distortion mosaic virus). Lots of papaya samples collected from Guangxi and Hainan (China) also showed positive for transgenic plants. It indicates that transgenic papayas have become the main cultivars in China. A PRSV isolate collected from Guangxi could infect transgenic papayas and caused typical symptoms whereas PSRV-DF and PRSV-SMN strains (from Taiwan) can not infect them. Molecular alignment and analysis of the coat protein gene revealed that the Guangxi isolate is somewhat different from the other reported PRSV isolates. Alignment of nucleic acid sequence demonstrated that this Guangxi isolate is 94.1% identical to the coat protein gene of transgenic papayas, and it is 96.3~96.5 % identical to the coat protein gene of PSRV-DF and PRSV-SMN strains. However, alignment of amino acid sequence did not show significant differences among them.
Subjects
木瓜輪點病毒
轉基因木瓜
聚合酵素連鎖反應
墨點雜合反應
即時定量聚合酵素連鎖反應
Papaya ringspot virus
Transgenic papayas
Polymerase chain reaction
Dot blot hybridization
Real-time PCR
Type
other
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