Establishment of the promoter assay system of arabidopsis gene PAP85 involved in replication of Tobacco mosaic virus
Date Issued
2011
Date
2011
Author(s)
Wu, Chao-Jung
Abstract
The genomes of plant viruses are small and encode limited genetic products. To complete their life cycles, plant viruses highly require assistance from host factors. In previous research, to study these host factors, Arabidopsis protoplasts infected by Tobacco mosaic virus (TMV) were used to screen the host genes responsive to TMV replication at initial stage of infection through microarray analysis (Chen, 2010). It is found that host gene PAP85 is continuously up-regulated during 0.5-24 hours post inoculation, and virus accumulation decreased both in PAP85 transient knock-down protoplasts and pap85-RNAi transgenic plants. In this thesis, we started to study how TMV induces the PAP85 in the initial stage during virus infection. Rapid amplification of cDNA ends (RACE) was conducted to determine the transcription start site of PAP85 and found that it is located at 24 bps upstream to translational start site. Computer assisted comparison analysis was conducted to identify the possible cis-acting elements, and CIACADIANLELHC, NODCON1GM, OSE1ROOTNODULE, TATABOX4, TATAPVTRNALEU, TBOXATGAPB. CIACADIANLELHC were identified that may be involved in regulation of PAP85. PAP85 promoter assay systems were initially established on transiently-expressed protoplasts; however, inconsistent results were obtained due to low transfection rates. The transgenic plants with different lengths (107, 1227 and 2000 bps) of PAP85 promoter fused GUS reporter gene were obtained. In plants transformed with pCambia1301-PAP85-P2000 and pCambia1301- PAP85-P1227, the GUS expression pattern is similar to the reported endogenous PAP85 expression. Besides, the knock-out mutants of 4 transcription factor-like genes (At1g09530, At2g38970, At4g29530, At5g41140) upregulated during TMV initial infection were obtained from the Arabidopsis Biological Resource Center (ABRC). After TMV inoculation, the virus accumulation decreased only in At5g41140 knockout plants, suggesting that At5g41140 was involved in virus accumulation.
Subjects
Tobacco mosaic virus
Arabidopsis thaliana
promoter
host factor
Type
thesis
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