The study of gibberellic acid insensitive involved in Bamboo mosaic virus replication in Nicotiana benthamiana

• Main Authors: Hsin-Yi Liu, 劉欣宜
• Other Authors: 蔡慶修
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/88268758104731508985

碩士 === 國立中興大學 === 生物科技學研究所 === 105 === Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus, a member of Potexvirus genus of Alphaflexiviridae family. In a previous study, 90 differentially expressed genes were identified from BaMV-inoculated N. benthamiana plants using the technique cDNA-amplified fragment length polymorphism (cDNA-AFLP). One of the differentially expressed genes ACCT7-1 was upregulated in BaMV-inoculated N. benthamiana plants. Furthermore, the full-length cDNA was cloned using rapid amplification of cDNA ends (RACE) and sequence. The identity of ACCT7-1 was revealed as a homolog of GA-INSENSITIVE (GAI), a member of DELLA family, when compared to the databases of National Center for Biology Information (NCBI). Therefore, ACCT7-1 is then designated as NbGAI. NbGAI was characterized by BaMV inoculated on virus-induced gene silencing (VIGS) leaves and protoplasts. The results showed that the accumulation of BaMV coat protein was decreased significantly in both knockdown leaves and protoplasts compared to the controls. These results can be inferred that NbGAI may play a positive role in assisting the virus replication. Furthermore, the fusion protein NbGAI with Orange fluorescent protein (OFP), NbGAI-OFP is expressed in plants and BaMV is then inoculated. The accumulation of BaMV coat protein is increased significantly compared with the control plants with the expression of OFP only. The results indicate that NbGAI play a role in assisting BaMV accumulation. NbGAI is a member of DELLA family and contains a nuclear localization signal (NLS). In this study, I have used confocal microscopy to confirm the nuclear localization of NbGAI in cell. In addition, the NLS of NbGAI is predicted by screening online software: cNLS Mapper. These results indicated that NbGAI could play an assisting role in BaMV replication.