Proteomic Analysis of Auxin Action in Stimulating Stigmatic Closure Response and Cloning of IAA Conjugate Hydrolase cDNA in Phalaenopsis

• Main Authors: Wang-Tin Lin, 林婉婷
• Other Authors: Yu-Kuang Chen
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/31237548015397358179

碩士 === 國立屏東科技大學 === 生物科技研究所 === 92 === Closing of the stigmatic cavity after pollination is a unique postpollination phenomenon in many orchids including Phalaenopsis. Among different plant growth regulators tested, only auxins such as IAA and NAA can induce the same response. Anatomical observation has shown that the closure of the stigmatic cavity is induced by column cell enlargement. IAA-induced stigmatic closure was inhibited by the protein synthesis inhibitor cycloheximide when applied simultaneously with IAA, but not by the RNA synthesis inhibitor actinomycin D. The application of cycloheximide at different times after IAA treatment revealed that stigmatic closure was no longer inhibited by cycloheximide when it was applied 10 hr after IAA treatment, indicating new proteins required for stigmatic closure have been synthesized at least partially within this time period. By two-dimensional electrophoresis analysis, changes in the protein expression profiles of columns after IAA treatment can be observed. Two major protein spots with differential expression after IAA treatments were identified to be thioredoxin h and ACC oxidase through LC-MS/MS characterization and database matching. Both proteins can be induced by IAA application and reached their maximal expression at 12 hr after IAA treatment. Treatments with IAA conjugates resulted in stigmatic closure response except IAA-Aspartate. In addition, the stigmatic response to different IAA conjugates varied in their efficiency, indicating that stigmata have the ability to differentiate among the IAA conjugates. Using 3’ and 5’ RACE, an IAA conjugate hydrolase gene (designated as TL-IAR) has been cloned from Phalaenopsis Tropican Lady. The length of the cloned IAR-like cDNA was 1,639bp, which contains a putative 1,335 bp ORF encoding a polypeptide of 444 amino acids. The 5’ UTR and 3’ UTR were 69 bp and 235 bp, respectively. The deduced amino acid sequence of the full-length TL-IAR has 81.3% identity to the IAR of rice, and the levels of identity to the Arabidopsis, IAR3, ILL5, and ILR1 were 75.1%, 71.6%, and 62.1%, respectively. RT-PCR analysis showed that TL-IAR was expressed in several organs, but it was not detected in pollinia. TL-IAR mRNAs gradually accumulated to high levels at the late stages of stigma maturation, and decreased in the stigmata of opened flowers.