Characterization of a novel early-gene during anter development in Lilium longiflorum；The lily ASR targeting to nucleus correlates with desiccation in developing pollen of lilium longiflorum.

• Main Authors: Chin-Hsien Wu, 吳志賢
• Other Authors: 王國祥
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/76488504325124328589

碩士 === 中興大學 === 生物科技學研究所 === 94 === The LLA-67 clone was identified from the suppression-subtractive cDNA library at the stage of microspore development in lily (Lilium longiflorum) anthers. Using 5''-RACE, a full-length of LLA-67 with a size of 565 bp was obtained. The LLA-67 encodes a protein of 101 amino acids, having a calculated molecular mass of 10.1 kDa and a calculated isoelectric point of 3.6. Sequence alignment indicates that LLA-67 shares no sequence similarity with known proteins. The protein contains a signal peptide at N-terminus, suggesting a secretory protein. Northern analysis indicates that LLA-67 transcripts are anther-specific and temporally expressed only at the phase of microspore development. In situ hybridization suggested that LLA-67 transcripts were only detected in tapetum, a confirmation of tapetum origin. Unlike other tapetum-specific genes induced by gibberellin, LLA-67 was not induced by GA3. LLA-23, an ASR (abscisic acid-, stress-, ripening-induced) protein was isolated from lily pollen. The ASR protein contains a nuclear localization sequence at C-terminus. Histological differences of various developmental stages of pollen grains were examined. At 6-cm buds, microspore is in a uni-nucleate form in the anther and amyloplasts of the vegetative cell begin to form. Mitosis occurs in microspores at 8-10-cm buds, giving rise to generative cells, and amyloplasts in the vegetative cells tend to aggregate. As the lily buds grow to 12 cm, aggregated form of amyloplasts becomes disperse. Immunogold labeling analysis indicated that ASR protein was not detected at microspores of 6-cm buds. At 8-10-cm buds, higher amount of ASR proteins were detected at the vegetative cytoplasm than at the vegetative and generative nuclei. As buds grow to 12-14 cm, ASR protein in the generative nuclei reaches to the highest amount, vegetative nuclei the next, and vegetative cytoplasm displays the least amount of ASR protein. Taken together, our results provide a clear-cut evidence that ASR protein enters into the nucleus at 12-cm buds a stage immediately before desiccation occurs in the anther (13-cm buds).