| Summary: | 碩士 === 中興大學 === 生物科技學研究所 === 94 === A suppression-subtractive cDNA library was successfully constructed at the stage of microspore development in lily (Lilium longiflorum) anthers. After primary screening near to ten thousand clones, 170 clones that showed positive signals at the microspore stage and no signal detation at the pollen maturation stage during development were identified. After secondary screening, classification and sequencing of these clones, we have finally identified 30 individual genes in which nine may be novel. After reverse northern blot analysis, most genes can be induced by GA3、MeJA and ABA, respectively. LLA-89 and LLA-115 were chosen for further investigation. Full lengths of the two cDNAs were obtained using 5′-and 3′-RACE. LLA-89 is a LIM4 cDNA that contains 493 bp. It encodes a protein of 100 amino acids, having a caculated molecular mass of 10 kDa and an isoelectric point of 3.2. The predicted LIM4 protein is rich in serine、proline and leucine. It was suggested that the protein may be related to the meiotic event of pollen mother cells. LLA-115 contains 545 bp encoding a protein of 112 amino acids. The protein shares 34% identity with a putative cell wall protein in Arabidopsis. LLA-115 protein is rich in glycine、serine、valine and has a caculated molecular mass of 11.6 kDa and an isoelectric point of 5.8. In situ hybridization indicated that LLA-115 mRNA is located in the tapetum. Both LLA-89 and LLA-115 proteins contain a signal peptide of 22 and 25 amino acids at the N-terminus respectively, suggesting the two proteins are secretory proteins. After secretion, the LLA-115 protein eventually deposits in the exine and becomes a component of pollen wall. The transcripts expressed by both genes are anther-specific and their expression only appears at the phase of microspore development. The two genes can be induced by GA3 and that the induction of LLA-89 is more sensitive than that of LLA-115.
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