Cloning and Identification of a LLA-32 cDNA Related to Desiccation and a cDNA Encoding Polygalacturonase during Development in Lilium longiflorum
| Autor: | Chyng-Wen Ko, 柯晴文 |
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| Rok vydání: | 2000 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 88 Pollen development is a series of complex differentiation. Within the lily anthers, pollen mother cells undergo meiosis to produce microspores at the tetrad stages. Consequently, each microspore goes on the first run of mitosis, resulting in the formation of bicellular and mature pollen. At the final stage of maturation, pollen exhibits various degrees of desiccation. During pollen development a subset of protein including the LLA-32 proteins accumulate upon desiccation. To screen and identify the clones encoding LLA-32 proteins in lily pollen, the purified LLA-32 protein was subjected to Lys-C digestion, producing five peptide fragments. The five peptides were sequenced and degenerate primers were designated from the suitable sequences resulted from peptide sequencing. By the use of RT-PCR, a piece of LLA-32 cDNA fragment (LLP-B3) was obtained and sequenced. The LLP-B3 cDNA is 343 bp long. Northern hybridization revealed that the LLP-B3 mRNA is abundant、organ-specific and expressed at the maturation stage of pollen development. Sequence analysis exhibits significant similarity between the amino acid sequence predicted from the LLP-B3 cDNA and a tomato LAT52 which is related to desiccation. The sequence of LLP-B3 is also similar to a group of allergenic proteins. Besides, a fragment of LLP-A1 cDNA was obtained during the screening process. Northern hybridization revealed that the LLP-A1 mRNA is abundant, organ-specific and expressed at the stage of pollen maturation, which are also characteristic of LLA-32 gene products. Therefore, the LLP-A1 cDNA was used as a probe to screen a cDNA library of lily pollen and a full-length cDNA was obtained. The cDNA has 1167 bp in length, containing 61 bp at the 5’-untranslated region, an 891 bp at the coding region encoding 296 amino acids, and 218 bp at the 3’-untranslated region. Sequence analysis exhibits significant similarity of the lily PG protein to a group of polygalacturonases of various species. The lily PG is a hydrophilic protein that contains a signal peptide at the N-terminus. It is a member of clade C polygalacturonase. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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