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標題: 百合花粉專一性基因編譯一嶄新、富含脯胺酸、甘胺酸和絲胺酸的蛋白質
A lily (Lilium longiflorum) pollen-specific gene encoding a novel proline, glycine and serine-rich protien
作者: 徐思維
Hsu, Ssu-Wei
關鍵字: 百合
出版社: 生物科技學研究所
LLP12-2是百合(Lilium longiflorum)花粉發育晚期表現的專一性基因。以百合葉片genomic DNA為模版,進行PCR(Polymerase chain reaction)分析得知LLP12-2基因包含一長度為669 bp的轉譯區,其間有兩個近5’端處之introns,其大小分別為72 bp及98 bp。LLP12-2基因可編譯出222個胺基酸,由序列分析可知,LLP12-2具有兩個(Gly-X)n motifs,這和glycine-rich protein相似。在胺基酸組成中,有三個胺基酸具有較高的含量:脯胺酸(15.8 %)、甘胺酸(11.3 %)和絲胺酸(10.8 %)。由胺基酸的序列比對可知,LLP12-2蛋白質無法在基因庫中比對到具有較高相似性的蛋白質,因此,LLP12-2蛋白質仍然是一個未知的蛋白質。以LLP12-2 cDNA為探針,進行genomic Southern的分析得知LLP12-2可能為單一基因。另外,利用大腸桿菌來表現LLP12-2蛋白質,經Ni-NTA瓊膠純化之後,將其施打至兔子中,以取得抗血清。以轉漬親和(blot-affinity)純化的LLP12-2抗體,進行西方墨點法(western blotting)的分析,結果得知LLP12-2蛋白質具有花粉專一性,只存在於花粉中,而其它的花器官和營養器官則沒有。LLP12-2蛋白質會在花粉成熟時期出現,並隨著花粉的發育和熟成而迅速累積,而且會受脫水乾燥所誘導,因此,此蛋白質可能與花粉成熟前的脫水現象有關。而在花粉萌發一段時間之後,仍留存有大量的LLP12-2蛋白質,由此可知,此蛋白質也可能與花粉的萌發和花粉管的生長有關。另外,次細胞分離蛋白質的分析顯示,LLP12-2蛋白質可能存在於花粉的細胞質部分。

LLP-12-2 is a gene that expresses specifically in the late stage of pollen development in lily (Lilium longiflorum) plants. Using the lily leaf genomic DNA as a template, polymerase chain reaction revealed that the LLP12-2 gene includes the coding region of 669 bp interrupted by two introns of 72 bp and 98 bp, respectively, near the 5' end of the gene. The gene encodes a protein that has 222 amino acid residues containing a couple of (Gly-X)n motifs, a characteristic of cell wall proteins, which is similar to the glycine-rich proteins (GRPs). The LLP12-2 protein is rich in proline (15.8 %), glycine (11.3 %) and serine (10.8 %). Amino acid sequence alignment indicated that LLP12-2 protein is an unknown protein because no significant similarity between LLP12-2 and other known proteins. Genomic Southern of lily leaf DNA indicated that LLP12-2 is a single gene. To obtain anti-LLP12-2 antiserum, the coding region of LLP12-2 cDNA was introduced into pET32a and expressed in the Escherichia coli. The overexpressed protein was purified by the Ni-NTA agarose. Using the blot-affinity purified anti-LLP12-2 antibody, the immunoblots indicated the LLP12-2 is a pollen-specific protein, which exists only in the pollen, but not in any other floral organs and vegetative organs of lily plants. Premature-drying suggested that the LLP12-2 protein is also a dehydration-induced protein. The LLP12-2 protein appeared in the late stage of pollen development and accumulated rapidly as pollen grew to mature prior to desiccation. The function of LLP12-2 protein may be involved in the pollen germination and pollen tube growth because it still exists in the pollen or pollen tube after germination. In addition, subcellular immunodetection of the LLP12-2 protein indicated that the protein may exist in the cytoplasm.
Appears in Collections:生物科技學研究所

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