鐵炮百合花藥專一基因之特性分析

Abstract

LLP40是經由抑制扣除雜合法(suppression-subtractive hybridization)從鐵炮百合花藥乾燥時期的cDNA集合庫中挑出的。LLP40經由5''-RACE和3''-RACE的延長，可獲得一長度為2085 bp的序列。經序列比對，LLP40 5’UTR部分為190 bp，3’UTR為90 bp，ORF為1803 bp，可轉譯出600個胺基酸，預測分子量約為64 kDa，等電點為5.2。LLP40在其N端具有一疏水性訊息胜肽(signal peptide)，經軟體分析為分泌性蛋白。經蛋白質序列比對後得知LLP40與一家族基因glyoxal oxidase有高度的相同性，其與水稻的glyoxal oxidase基因有54%的相同度(identity)，而玉米則是49%。LLP40 N端擁有高度的保留區域(conserved domain)。Glyoxal oxidase已知與木質素(lignin)的降解有關，並受到二價金屬離子調控。利用北方墨漬法分析，LLP40為花藥專一性基因，在花藥成熟晚期大量表現。14~15 cm百合花藥開始表現LLP40 mRNA，隨著花藥成熟，在15 cm花藥大量累積，且至萌發後32小時仍持續表現，顯示LLP40可能對花粉管的生長很重要。為了更進一步了解LLP40對花粉生長的影響(phynotype)，乃利用花粉專一的Zm 13啟動子起動建構了三種不同的重組基因構築，分別為GFP-LLP40，LLP40-GFP與只有LLP40的載體。以基因槍方式送入百合花粉內做短暫性表現，藉以觀察LLP40對花粉萌發與花粉管延長的影響。在螢光顯微鏡觀察下，我們可以觀察到LLP40可能存在於花粉管的細胞質中，花粉管生長並沒有明顯的差別。

LLP40 clone has been identified from a suppression subtractive cDNA library constructed from mRNA isolated at the desiccation stage of lily (Lilium longiflorum) anthers. Using 5''-RACE and 3''-RACE, the LLP40 cDNA with a length of 2,085 bp was obtained. LLP40 contains 190 bp at 5''UTR, 90 bp at 3''UTR, and 1803 bp of ORF (open reading frame) which encodes 600 amino acids with a predicted molecular mass of 63.8 kDa, and its isoelectric point is 5.157. The LLP40 protein has a signal peptide at N-terminus suggesting that it is a secretory protein. Sequences analysis revealed that LLP40 is highly related to a family gene of glyoxal oxidase, characterized as a lignin degradation protease. LLP40 shared 54% with rice and 49% identity with maize glyoxal oxidase. Glyoxal oxidases contain a conserved domain, reported to bind Cu2+ or Fe2+ cation. Northern blot analysis showed that LLP40 mRNA is anther- specific. The LLP40 mRNA was significantly detected in the anther of 14~15 cm and accumulated to the maximum level at 15 cm anthers during maturation. The LLP40 mRNA remained its level of accumulation in the growing pollen tube 32 h after germination. It suggests that LLP40 may be importantly involved in tube growth. To explore whether LLP40 may affect tube growth, particle bombardment of GFP-LLP40 and LLP40-GFP driven by pollen-specific promoter Zm13 showed that LLP40 is located in the cytoplasm of pollen grains and no altered phenotype was obtained.