百合花粉專一性LLP90和LLP13-2cDNAs之特性分析

Abstract

中文摘要 百合 (Lilium longiforum) 花粉的LLP-90 cDNA選殖體是由鐵砲百合成熟花粉cDNA集合庫中所挑出的，由於含有229 bp並非全長，因此利用5′-和3′-RACE (rapid amplification of cDNA ends) 方式找出了包含ㄧ個2,259 bp的轉譯區，可轉譯出752個胺基酸的蛋白質。經過序列比對後發現百合LLP-90蛋白質與其他OPT (oligo peptide transporter) 家族有61∼45％的一致性。LLP-90蛋白質預測分子量為82.7 kDa，pI值為9.1。其胺基酸序列具有兩個OPT family高度保留的區域，分別是NPG和KIPPR motif，以及含有十二個穿膜的序列區域。北方墨點法顯示LLP-90 mRNA具有花粉專一性，且只在花粉成熟期才開始表現，經過花粉乾燥測試發現LLP-90 mRNA並不會因為乾燥而誘導產生，在花粉管萌發八小時後LLP-90 mRNA稍微下降，而十六與二十四小時相較於八小時有稍微提升的情形。ABA處理則會誘導LLP-90 基因的表現。 百合LLP13-2 cDNA長度為1789bp，具有一open reading frame 可轉譯出375個胺基酸的蛋白質。為了取得抗百合LLP13-2抗體，利用大腸桿菌將LLP13-2全長與前半段cDNA黏接到表現載體大量表現LLP13-2融合蛋白質，以Ni-NTA 與GS-bead瓊膠純化，再施打入大鼠體內製造抗LLP13-2多株抗體，所得到的全長與前半段的多株抗體可以辨認大量表現的LLP13-2蛋白質。

Abstract LLP-90 cDNA clone was selected from the cDNA library of lily (Lilium longiflorum Thunb. cv. Snow Queen) mature pollen.The cDNA which is 229 bp in this clone and is not full length.Using 5'- and 3'-RACE (Rapid amplification of cDNA ends) analysis, a full-length cDNA with a size of 2259 bp was obtained.Alignment of lily LLP-90 with known sequences in Genebank shows the protein is peptide transporter exhibiting 61-52% sequence identity compare to the other OPT (oligo peptide transporter) family.The predicted amino acid molecular weight is 82.7kDa and pI is 9.1.There are two highly conserved motifs, NPG and KIPPR motifs in OPT family and 12 transmembrane region.The Northern-blot indicated that LLP-90 mRNA was pollen specific, and began expressing in the pollen maturation stage. Premature drying analysis exhibited LLP-90 mRNA was not induced by dessication treatment. When germinated, LLP-90 mRNA expression level in 8 hour was slightly lower than mature pollen, and the mRNA expression level was more higher in 16 and 24 hour than 8 hour. On the other hand, LLP-90 mRNA was induced by ABA. Lily LLP13-2 gene contains a coding region of 1789 bp encoding 375 amino acids.To obtain LLP13-2 antiserum, LLP13-2 cDNA full length and half length coding regions were introduced into vector and expressed in Escherichia coli BL21 from which fusion proteins were isolated and purified by Ni-NTA and GS-bead agarose. These antibodies can recognize LLP13-2 overexpression proteins.