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Characterization of two unknown anther-specific genes in Lilium longiflorum
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|摘要:||LLP-14和LLP-69是利用抑制扣除雜合法(suppression-subtractive hybridization)從鐵炮百合花藥乾燥時期的cDNA集合庫中所挑出的cDNA片段。利用5′-RACE和3′-RACE得到LLP-14 cDNA長為1,600 bp，可轉譯出492個胺基酸的酸性蛋白質，預測分子量大約為54 kDa。藉由北方墨漬法預測LLP-14 mRNA全長約1,700 bases，可知目前LLP-14 cDNA仍未完整。利用Kyte-Doolittle軟體測出N端具有19個胺基酸的疏水性訊息胜肽(signal peptide)，LLP-14為親水性的蛋白。經過序列比對發現LLP-14蛋白與葡萄(70%)、水稻(66%)、和阿拉伯芥(62%)蛋白質具有相當高的相同度(identity)，屬於未知功能的蛋白質(unknown protein)。利用5′-RACE和3′-RACE得到LLP-69 cDNA全長為1,934 bp，可轉譯出510個胺基酸的偏鹼性蛋白質，預測分子量為58 kDa。利用北方墨漬法預測全長約2,700 bases，可知目前LLP-69 cDNA仍未完整。利用Kyte-Doolittle軟體預測，發現LLP-69也是親水性的蛋白，沒有訊息胜肽序列，但在蛋白質N端和C端有疑似的進核序列(nuclear localization signal)。經過序列比對之後發現此蛋白和水稻的SF16蛋白有52%的相同性，也和阿拉伯芥的calmodulin 結合蛋白有50%的相同度。利用北方墨漬法分析，顯示出LLP-14和LLP-69都為花藥專一性的表現基因，而且都只表現在花粉成熟時期。LLP-14和LLP-69兩基因的表現，在花粉萌發24小時後仍能偵測到LLP-14的mRNA，而LLP-69的mRNA在花粉萌發32小時仍能偵測到，因此推測LLP-14和LLP-69可能和花粉管的生長有關。利用基因槍將不同的構築載體以花粉專一性啟動子Zm13送入百合花粉中發現GFP-LLP69的融合蛋白會集中在花粉管的營養核(vegetative nucleus)和生殖核(generative nucleus)中。為了進一步探討LLP-69蛋白質，因此選殖可轉譯區全長、LLP-69C以及LLP-69N cDNAs片段放入pET29a載體在大腸桿菌BL21(DE3)中表達蛋白質，以瓊膠純化後的LLP-69C蛋白質打進兔體內產生抗血清，以便進一步分析LLP-69蛋白質。|
LLP-14 and LLP-69 clones have been identified from a suppression subtractive cDNA library constructed from mRNA isolated at the desiccation stage of lily (Lilium longiflorum) anthers. The method of 5'- and 3'-RACE PCR was used to obtain the LLP-14 cDNA with a size of 1,600 bp that encodes an acidic poly peptide of 492 amino acids with a calculated molecular mass of 54 kDa. Northern blot analysis showed that LLP-14 mRNA is about 2,690 bases, indicating that the obtained LLP-14 cDNA sequence is incomplete. The hydrophilic LLP-14 protein contains a signal peptide at the N-terminus. Sequence alignment reveals resemblance to LLP-14 is a grape hypothetical protein (70% identity), a rice hypothetical protein (66% identity) and an Arabidopsis unknown protein (62% identity). On the other hand, the method of 5'- and 3'-RACE PCR was used to obtain the LLP-69 cDNA with a size of 1,934 bp that encodes a basic poly peptide of 510 amino acids with a calculated molecular mass of 58 kDa. Northern blot analysis showed that LLP-69 mRNA is about 2,700 bases, indicating that obtained LLP-69 cDNA sequence is incomplete. The hydrophilic LLP-69 protein has a NLS (nucleus localization signal) sequence at N-terminus and C-terminus. Sequence alignment reveals resemblance to LLP-69 is a rice SF16 protein (52% identity) and an Arabidopsis calmodulin binding protein (50% identity). Northern blot analysis indicated that both LLP-14 and LLP-69 mRNAs were specifically expressed in the anther at the stage of pollen maturation during anther development. LLP-14 remained mRNA its level of accumulation in the germinating pollen even 32 hours after germination, and LLP-69 mRNA remained mRNA its level of accumulation in the germinating pollen even 32 hours after germination, indicating that LLP-14 and LLP-69 may play a critical role during pollen tube growth. Particle bombardment of GFP-LLP-69 has demonstrated that LLP-69 protein is located at vegetative and generative nuclei of pollen grains. The coding region of LLP-69, LLP-69C and LLP-69N cDNAs were introduced into pET29a vector and expressed in Escherichia coli BL21 (DE3) . Only LLP-69C fragment was successfully expressed by E.coli. The overexpressed protein was isolated, purified and injected into rabbits to obtain antiserum for further analysis.
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