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標題: 鐵炮百合LLP-Rop1基因於花粉生長之功能性分析
Functional Analysis of a Lily (L. longiflorum) LLP-Rop1 Gene in Pollen Growth
作者: 鄭兆伶
Cheng, Chao-Lin
關鍵字: pollen tube growth;百合花粉
出版社: 生物科技學研究所
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利用抑制扣除雜合 (suppression subtractive hybridization) 的方式在鐵砲百合 (Lilium longiflorum) 成熟花藥篩選出LLP-Rop1。藉由5''-及3''-RACE的方法得知LLP-Rop1之全長為594 bp。LLP-Rop1 cDNA可轉錄出197個氨基酸,估計其蛋白質分子量約為21.7 kDa,等電點為9.1。經由序列比對發現,LLP-Rop1屬於Ras superfamily 中的Rop (Rho-like GTPases of plants) GTPases 之一員。LLP-Rop1是屬於晚期基因,不含插入子 (intron)。LLP-Rop1 mRNA會在百合的根、莖、及成熟的花絲、花藥中存在。花粉萌發後的24小時內皆可偵測到LLP-Rop1 mRNA的存在,顯示其在花粉管發育伸長中扮演重要角色。為了進一步研究LLP-Rop1的功能,分別創造持續性大量表現 (constitutively active, CA-llp-rop1Q64L) 和顯性抑制型 (dominantly negative, DN-llp- rop1D121A) 的定點突變體,構築在含有Zm13 啟動子 (promoter) 以及帶有 eCFP (enhanced cyan fluorescent protein) 之質體上,以電穿孔 (electroporation) 的方式送入百合的花粉管中。結果顯示,野生型 (eCFP-LLP-Rop1) 和持續性大量表現型的突變 (eCFP-CA-llp-rop1 Q64L) 會抑制花粉管的延長和造成管尖膨大的現象。而且兩者的融合蛋白分佈在膨大的花粉管尖及次尖端區 (subapical region)。顯性抑制型的突變 (eCFP-DN-llp-rop1 D121A),則無花粉管膨大的現象,但抑制花粉管的延長。因此藉由大量表現LLP-Rop1所造成花粉管的去極性 (depolarized) 生長和花粉管尖端的膨大可証實LLP-Rop1在花粉管的伸長上扮演著重要的角色。
Rop蛋白質會和RIC (ROP-interactive CRIB-motif containing protein) 結合以調控下游的訊息傳導。為了進一步的了解LLP-Rop1和RIC相互關係,利用雷射共軛焦掃描顯微鏡觀察其分佈的相關性。將百合的LLP12-2 (Group Ⅱ RIC) cDNA的全長構築在含有Zm13 啟動子以及eYFP (enhanced yellow fluorescent protein)之質體上。以電穿孔的方式和ECFP-LLP-Rop1之質體共同表現於百合的花粉管中。發現LLP-Rop1和LLP12-2兩蛋白都分佈於花粉管尖及次尖端區。進一步的FRET (fluorescence resonance energy transfer) 實驗驗證LLP12-2會直接和LLP-Rop1發生相互作用。

LLP-Rop1 was identified from a suppression subtractive cDNA library from mature pollen of Lilium longiflorum. The full length of LLP-Rop1, 594 bp, was obtained using 5''- and 3''-RACE. LLP-Rop1 encodes 197 amino acids, having a calculated molecular mass of 21.7 kDa and an isoelectric point of 9.1. Sequence alignment revealed that LLP-Rop1 is a member of Rop (Rho-like GTPases of plant) GTPases of Ras superfamily. LLP-Rop1 is a late gene that contains no intron. Its mRNA was detected in roots, stems, mature filaments, and pollens. LLP-Rop1 mRNA remained its level of accumulation 24 h after germination suggesting of a critical role in tube growth. To investigate the function of LLP-Rop1, constitutively active (CA) mutant, CA-llp-rop1Q64L, and dominantly negative (DN) mutant, DN-llp-rop1D121A, were created and of which the mutant gene was fused with Zm13 promoter and eCFP (enhanced cyan fluorescent protein) and expressed them in lily germinating pollen by electroporation. It showed that eCFP-LLP-Rop1 and eCFP-CA-llp-rop1Q64L caused inhibition of tube elongation and formed a swelling tip. In addition, the fusion proteins were localized to the apical and subapical region of bulbous pollen tubes. However, eCFP-DN-llp-rop1D121A caused the inhibition of pollen tube growth but no occurrence of swelling in the tube tip.
It's known that AtRop proteins can target various RIC (ROP-interactive CRIB-motif containing protein) to control specific downstream signaling of Rop-dependent pathway. To study the interaction between LLP-Rop1 and LLP12-2 (Group Ⅱ RIC), LLP12-2 was fused with eYFP (enhanced yellow fluorescent protein) driven by Zm13 promoter. Both eCFP-LLP-Rop1 and eYFP-LLP12-2 were cooverexpressed in pollen tube using electroporation. It showed that LLP-Rop1 and LLP12-2 were colocalized at the apical and subapical regions of pollen tubes, implying the possibility that LLP12-2 is a target protein of LLP-Rop1. Furthermore, the analysis of FRET (fluorescence resonance energy transfer) was demonstrated that LLP12-2 is a direct target of LLP-Rop1 in vivo.
其他識別: U0005-0808200610305300
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