Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90086
標題: Characterization and function analysis of a tapetum/microspore-specific gene in Lilium longiforum
鐵炮百合絨氈層/小孢子基因LLA139之特性及功能分析
作者: 張詔雁
Zao-Yen Chang
關鍵字: 絨氈層
小胞子
小分子富含半胱胺酸蛋白
tapetum
microspore
small cysteine-rich protein
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摘要: 鐵炮百合(Lilium longiflorum) LLA139是利用扣除抑制雜合(suppression subtractive hybridization)的方法自花藥小胞子發育時期cDNA集合庫中選殖出的基因。LLA139 cDNA可轉譯出102個胺基酸,扣除N端30個胺基酸的訊息胜?,估計成熟蛋白的分子量為7.3 kDa。推測其可形成兩個finger motifs的蛋白質。序列比對得知與LLA67及HvLEM1兩蛋白質分別有42%及47%的相同度。特別的是三者蛋白質序列之半胱胺酸呈現高度保守,屬於小分子富含半胱胺酸的蛋白質,但三者功能皆尚未知。原位雜合及北方墨漬分析顯示LLA139為絨氈層和小孢子特有的基因,且不受外加激勃素的誘導。利用激勃素生合成抑制劑uniconazole和乙烯競爭抑制劑 2,5-norbornadiene (NBD)處理後LLA139 mRNA會明顯增加,顯示LLA139分別受乙烯和激勃素的逆調控。然而,同時添加uniconazole和NBD時,LLA139 mRNA的量和未添加的對照組相比反而減少,顯示乙烯與激勃素之間的相互對話(cross-talk)反而使發育花藥中的LLA139基因表現下降。為了進一步探討LLA139的功能,利用絨氈層專一的TAP啟動子建構了三種不同的重組基因構築,分別為full-length (TAP::FL)、刪除N-finger motif (TAP::△N)、刪除C-finger motif (TAP::△C)的載體,以農桿菌方式感染轉型至阿拉伯芥,篩選出T2 homologous子代植株,觀察LLA139蛋白對阿拉伯芥轉殖株花藥發育的影響。三個轉殖株莢果皆很明顯的較野生型短小,然而只有LLA139::△C轉殖株的花粉萌發率較野生型少。藉由掃描式電子顯微鏡觀察,三個轉殖株花粉外壁皆有異物堆積的現象,特別是LLA139::△C花粉外壁部分區域的網格狀結構喪失,呈現平滑的表面。為了找到LLA139的啟動子,進行兩次TAIL-PCR (thermal asymmetric interlaced PCR)向5'UTR總共延伸了1953 bp。LLA139啟動子具有花藥專一性的調控區,包括9個AGAAA、15個GTGA motif、2個LAT56/59 box以及多個植物荷爾蒙調控區。將LLA139基因的啟動子依不同長度之刪除片段分別構築含有β-glucuronidase (GUS)基因轉殖至阿拉伯芥中,發現所有轉殖株GUS訊號皆專一表現在花藥當中。以切片觀察,進一步得知GUS訊號專一表現在絨氈層中。最小刪除片段373 bp中具有一個預測的LAT56/59 box及兩個GTGA motif可能為調控小孢子/絨氈層專一表現的調控區。另外,LLA139f-1612::barnase植株花藥在塑膠切片下觀察,可以觀察到阿拉伯芥轉殖株之花粉發生空泡的異常現象,而且後續裂孔及藥隔的發育皆受到影響,導致花藥無法正常開裂。轉殖株果莢長度短亦無種子產生。將LLA139f-1612::barnase植株雌蕊與野生型花粉雜交後,果莢長度復原,種子產量也恢復正常。LLA139為百合花藥中絨氈層及小孢子專一性的小分子富含半胱胺酸蛋白基因,其啟動子能夠造成雄不孕,具有實際的農業應用價值。
LLA139 transcript was identified from a suppression subtractive cDNA library at the microspore satge of lily (Lilium longiflorum) anthers. The LLA139 cDNA encodes 102 amino acids. Taking out the signal peptide of 30 amino acids at the N-terminus, the estimated molecular mass of mature LLA139 protein is 7.3 kDa. The protein may form a structure of two finger motifs. Sequence analysis indicates that the protein shares 42% and 47% identity with LLA67 and HvLEM1 protein, respectively. Particularly the cysteine residues in the sequence of these proteins are highly conserved. They are classified as small cysteine-rich proteins, but the function is still unknown. In situ hybridization and Northern blot analysis showed that LLA139 mRNA was expressed at the tapetum and microspore. The LLA139 gene was not induced by exogenous gibberellins (GA). With the treatment of uniconazole, an inhibitor of GA biosynthesis and 2,5-norbornadiene (NBD), an inhibitor of ethylene action, we revealed that the level of LLA139 mRNA was significantly elevated, suggesting LLA139 is negatively regulated by either GA or ethylene. However, with the treatment of both uniconazole and NBD, the level of LLA139 mRNA was reduced when compared with the control without treatment. It suggested that a cross-talk between GA and ethylene occurring in the developing anther reduced the expression of LLA139. To further explore the function of LLA139, three constructs: TAP::LLA139 full length (TAP::FL), TAP::deleted N-finger motif (TAP::△N), and TAP::deleted C-finger motif (TAP::△C) were ligated with a rice tapetum-specific promoter (TAP) and transformed into Arabidopsis thaliana by Agrobacterium-mediated transformation system. The T2 homologous lines were obtained and their phenotypes were exemined. The three transgenic lines had shorter siliques than wild-type. The germination percentage of TAP::△C pollen is significantly less than that of the wild type. Scanning electron microscopy revealed the observed amorphous extrabacular protrusions on the exine surface of these transgenic lines. In particular, the TAP::△C pollen showed patches of smooth exine without a visible exine network. A region of LLA139 promoter sequence with 1953 bp upstream from start codon was identified using two runs of TAIL-PCR approach. The LLA139 promoter contains putative anther-specific cis-elements, including nine AGAAA, fifteen GTGA and two LAT56/59 boxes. The LLA139 promoter was subjected to a series of 5'-deletion by which the full-length and three 5'-deleted fragments were fused with the GUS (β-glucuronidase) reporter gene, and introduced into Arabidopsis for functional assays. The LLA139 transgenic lines with various 5'-deleted fragments exhibited GUS signals in the anther, indicating of anther-specificity. The cross-sections of LLA139f-1612::GUS and LLA139f-373::GUS detected GUS signal only in the tapetum. The GUS signal in the anther of LLA139f-373::GUS line is reflected by the sequence contains two GTGA motifs and a LAT56/59 box. In addition, the anthers of the LLA139f-1612::barnase transgenic lines contained vacuolated pollen which differred from the normal pollen of wild-type. The development of stomium and septum in LLA139f-1612::barnase transgenic lines were accordingly affected, resulting in an inability of dehiscence. The LLA139f-1612::barnase transgenic siliques were short and seedless. When the female organs of LLA139f-1612::barnase transgenic lines were cross-pollinated with wild-type pollen, the siliques were reverted to the normal size with regular seed production, similar to wild type. LLA139 gene is a tapetum and microspore-specific gene that encodes a small cysteine-rich protein in Lilium longiflorum, and the identified promoter that causes male sterility has a practical value of agricultural application.
URI: http://hdl.handle.net/11455/90086
文章公開時間: 2017-08-31
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