Characterization and function analysis of a tapetum/microspore-specific gene in Lilium longiforum

• Main Authors: Zao-Yen Chang, 張詔雁
• Other Authors: Co-Shine Wang
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/87511205740085610131

碩士 === 國立中興大學 === 生物科技學研究所 === 102 === 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.