| Summary: | 碩士 === 國立中興大學 === 生物科技學研究所 === 96 === NAC-like genes, a class of plant-specific transcription factors, are characterized by having a highly conserved 150 amino acid NAC (for NAM, ATAF1, 2, and CUC2) domain at their N-terminal of proteins and have been thought to be involved in the regulation of diverse plant development processes. In this study, five Arabidopsis NAC-like genes AtNACL5, AtNACL7, AtNACL9 in ATAF subgroup and AtNACL11, AtNACL15 in NAC2 subgroup were isolated and analyzed. Further promoter assay by transforming constructs fusing the promoter of these genes with report GUS gene in Arabidopsis indicated that AtNACL5, AtNACL7 and AtNACL15 were highly expressed in roots, cotyledon, shoot apical meristem, and flowers where AtNACL9 and AtNACL11 were only weakly expressed. Further analysis indicated that GFP tagged AtNACL9 and AtNACL15 proteins were able to enter nucleus. Further functional analysis by ectopic expression of the sense and anti-sense for these genes were performed and phenotypic analyzed. Novel phenotypes were observed in transgenic plants. For example, severed alteration of shoot, inflorescence and flower formation was observed in the AtNACL9 RNAi transgenic plants. These results revealed that AtNACL1 and AtNACL9 genes might have redundant function in the regulation of both shoot and flower development in Arabidopsis.
ABCDE model predicts the formation of any flower organs by the interaction of five classes of homeotic genes in plants. Most ’’ABCDE’’ function genes are MADS box genes. MIKC-type MADS-box genes encode a protein consisting of the MADS (M) domain, intervening (I) domain, keratin-like (K) domain, and C-terminal (C) domain. Among these domains, I and K domains are most well known for determining the pattern of homodimerization or heterodimerization of MADS-box proteins. The K domain is involved in protein–protein interaction and is characterized by three strings of heptad repeats (abcdefg)n which are potentially forming coiled coils, with hydrophobic amino acids predominantly in positions a and d. Lily (Lilium longiflorum) B functional MADS box protein LMADS1 has been shown to be able to form homodimers. This is different from the B functional MADS box proteins such as AP3 and PI which can only form heterodimers. Amino acids that responsible for the homodimerization of LMADS1 protein in lily were identified (R136C and R226Q) through mutagenesis assay. To further confirm whether the polarity or charge of the amino acids influenced LMADS1 protein dimerization, different point mutations on R136 were generated and homodimerization analyzed. The result indicated that the ability for homodimerization was recovered in the mutants of R136D、R136K、R136S and was partially recovered in the mutants of R136M. This result revealed that neither the polarity nor the charge of the amino acid in 136 was the factor for homodimerization of LMADS1. The result was correlated with the prediction of the stable formation of the second coiled-coil structure in K domains of LMADS1 by COILS program. The more stable of the second coiled-coil structure the more ability for LMADS1 protein to form homodimers. Furthermore, COILS program also predicted the importance of the two other amino acids beside K2 domain in playing critical role for LMADS1 dimerization.
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