Molecular cloning and functional characterization of wound-inducible NAC genes in sweet potato (Ipomoea batatas cv. Tainong 57)

Abiotic and biotic stresses cause major losses in crop productivity worldwide. Multiple signaling pathways regulate the stress responses in plants. Transcription factors are one of the groups that are involved in the regulation of signal transduction and gene expression. NAC family is composed of plant-specific transcription factors, and contains 75 and 105 members in rice and Arabidopsis, respectively. We are interested in the NAC genes in sweet potatos (Ipomoea batatas cv. Tainong 57) that may participate in response to wounding, and isolated three sweet potato NAC genes from a phage cDNA library. These genes belong to an ATAF subfamily, which contains genes that are responsive to defenses and stresses. The cDNA and protein sequences of the three genes are highly similar to each other. The cDNA sequences of IbNAC1-1 and IbNAC2-18 contain 900 bp open reading frame (ORF), which encode proteins of 300 amino acids, and include the five conserved blocks of homology that characterize the NAC family. IbNAC97 contains 696 bp ORF, which encodes a protein of 232 amino acids, but only contains three conserved blocks of NAC domain. The C-termini of three proteins are not various, but are very similar. The Southern blotting analysis revealed that there were multiple members in sweet potato NAC family. The gene expression pattern showed that the three genes are abundant in root and tuber, and could be induced by wounding and lots of stress treatments. However, the expression level of IbNAC97 was low. After wounding, the three genes were induced at highest level within thirty minutes, and extended to six hours. Drought and high salinity could induce three genes within thirty minutes, and extended to twelve hours. The cold treatment induced latent expression of three genes. IbNAC97 was much more induced by cold treatment than other stress treatments. Three genes were induced instantly by MeJA, and for a moment by ABA. SA induced gene expressions in thirty minutes and to two hours. The IbNAC1-overexpression Arabidopsis plants were more sensitive to ABA and high salinity, flowered earlier than wild-type plants, and had abnormal flower structure. In conclusion, the three NAC genes reported here are all stress-induced ones in NAC gene family and may be involved in stress signal transduction pathways.口試委員會審定書 謝 i文摘要 iibstract iiibbreviations vontents viable contents viiigure contents viippendix contents viintroduction 1egulatory networks of plant defense and stress signals 1unction of stress-inducible genes 1lant transcription factors 2AC gene family 3ethods and materials 5lant growth and treatments 5DNA library construction 6creening of recombinant cDNA library 6DNA sequencing 7xpression analysis 7omputational analysis 8outhern blotting 8ector construction and generation of transgenic Arabidopsis 8esults 10DNA library construction 10solating NAC genes from cDNA library 10olecular characterization 11nalysis by Southern blot hybridization 12xpression of NAC genes in sweet potato 12verexpression of sweet potato NAC gene in Arabidopsis 13iscussion 15eferences 19able contentsables 26able 1 26able 2 27igure contentsigures 28igure 1 28igure 2 29igure 3 30igure 4 32igure 5 34igure 6 35igure 7 37igure 8 38igure 9 39igure 10 40igure 11 41igure 12 42igure 13 44ppendix contentsppendix 45ppendix Table 1 45ppendix Table 2 46rotocols 4