The Functional And Transcriptional Analysis Of The PwNAC1 InPicea Wilsonii And Its Homologous Gene ANAC018 In Arabidopsis Thaliana

NAC transcription factor is a kind of plant-specific transcription factor which constitute a major transcription factor family renowned for their roles in several developmental programs. Functional studies of these proteins showed that they are involved in different biological processes, including auxin signaling, embryo and shoot meristem development, abiotic stress tolerance and biotic stress response. Here, according to research of PwNACl in Picea wilsonii, we conducted a study on the functional studies of its homologous gene ANAC018 in Arabidopsis thaliana. We identified a NAC gene from Arabidopsis thaliana, ANAC018 gene, and transformed it into Arabidopsis to detect its function in plant. Our data showed that over-expression of ANAC018 altered the flowering time under red light, altered the fruit senescence time and conferred an enhanced tolerance to salt and drought stress in Arabidopsis. The results were showed as follows:1.To determine whether ANAC018 or PwNACl functions as transcription factor, we observed the localization of ANAC018 and PwNAC1 by transient expression in Arabidopsis. The PwNAC1/ANAC018-GFP fusion protein were predominately localized in the cell nucleus, while the GFP protein alone was detected both in cytoplasm and nucleus, as expected.2.Transactivation of ANAC018 and PwNACl were not detected in yeast, suggesting that ANAC018 and PwNAC1 were likely to act as transcription inhibitor. The C-terminal domain of ANAC018 and PwNAC1 have the activation activity, but the full length of ANAC018 and PwNAC1 have no activation activity. Furthermore, to identify the repression domain of ANACO 18 and PwNAC1 protein which was truncated into 9 fragments and showed that a putative NARD in 128/138 and 126/136 of N-terminal domain which suppressed the activation activity of C-terminal domain, respectively.3.ANAC018 and ANAC029 can interaction in vitro through the method of Yeast Two Hybrid System, but PwNAC1 cann’t interaction with itself, suggesting that ANAC018 act as transcription activator by interacting with other genes or transcription factors and PwNACl cann’t form a homodimer.4.The tissue-specific expression patterns of ANAC018 homologous genes from Arabidopsis were examined by RT-qPCR assays. The expression of ANAC018 was only up-regulated after the maturity of Arabidopsisi, The expression of ANAC018 was up-regulated under Eth treatment.5.To determine whether ANAC018 functions in plants response to salinity and osmotic tolerance, ANAC018 was transformed into Arabidopsis. Overexpression of ANAC018 in Arabidopsis plants increased tolerance to salinity stress and Mannitol treatment, and enhanced the POD and SOD activity.6.To investigate the mechanism of ANACO18 involved in senescence, we selected several SAG genes and examined their expression level by RT-qPCR in ANAC018 over-expression line and mutation plants. Our results suggest over-expression ANAC018 depressed several SAGs (SAG 12/SAG 18/ SAG20/SEN4) expression. Furthermore, we truncated the leaves and cultured in 3μM ABA for 3 days, the leaf senescence of over-expression line was lower than mutation.7.ANAC018 alters flowering time under red light in transformed Arabidopsis. Over-expression ANAC018 could repress some genes expression, such as SOC1 and FT, which advanced flowering, while enhanced gene expression that inhibited flowering.