JIA-2018-09

2011 CHEN Ke-qin et al. Journal of Integrative Agriculture 2018, 17(9): 2007–2014 protoplasts, and the GFP empty vector was used as a control (Fig. 2-A). Then, we performed a yeast two-hybrid assay to detect the transcription activation of CpSND1. Constructs of the CpSND1 gene as a full-length cDNA or with N- and C-terminal deletions were tested for transcriptional activation activity in yeast cells (Fig. 2-B); the full-length cDNAand the C-terminal of CpSND1 exhibited transcriptional activity in yeast cells. These results demonstrated that the CpSND1 protein is targeted to the nucleus and is able to activate transcription in yeast cells, indicating that it is a transcription activator. 3.3. Overexpression of CpSND1 induced stunted growth in Arabidopsis To determine the biological function of CpSND1 , the recombinant plasmid pRI101- CpSND1 was introduced into A . thaliana . In transgenic Arabidopsis , the level of CpSND1 sharply increased (Fig. 3-A). In addition, the plants that overexpressed CpSND1 appeared to have restricted growth, curled leaves, sepal dysplasia and sterility (Fig. 3-B and C). These phenotypes induced by CpSND1 were similar to those induced by AtSND1 (Zhong et al . 2006). To study whether CpSND1 could participate in secondary wall biosynthesis, RT-PCR was performed to detect the expression of the synthesis genes during secondary wall formation in transgenic Arabidopsis . In transgenic lines 1, 4 and 5, the biosynthesis genes of lignin, cellulose and xylan were significantly induced (Fig. 3-D). To determine whether CpSND1 functioned in regulating secondary wall thickening in stems, transverse sections were taken from the basal stems of wild-type (WT) and transgenic plants. Compared with WT plants, CpSND- overexpressing plants had thicker secondary cell walls in the xylem, as the red arrows indicate, and lignin accumulated in xylem cells, which were stained by sarranine (Fig. 3-E). III CpSND1Δ N II CpSND1Δ C A Empty vector CpSND1-GFP GFP Bright field Nucleus marker Merged B I II III III II I –T/–H/–A/+X-alpha-gal –T I CpSND1 1–150 aa DNA-binding domain Activity domain 151–423 aa Fig. 2 Subcellular localization and transactivation assay of CpSND1. A, 35S:: CpSND1 -GFP fusion gene and 35S:: GFP control plasmid were transformed into rice protoplasts. SND1, secondary wall-associated NAC domain protein1; GFP, green fluorescent protein. The transformed cells were imaged by confocal microscopy after transformation for 16 h. B, the full-length, N-terminal and C-terminal sequences of CDS (coding sequence) of CpSND1 were fused in-frame, separately, to the GAL4 DNA-binding domain in pGBKT7 and transformed into Y2H Gold yeast cells. The transformed cells were plated onto SD/–T (growth control) and SD/–T/–H/–A/+X-alpha-gal medium.