JIA-2018-09

2009 CHEN Ke-qin et al. Journal of Integrative Agriculture 2018, 17(9): 2007–2014 using ClustalX 1.83, and then a phylogenetic tree was derived from the multiple alignment using the neighbour- joining method in MEGA6. Analysis of the conservation of the NAC domain in hawthorn SND1 was determined by using the NCBI CDD tool (http://www.ncbi.nlm.nih.gov/Structure/ cdd/wrpsb.cgi). The respective domains of NAC proteins were aligned using DNAMAN software. 2.4. Cellular localization assays CpSND1 -coding sequences were amplified from the cDNA and then inserted into the C-terminal side of green fluorescent protein (GFP) at the Xba I and Xma I sites in the vector pGPTII-GFP. The correct recombination plasmid was sequenced to ensure insert accuracy. pGPTII-GFP (positive control) and fusion constructs pGPTII- CpSND1 -GFP were employed for localization studies. The constructs were transiently expressed in rice protoplasts, and then the GFP fluorescence was observed using a laser scanning confocal microscope. All the primers are listed in Appendix A. Transferred protoplasts were detected under a Nikon E600 fluorescence microscope (Nikon, Japan), and GFP fluorescence was visualized with a filter setting consisting of an excitation filter of 450–490 nm, a dichroic mirror of 510 nm, and a barrier filter of 520–560 nm. Images were captured with a SPOT2 Slider charge-coupled device camera and associated software (Sterling Heights, MI, USA). 2.5. Transcriptional activity assay in yeast For the transactivation activity assay, the full-length CpSND1 , the N-terminal NAC domain of CpSND1 ( CpSND1-N ) and the C-terminal of CpSND1 ( CpSND1-C ) were amplified and inserted into vector pGBT9 (BD) (Clontech, Palo Alto, CA, USA) at the Eco RI and Sal I sites. All primers are listed in Appendix A. All the fusion constructs were introduced into the yeast strain Y2H GOLD according to manufacturer’s instructions, and the transformed yeast cells were grown on SD/–T (growth control) and SD/–T/–H/–A/+X-alpha-gal plates to check transactivation activity. 2.6. Quantitative RT-PCR Total RNAwas extracted from the Arabidopsis leaves using TRIzol Reagent and precipitating with lithium chloride and chilled absolute ethanol. First-strand cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNAEraser (Perfect Real Time) (TaKaRa, Dalian, China). The reverse transcription products of cDNA, diluted four times, were used as the template for quantitative PCR. Reactions were set up with SYBR Green Fast qPCR Mix (TaKaRa) according to the manufacturer’s instructions in a total volume of 20 μL with each primer at 0.2 μmol L -1 . The amplification programme was as follows: one cycle of 30 s at 95°C followed by 40 cycles of 5 s at 95°C and 10 s at 60°C. All reactions were run in triplicate, and average values were calculated. Relative expression levels of target genes and SD values were calculated using the 2 −ΔΔC T method (Livak and Schmittgen 2001). All the primer sequences are listed in Appendix A. 3. Results 3.1. Isolation and sequence characteristics of haw- thorn SND1 From early transcriptome data of soft-endocarp and hard-endocarp hawthorns (Dai et al . 2013), we found four NAC family transcription factors that were strongly down-regulated in the fruits of soft-endocarp hawthorn compared to the fruits of hard-endocarp hawthorn. We suspected that they might participate in the biosynthesis of lignin or secondary cell walls. Therefore, according to the conservative NAC domain of these TFs, we aligned other NAC TFs in other species, which all contained a similar NAC DNA-binding domain, by the NCBI (https://www.ncbi . nlm.nih.gov/pubmed) and PLAZA (https://bioinformatics. psb.ugent.be/plaza/ ) web blast. Because AtSND1 was the first key switch involved in secondary wall formation, 8_Unigene_BMK.37276 (log 2 (S7/H8)=–6.31) gene which was the most homologous to AtSND1 (Fig. 1-A), was named as CpSND1 . The sequence alignment results also indicated that there were six NAC genes in Brassica rapa , two genes in Populus trichocarpa , four genes in Gossypium raimondii , one gene in Fragaria vesca , one gene in Citrus sinensis , two genes in Malus domestica and one gene in Prunus persica closer to AtNST1 - AtNST3 . In addition, four NAC TFs of G . raimondii , one of F . vesca , one of C . sinensis , two of M . domestica , one of P . persica and two of B . rapa were closer to AtSND1 . The amino acid sequence alignment showed that all of the SND1 homologous genes had a highly conserved NAC DNA-binding domain (Fig. 1-B). These bioinformatic results revealed that CpSND1 may have a similar function as AtSND1. 3.2. CpSND1 is localized in the nuclei and acts as a transcription activator in yeast cells To detect whether CpSND1 is a transcription factor localized in nuclei, the coding sequence of CpSND1 was fused to GFP and expressed under the control of the constitutive CaMV35S promoter. Merged images of GFP fluorescence showed that CpSND1-GFP localized in the nuclei in rice