Total lignin content in pomegranate seed coat and expression analysis and preliminary functional analysis of PgSND1 gene

Abstract: 【Objective】In order to study the mechanism of the formation of seed hardness and the functionof PgSND1 on lignin synthesis in pomegranate seed coat.【Methods】We measured the seed hardness andlignin content of pomegranate in different cultivars and different development stages，and analyzed the ex⁃pression pattern of PgSND1 in different tissues，cultivars and development stage of pomegranate，and howPgSND1 functions as a transcription factor.【Results】There was a positive correlation between the hard⁃ness and the lignin content of‘Tunisia’soft seed and‘Sanbai’at different developmental stages，and thecorrelation coefficient was 0.964 1. PgSND1 was isolated from the‘Tunisia’pomegranate genome. Weused the ClustalW software and MEGA5.0 software to systematically analyze the relationship between thePgSND1 and the Arabidopsis thaliana NAC family members. The results showed that PgSND1 was in thesame subgroup as AtNST1-3，and was in the same branch as AtNST3（SND1），indicating that their closerelationship. The open reading frame of PgSND1 was 1 245 bp，and encode a protein with 415 amino ac⁃ids. Phylogenetic analysis showed that PgSND1 shares 48.324% similarity with AtNST3. PgSND1 has aconserved NAC-like domain that contains five subdomains at the N-terminus，and contains a conservedLP and WQ motif at the C-terminus，which are similar to NST1-3 in Arabidopsis thaliana. We create thefusion construct of pGBKT7- PgSND1, pGBKT7- PgSND1N and pGBKT7- PgSND1C. The fusion vectorwere transformed into yeast strains AH109 and Y187（containing the ADE2 and LacZ reporter genes），re⁃spectively. The full length of PgSND1 and the C-terminus of PgSND1 showed transcriptional self-activa⁃tion activity，and the N-terminus of PgSND1 lacks the activity of transcriptional self-activation. PgSND1can interact with itself in the yeast cell，therefor，PgSND1 functions by forming homodimers. The expres⁃sion level of PgSND1 was higher in leaves，roots and stems of‘Tunisia’，but it was little expressed at 30days after Booting，and the expression level of PgSND1 increased at 60 days and 120 days after booting.While，PgSND1 was little expressed in‘Sanbai’.【Conclusion】We speculated that PgSND1 may inhibitethe synthesis of lignin in pomegranate seed coat.