Abstract:
Anthocyanidin reductase (ANR) is one of the key enzyme in the flavonoid biosynthetic pathway, and its catalytic activity is important for the synthesis of plant anthocyanin. In this study, specific primers were designed according to the transcriptome data of
Lonicera japonica Thunb., and the CDS, gDNA and promoter sequences of
ANR genes from
Lonicera japonica Thunb.
and Lonicera japonica Thunb. var.
chinensis (Wats.) Bak. were cloned. The results showed that the CDS sequences of
LjANR and
rLjANR were 1 002 bp, the gDNA sequences were 2 017 and 2 026 bp respectively, and the promoter sequences were 1 170 and 1 164 bp respectively.
LjANR and
rLjANR both contain 6 exons and 5 introns, which have the same length of exons and large differences in introns. The promoter sequences both contain a large number of light response, hormone response and abiotic stress response elements. Bioinformatics analysis showed that both
LjANR and
rLjANR encoded 333 amino acids and were predicted to be stable hydrophobic proteins without transmembrane segments and signal peptides. The secondary structures of LjANR and rLjANR were predicted to be mainly consisted of
α-helix and random coil. Sequence alignment and phylogenetic analysis showed that LjANR and rLjANR had high homology with
Actinidia chinensis var.
chinensis,
Camellia sinensis and
Camellia oleifera, and were closely related to them. The expression levels of
LjANR and
rLjANR were the highest in flower buds and the lowest in roots. The expression patterns at different flowering stages were similar, with higher expression levels in S1 and S2 stages and then gradually decreased until reaching the lowest level in S4 stage, after a slow increase in S5 stage, the expression levels decreased again. The expression levels of
ANR genes in the two varieties showed significant differences in roots, S2 and S5 stages, while the differences in stems, flower buds, S1, S3 and S6 stages were extremely significant. The prokaryotic expression vector pET-32a-LjANR was constructed for protein expression. The target protein was successfully expressed of about 59 kD. This study lays a foundation for further study on the function of
ANR gene and provides theoretical guidance for breeding new varieties of
Lonicera japonica Thunb.