Background
As a class of natural antioxidants in plants, fruit flavonol metabolites are beneficial to human health. However, the regulatory networks for flavonol biosynthesis in most fruits are largely unknown. Previously, we reported a spontaneous pear bud sport ‘Red Zaosu’ (
Pyrus bretschneideri
Rehd.) with a high flavonoid content in its fruit. The identification of the flavonol biosynthetic regulatory network in this mutant pear fruit is crucial for elucidating the flavonol biosynthetic mechanism in fruit.
Results
Here, we demonstrated the
PbMYB12b
positively regulated flavonols biosynthesis in ‘Red Zaosu’ fruit. Initially, we investigated the accumulation patterns of four major quercetin glycosides and two major isorhamnetin glycosides in the fruit of ‘Red Zaosu’ and its wild-type ‘Zaosu’. A PRODUCTION OF FLAVONOL GLYCOSIDES (PFG)-type MYB transcription factor
PbMYB12b
was also screened for because of its correlation with flavonol accumulation in pear fruit. The biofunction of
PbMYB12b
was verified by transient overexpression and RNAi assays in pear fruit and young leaves. Overexpression of
PbMYB12b
enhanced the biosynthesis of quercetin glycosides and isorhamnetin glycosides by positively regulating a general flavonoids biosynthesis gene
PbCHSb
and a flavonol biosynthesis gene
PbFLS
. This finding was also supported by dual-luciferase transient expression assay and transient β-glucuronidase (GUS) reporter assay.
Conclusions
Our study indicated that
PbMYB12b
positively regulated flavonol biosynthesis, including four major quercetin glycosides and two major isorhamnetin glycosides, by promoting the expression of
PbCHSb
and
PbFLS
in pear fruit.
Electronic supplementary material
The online version of this article (10.1186/s12870-019-1687-0) contains supplementary material, which is available to authorized users.
Differential expression of genes is crucial to embryogenesis. The analysis of gene expression requires appropriate references that should be minimally regulated during the embryonic development. To select the most stable genes for gene normalization, the expression profiles of eight commonly used reference genes (ACTB, GAPDH, rpL17, alpha-Tub, EF1-alpha, UbcE, B2M, and 18S rRNA) were examined during Japanese flounder (Paralichthys olivaceus) embryonic development using quantitative real-time polymerase chain reaction. It was found that all seven mRNA genes appeared to be developmentally regulated and exhibited significant variation of expression. However, further analyses revealed the stage-specific expression stability. Hence when normalization using these mRNA genes, the differential and stage-related expression should be considered. 18S rRNA gene, on the other hand, showed the most stable expression and could be recommended as a suitable reference gene during all embryonic developmental stages in P. olivaceus. In summary, our results provided not only the appropriate reference gene for embryonic development research in P. olivaceus, but also possible guidance to reference gene selection for embryonic gene expression analyses in other fish species.
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