Cloning And Functional Analysis Of The Yellow Petal Gene GaMYB113 In Asiatic Cotton

Flavonols,a group of flavonoids,are important secondary metabolites,involved in many physiological processes in plants including hormone signal transduction,pollen tube growth and response to stresses.In addition,flavonols are important colorants in plant flowers.Petal color is an important agronomic traits.Different colors play a key role in the processes of flowering,pollination,and fruiting through special interactions with different pollinators(such as bees,butterflies,and birds).In addition,flower color have high ornamental and commercial value of ornamental crops.In this study,using a recombined inbred line population,the yellow petal gene in Asiatic cotton was cloned and its functions were further analyzed.The main results are as follows:1.The yellow pigmentation in Asian cotton petals is mainly caused by flavonol accumulation.From the Asian cotton recombinant inbred line population,each 10 yellow and white petal lines were selected,and the petals of 1 day before flowering were sampled for metabolomic and transcriptomic analysis.A total of 144 flavonoids were identified in the petals,and 50 metabolites(36 up-and 14 down-regulated in yellow petals)had significantly different relative content between white and yellow petals.LC-MS/MS analysis indicated that the major flavonols in Asiatic cotton petals are gossypetin and6-hydroxykaempferol,with the levels in yellow petals 13.5 and 2.3 fold higher than white petals,respectively.Transcriptomic analysis revealed that the expression of flavonol structural genes,especially the flavonol-specific flavonol synthase gene GaFL(Ga05G2477)and UDP-glucosyl transferase gene(Ga12G0252),was significantly increased in the yellow petals.These results demonstrated that flavonols are the main colorants of Asian cotton yellow petals.2.GaMYB113 is the yellow petal gene in Asiatic cotton.With the whole genome re-sequencing method,the yellow petal gene of Asiatic cotton were located in an interval of around 3 Mb(22895333-25611091)on chromosome 11.In this interval,there were 48 protein-coding genes,among which GaMYB113(Ga11G1228)was annotated as a transcription factor with significantly higher transcript level in yellow petals.RT-PCR analysis indicated that the high expression level of GaMYB113 concurred with yellow pigmentation in petal.Comparing cloning revealed a retrotransposon inserted in the second exon of GaMYB113 in white petal lines,resulting in abnormal splicing.Transgenic experiments showed that overecpression of GaMYB113 led to increased flavonol biosynthesis and accumulation in Arabidopsis and cotton roots.Based on the above results,we concluded that GaMYB113 is the yellow petal gene of Asiatic cotton.3.GaMYB113 activates the flavonol biosynthesis pathway transcriptionallySequence analysis indicated that GaMYB113 belong to the SG6 subgroup of MYB proteins.GaMYB113 showed transcription activation in yeast and located mainly in nucleus in tobacco cells.RT-PCR analysis showed that the flavonol structural genes GaFL and GaUGT88A1 were coexpressed in various tissues and lines of Asian cotton.Furthermore,the dual luciferase experiment in tobacco proved that GaMYB113 can directly bind to the promoter of GaFL,and activate the expression of downstream genes.In summary,the GaMYB113 gene may activate flavonol biosynthesis pathway in the Asian cotton,promote flavonol biosynthesis accumulation in patals,and finally lead to the yellow petal trait.