| Being a perennial aquatic ornamental plant,lotus(N.nucifera)is among the top ten famous flower plants in China.Its flower colors include red,pink,white,variegated and brocade,with red and brocade being the major ones in ornamental horticulture usage.Therefore,it is very important to elucidate the mechanism underlying the coloration of lotus flower,especially on the purpose of new bright-colored cultivar selection in lotus breeding.Based on previous studies,it has been known that the red color of lotus is mainly determined by the content of anthocyanins.The biosynthesis of anthocyanins is jointly regulated by two types of genes,including structural and regulatory genes.Currently,key structural and some regulatory genes for anthocyanin biosynthesis in lotus have been identified,including Nn ANS,Nn UFGT,and a b HLH transcription factor Nn TT8.However,the identification of the key MYB transcription factors that regulate lotus color is rarely reported.In this study,we characterized a MYB gene named as Nn MYB5 through transcriptomic analysis,which was further verified to be involved in the regulation of anthocyanin biosynthesis.The main research contents and results are as follows:1)Transcriptomic analysis focusing on the coloration process of lotus flower: A red color flower lotus‘Yehonglian’was selected to conduct the transcriptomic analysis with its flower and leaf buds at three different developmental stages.Analysis showed that the content of anthocyanins in these buds were increased among with their development.A total of 13022 differentially expressed genes(DEGs)were identified through transcriptomic analysis,with 43 DEGs being significantly enriched in the anthocyanin pathway.The expression levels of the downstream pathway structural genes DFR,ANS and UFGT were consistent with the accumulation of anthocyanin,which may be the rate-limiting structural enzyme genes of the anthocyanin pathway.Furthermore,there were 6 MYB,5 b HLH and 3 WD40 transcriptional factors encoding genes being identified as DEGs.2)Screening the candidate MYB gene involved in anthocyanin biosynthesis: Among the6 differentially expressed MYB genes,there is one with number in database as NNU＿03019showing an expressional pattern consistent with the accumulation of anthocyanin in both flower and leaf buds.Phylogenetic analysis showed that it is a homolog of At MYB5 from Arabidopsis,and hence was named as Nn MYB5.Subcellular analysis showed that Nn MYB5 localizes in nucleus.3)Functional analysis of Nn MYB5 in anthocyanin biosynthesis: The function of the identified Nn MYB5 transcription factor was further verified through constructing the transgenic Arabidopsis overexpressing Nn MYB5.Phenotypic analysis showed that anthocyanin accumulation was evident in the leaf veins and stems of the transgenic Arabidopsis.And realtime quantitative PCR analysis showed that the overexpression of Nn MYB5 promoted the expression of structural genes of the anthocyanin pathway.Meanwhile,yeast two-hybrid and bimolecular fluorescence complementation experiments proved that Nn MYB5 could interact with Nn TT8,but no WD40(NNU＿18960)protein was found to interact with it.In addition,yeast one-hybrid showed that Nn MYB5 can specifically bind to the promoter of anthocyanin biosynthesis gene Nn UFGT and promote anthocyanin biosynthesis by regulating the expression level of Nn UFGT.Taken together,a MYB transcriptional factor gene Nn MYB5 was screened through transcriptomic analysis of lotus flower and leaf buds during the coloration process,and proven to be a positive regulator of anthocyanin biosynthesis,which may affect anthocyanin accumulation by activating the expression of downstream structural genes.The research results of this subject could not only deepen our understanding on the regulation mechanism of anthocyanin biosynthesis in lotus,but also lay a theoretical foundation for the breeding of new lotus cultivars with bright flower colors. |
