Transcriptome Analysis Of Flowers And Functional Studies Of MYB Genes Of Flavonoid Biosynthetic Pathway In Chinese Narcissus (Narcissus Tazetta Var.chinensis)

Narcissus tazetta var.Chinensis is one of the ten most famous traditional flowers in China.So far,there are still a few narcissus varieties in China because of the absence of various colors.It will be an effective way to innovate narcissus colors and cultivate new varieties through further research on the molecular mechanism of Chinese narcissus color formation and molecular transformation.In this study,database of RNA-seq of Zhangzhou narcissus was used to screen MYB transcription factors involved in regulation of flavonoid biosynthesis.Functional verification were done by expression analysis and transformation.The results will provide theoretical basis for the color formation in Chinese narcissus,Following are the main results:1.In this experiment,Chinese Narcissus 'Jinzhanyintai' was used as the experimental material to extract RNAs from the early flowering season.High-throughput sequencing was performed using Illumina Hiseq.We obtained 23,081,029 total clean reads in the flower and thenassembled into 55,418 unigenes.There were 11,053 unigenes has a length more than 1kb.Unigene average reading length is 669bp,N50 reading length is 1,049bp?Functional annotation of unigene,including alignments with the NR,Swiss-Prot,KEGG,COG,KOG,GO,and Pfam databases,we finally got 32,414 annotated unigene.Gene structure analysis based on the unigene library was performed,in which 3,276 SSR markers were obtained by SSR analysis.The flower transcriptome was analyzed with the previously sequenced bulb transcriptome,7,059 differentially expressed genes(DEGs)were identified,of which 3663 DEGs were up-regulated and 3396 DEGs were down-regulated,and were annotated to 126 KEGG metabolic pathways.2.By analysis of the differentially expressed genes in between the flower transcriptome and bascal plate transcriptome,two R2R3-MYB transcription factors were obtained associated with flavonoid metabolism.Preliminary sequence analysis showed that one of them was related to the inhibition of anthocyanin synthesis and was named NtMYB7;the other was related to activation of flavonoids and named NtMYB8.3.The full length sequences of two MYB genes related to flavonoid metabolic pathway had been cloned.The open reading frame of NtMYB7 and NtMYB8 are 753bp and 807bp which were capable of encoding 250 and 268 amino acids respectively.NtMYB7 and NtMYB8 assession number is MF522209 and MF522208.Multiple alignments showed that both of them contain R2-domain and R3-domain.Phylogenetic tree analysis showed NtMYB7 was closer to repressor genes of anthocyanin and NtMYB8 was clustered with activator of proanthocyanin.The expression level of MYB genes in different organs of narcissus had been conducted,the results suggested that the expression of NtMYB7 was highest in basal plates.The expression level in basal plates was nearly 20 times of that of in the petals during the flowering stage.In flower petals,the relative expression of NtMYB7 gene was gradually increased during the opening of flowers,and the expression in the full flowering stage was the highest.In the corona,the expression level of NtMYB7 was the highest in the early stage,and the expression level of NtMYB7 was lower in bud period stage and full-bloom stage stage.Qpcr results showed NtMYB8 had more transcriptes in flowers than leaves and basal plates.The expression of NtMYB8 in the buding stage corona was nearly 17 times of that in the basal plates.The expression of NtMYB8 in the corona decreased significantly during the process of flowering.4.The expression vectors of pSAK277-NtMYB7 and pSAK277-NtMYB8 were constructed and transformed into Agrobacteria for tabacoo transient expression.In the transient expression of tobacco,NtMYB7 down-regulated the expression of anthocyanin synthesis structural genes and LAR gene of proanthocyanidin branch activated by StMYB in tobacco leaves.Our results suggest that NtMYB7 is a repressor of flavonoid metabolic pathway in Chinese narcissus.Quantitative PCR analysis showed that NtMYB7 significantly repressed the expression of tobacco FLS gene which belongs to flavonol metabolic branch.There was no change in tobacco leaves injected with NtMYB8 gene alone.Quantitative PCR results showed NtMYB8 significantly up-regulated the expression of FLS?LAR and ANS genes.NtMYB8 and StBHLH may promote the synthesis of flavonols?procyanidins and anthocyanins.NtMYB8 has the highest expression level in narcissus,it is speculated that it may simultaneously activate the synthesis of flavonols?procyanidins and anthocyanins in Chinese narcissus coronas.Further study is required to check whether NtMYB8 can activate flavonoid biosynthesis pathway in narcissus considering the differences between narcissus and tobacco.5.At the same time,NtMYB8 gene and NtMYB7 gene were transformed into tobacco for stable transformation,and transgenic plants were obtained.The flowering phenotype analysis showed that the transgenic flower color became lighter.It was further speculated that NtMYB7 gene may inhibit anthocyanin metabolic pathways.NtMYB8 gene may promote the flavonol branch mainly,which may compete with the anthocyanin branch.Then it resulting in lighter tobacco color.And its gene function needs further verification in subsequent experiments.