Identification Of Transcription Factor Genes Affecting Flavonoid Pathway In Strawberry Fruits

Strawberry(Fragaria×ananassa Duch.)which is a kind of herbaceous perennial,belonging to Rosaceae,is an attractive fruit for human beings not only because of its bright color,palatable flavor and outstanding aroma,but also because of its remarkable nutritional value and a variety of phytochemical constitute.Flavonoids which are characterized by a C6-C3-C6 skeleton are the major class of these compounds contributing to the antioxidant capacity of strawberry,the best known phenolic compounds.Flavonoids in strawberry are consisted by anthocyanins,proanthocyanins and flavonols.Anthocyanins are the main pigments determining the color of the berry with flavonols as a sort of co-pigments,while the high content of proanthocyanins in young berries can help them defending the pathogens such as powdery mildew,and affect the taste and flavor of ripe berries.Nevertheless,they play a prominent role in human health after ingestion with the consumption of strawberries.Considering these important roles flavonoids playing for the values of strawberry,the study about flavonoid pathway in strawberry is with high necessity.For finding the unknown transcription factors of flavonoid pathway in strawberry fruit,a group of 16 genotypes in total was used,which has two parent lines ‘Florida Elyana' and‘Mara des Bois' with significant differences and their 14 progenies with different genotypes of them,and the researches were performed and results were gained in this study as following:1.The transcriptomes of ripe fruits from the 16 genotypes were obtained by RNAseq,thecorrelations of expression profile in this group between 15 flavonoid genes(PAL1?PAL2?C4H?4CL?CHS1?CHS2?CHI?F3H?F3'H?FLS?DFR?LAR?ANS?ANR and UFGT)were analyzed to get more information about their relationships andto verify the validity of these data from this particular group.The relationship betweengenes' expression profiles was determined by Pearson's r and P values.Correlationswere considered positive when r > 0.65 and P < 0.05.2.To find the candidate genes,the correlations of expression profiles betweentranscription factor genes and the 15 flavonoid genes were calculated.According to th‘guilt by association' principle,13 transcription factor genes were defined as candidategenes because they shared at least 4 positive correlations with flavonoid genes.3.Primers were designed for cloning the full and special cDNA sequences of 13candidate genes and 11 overexpression vectors and 13 RNAi vectors were constructed.4.After that,the functions of these candidate genes in developing fruits were identifiedby transient expression induced by agrobacterium,and 3 were found with capability toaffect the expression of flavonoid genes.The expression of LAR and F3'H wereupregulated in FaTCP11 overexpression fruits;the expression of FLS and LAR wereupregulated in FaPCL1-like overexpression fruits;and in the FaSCL8 silenced fruits,the expression of PAL2,CHS,CHI,F3 H,UFGT and MYB10 were significantlydecreased while the expression of F3'H,ANR,MYB9 and MYB11 were upregulated.5.The expression profiles of these 3 genes in 4 development stages of fruits wereexamined by qRT-PCR: they were all mainly expressed in receptacles and lessexpressed in achenes;FaTCP11 and FaPCL1-like were more expressed in unripefruits while FaSCL8 was highly expressed in ripe fruits.6.For future study plan,the conserved domains of each transcription factor wereanalyzed by alignment with their homologues,and the potential mechanisms about howthey regulate the strawberry flavonoid pathway were predicted: FaTCP11 andFaPCL1-like can recognize and combine DNA with specific sequences using their TCPand GARP conserved domain respectively,and they may affecting the flavonoidpathway by interacting with MYB and bHLH factors;FaSCL8 can form homodimer tocombine DNA,and it may participate the signal regulation of strawberry fruits toupregulate the transcription of MYB10 and downregulate the transcription of MYB9 andMYB11.