Study On The Regulation Of Anthocaynin Biosynthesis Related Genes And Volatile Profile In Fruits Of Fragaria Pentaphylla

Fragaria ×ananassa is a perennial herb belonging to Fragaria genus in Rosaceae family.F.× ananassa is hightly welcome for its high vitamin C and rich in anthocyanins.F.× ananassa is known as its large size and attractive color,but lack flavor and fragrance.Fragaria pentaphylla,one of the wild strawberry species,produces white or red fruits.The white fruits has stronger aroma than the red one.In this study,the synthesis of the anthocyanins and aroma and the gene expression and regulation were analyzed by using RNA-seq,GC-MS,RNAi and real time qPCR techniques.The results were shown in the followings.1.2271 genes were found significant difference expression between the red and white fruit of F.pentaphylla,of which 1164 genes up-regulated in red fruit,others were found down-regulated.Most of those genes concentrated in plant secondary metabolism pathway and 15 significant difference express genes were found to be related with anthocyanin biosynthesis,suggesting that these genes may play an important role in the regulation of anthocyanin biosynthesis.2.Three extraction method(microwave extraction,ultrasonic extraction and room temperature leathing)were used to extract anthocyanins from plants fruits,compared with ultrasonic extraction and room temperature leathing,microwave extraction was demonstrated to be a fast and efficient extraction method for extracting anthocyanin from plant fruits,however,under certain conditions,microwave extraction can be resulting in the degradation of anthocyanin.Finally,we chose room temperature leathing method as the extract anthocyanin from strawberry fruits and optimized the extraction condition,the optimal conditions were:75%methanol + 1%hydrochloric acid as extraction agent;solid to liquid ratio of 1:6(m/v);extraction time 40 hours.Under these conditions,the yield of anthocyanin from strawberry fruit was 13.78±2.40 mg/100 g FW.3.The relationship between anthocyanin accumulation and expression of anthocyanin biosynthesis related genes were analyzed during fruit maturation of F.pentaphylla.Expression of transcription factor of MYB10 and four structure genes(CHS?DFR?ANS and UFGT)were found to be significant related with the accumulation of anthocyanin,suggesting those genes may play important role in the anthocyanin biosynthesis in fruit of F.pentaphylla.4.The DFR gene from F.pentaphylla(FpDFR)contained a 1050 bp open reading frame which encodes a protein of 349 amino acid residues.The FpDFR cDNA shared significant homology with DFR cDNA of known Fragaria species.Secondary structure analysis of the predicted DFR revealed that it predominantly consists of ?-pleated sheets,followed by a-helices and random coils.The expression of the FpDFR gene in fruits was strongly associated with anthocyanin accumulation in fruits of F.pentaphylla.The full-length sequence of FpDFR is 2080 bp separated by five introns and shared 95%homology with that of F.vesca.Twenty-seven SNPs were observed in the FpDFR gDNA sequences from the red and white genotypes.Among them,70.4%were located in introns and 29.6%in exons.In the coding region,12.5%SNPs are missense SNPs,while the others are synonymous.One missense mutation SNP showed significant correlation with the red color trait,while the other two synonymous mutation SNPs were significantly association with the white trait.5.We construct MYB10 interference vector,and transformed it into agrobacterium to infect the red fruits of F.pentaphylla.After infection,MYB10 show sharply drop expression,which was 0.54 times less than control red fruits.Meanwhile,a significant down-regulation of CHS,DFR,ANS and UFGT,and a up-regulation of CHI?F3H and F3'H were found in the RNAi infection fruits.Determination of anthocyanin showed that the anthocyanin content significantly decreased 26.5%after RNAi interference,but the content of flavonoids was not significantly changed.6.A total of 103 volatile compounds were identified in F.pentaphylla,while 71 in F.×ananassa.Besides,the predominant volatiles in white strawberry were 3(2H)-Furanone 4-methoxy-2,5 methyl,Butanoic acid 2-methyl methyl,2-Hexenal and 2-Hexen-l-ol acetate;In red strawberry were 2-Hexenal,Hexanol,2-Hexen-l-ol acetate and 3(2H)-Furanone 4-methoxy-2,5 methyl;While in cultivated species,they were Butanoic acid ethyl ester,2-Hexenal,Butanoic acid 2-methyl,2,5-Dihydroxyacetophenone and Hexanoic acid ethyl ester.Furthermore,Cyclopropane propyl was first time reported in white fruits with high amount.