| Prunus armeniaca L.is a Stone-fruit of the genus Prunus Linn.of the family Rosaceae.Xinjiang apricot is rich in germplasm resources,with many types of color variation such as white,yellow,orange,and red.It is an ideal model material for studying the mechanism of fruit color formation.Anthocyanins are water-soluble flavonoid pigments synthesized from flavonoid metabolic pathways.They are widely found in plants.They have many nutritional functions such as anti-oxidation,anti-aging,and prevention of cardiovascular diseases.The type and content of anthocyanins determine the coloration of fruits,and the transcription factors regulating anthocyanin metabolic pathways have been reported in various plants,but the transcription factors associated with anthocyanin synthesis in apricot fruits are not known.In the study,using red varieties(“Danxing”,“Hongyu”),light-colored varieties(“Baixing”,“Luntaixiaobai”)and bagged fruits(“Baixing”,“Danxing”)as materials,the changes of anthocyanins during fruit development were identify by high performance liquid chromatography(HPLC).Anthocyanin synthesis-related transcription factors were mined from apricot transcriptome data by the weighted gene co-exoression network analysis(WGCNA),and phylogenetic tree and protein structure analysis were used to predict their function.Based on gene cloning of the full length,the function of MYB in the regulation of anthocyanin content in apricot fruit were analyzed by subcellular localization,gene expression pattern analysis,and investigation of relationship between transcription factors and anthocyanin synthesis.The main findings are as follows:1.Cyanidin-3-O-rutinoside,keracyanin chloride and peonidin-3-O-rutinoside were detected in apricot fruit tested.The contents of total anthocyanins in “Danxing” and“Hongyu” were 35.05 mg/kg and 39.17 mg/kg respectively,which were much higher than 0.09 mg/kg and 0.07 mg/kg in “Baixing” and “Luntaixiaobai”.Among t,akeracyanin chloride is dominated in red cultivars,accounting for 58.8% and 62.7% of the total anthocyanins in “Danxing” and “Hongyu” respectively,while keracyanin chloride was not detected light-colored cultivars.The anthocyanin accumulated in "Danxing" during the mature period,and accumulated in "Hongyu" during the middle development period.2.A transcription factor Unigene1373 which is associated with anthocyanin biosynthesis was identified by co-exoression network analysis and phylogenetic tree analysis.3.The full length of this gene was cloned by rapid-amplification of cDNA ends(RACE),which include 675 bp sequence and containing a complete open reading frame.It encodes 225 amino acids.Analysis of the protein domain indicated that the sequence encodes a R2R3-MYB transcription factor and the transcription factor is inferred that associated with the synthesis of anthocyanins.So,we named the gene PaMYB10.4.A green fluorescent protein(GFP)fusion expression vector p163-GFP-MYB10 was constructed.Using empty vector p163-GFP as control,and then were thansformed into the onion epidermal cells by particle bombardment,and subcellular localization of p163-GFP-MYB10 protein was observed by laser scanning confocal microscopy.The results indicated that the expression site of this gene is in the nucleus.5.The expression levels of PaMYB10 and the structural genes of anthocyanin biosynthesis were analyzed by qRT-PCR during fruit development and ripening for different cultivar.The results showed that the relative expression levels of the genes in the red cultivars were significantly higher than those in the light color cultivars(P <0.05).The expressions of PaMYB10,CHI,CHS,LDOX,UFGT1 and UFGT2 in "Daixing" increased during the whole developmental peroid,and their expression levels have a significant positive correlation with the content of anthocyanins.The expression pattern of PaMYB10 and most of the structural genes of "Hongyu" showed a "double-peak" pattern and reached the highest value at the green maturation stage(S4).Each gene was slightly expressed in the period from fruitlet(S1)to turning(S3)in "Baixing" and "Luntaixiaobai",and only a few genes were detected during the later fruit development period.This change is consistent with the content of anthocyanins accumulation.6.Bagging affected the color of the fruit.The bagging of “Baixing” reduced the L*from 72.98 to 63.76,and its brightness decreased significantly.The color saturation decreased from-1.66 to-2.43.Bagging prevents the skin of "Danxing" from redappearence.HPLC analysis revealed that the anthocyanin content in bagged fruits was greatly reduced,"Danxing" decreased from 35.05 mg/kg to 2.17 mg/kg,and "Baixing" decreased from 0.04 mg/kg to 0.02 mg/kg.The results of qPCR showed that the expression levels of PaMYB10 and anthocyanin synthesis-related genes were also significantly lower than those in unbagged fruits(P < 0.05).In summary,the transcription factor PaMYB10 isolated from apricot fruit is involved in the regulation of anthocyanin synthesis and affects the formation of fruit color. |
