Expression analysis of FaMYB10 transcription factor and structural genes related to anthocyanin biosynthesis in strawberry (Fragaria × ananassa) fruit

Abstract:【Objective】Fruit color is an important trait for strawberry fruit quality and commercial value. The color of fruits is determined by the content and components of anthocyanins. In plants, several enzyme genes and transcription factors are involved in anthocyanin biosynthesis, but the molecular mechanism underlying the variation in the color of strawberry fruits is still unclear. The study aimed to analysis the expression patterns of structural genes and FaMYB10 in the fruits with different color of strawberry and to verify, the function of the FaMYB10 transcription regulator. It will provide the reference for the molecular mechanism of anthocyanin synthesis in strawberry fruits.【Methods】Fruits from the three octoploid cultivated strawberry varieties (Fragaria × ananassa‘Benihoppe’with red skin and flesh;‘Xiaobai’with red skin and white flesh;‘Snow White’with white skin and flesh) were used as materials. Extraction of total anthocyanin was carried out by high performance liquid chromatography. The total RNA of fruit was extracted using an E.Z.N.A Plant RNA Kit. The first-strand cDNA was syn-thesized from the total RNA using a PrimerScriptTM RT Reagent Kit with gDNA Eraser according to the manufacturer’s instructions. Then, RNA- seq was used to analyze the transcriptomic change during three development and ripening stages of the three varieties. In addition, the expression levels of eight candidate genes were conducted by qRT-PCR. Homologous recombination technique was performed to insert the FaMYB10 gene in the PBI121-GFP vector. After the constructed over-expression vector was transferred into Agrobacterium GV3101. A. tumefaciens strain GV3101 was resuspended in the Agro- bacterium infiltration buffer (10 mmol·L-1 MgCl2, 10 mmol·L-1 MES, pH 5.6, 200 μmol·L-1 acetosyrin- gone) to a final OD600 of 0.6- 0.8. The PBI121- FaMYB10- HY- GFP vector Agrobacterium tumefaciens was injected using a syringe with needle into the initial ripening fruits in vitro until the whole fruit was permeated. The fruit coloration was observed three or five days after post-injection.【Results】The total anthocyanin contents varied significantly with fruit developmental stages. In the ripening stage, the to- tal anthocyanin contents of‘Benihoppe’was rapidly increased from 7.92 mg · kg- 1 to 175.60 mg · kg- 1, and the total anthocyanin contents of‘Xiaobai’was 15.2 mg·kg-1, which was only 8.66% of the‘Beni- hoppe’. However, the total anthocyanin contents were very low in‘Snow White’. The results of tran- scriptome analysis showed that the expression levels of genes related to the anthocyanins biosynthesis were significantly different between the red and the white strawberries. The expression of early biosyn- thetic genes including FaPAL (gene22493), FaCHI (gene25025 and gene25539), and FaCHS(gene3479) and the late biosynthetic genes including FaDFR (gene236, gene5738, gene7069 andgene21883), FaANS (gene14037 and gene15699), FaUFGT (gene21489 and gene21620), Fa3GT(gene9227, gene16078, gene22086 and gene22630), FaGST (gene2828, gene5140, gene9114 andgene2665) was significantly higher in the ripe fruits of‘Benihoppe’and‘Xiaobai’compared with the fruits of‘Snow White’. According to the results of qRT-PCR, the expression of FaMYB10 significantly increased in the ripe fruits of‘Benihoppe’,‘Xiaobai’and‘Snow White’compared with the fruits in initial developmental stage. Interestingly, in the full ripe stage of the fruits, the expression level ofFaMYB10 gene in the‘Snow White’and‘Xiaobai’was significantly higher than that of the red variet- ies‘Benihoppe’, and its transcription level was not consistent with changes of the color phenotype of the fruits. Meanwhile, qRT-PCR analysis of eight candidate genes was consistent with the transcriptome results. By analysing the coding region sequences of the FaMYB10 gene in the red and the white straw- berry varieties showed that there were two sequence types of the FaMYB10-XB-1 and the FaMYB10- XB-2 was in‘Xiaobai’and the FaMYB10-SW in‘Snow White’. The coding sequences of FaMYB10- XB- 2 and FaMYB10- SW possessed GA and ACTTATAC base insertion respectively, which encoded truncated proteins. Meanwhile, the over-expression of FaMYB10 in the strawberry induced coloration of the fruits compared with those of the control. It suggested that FaMYB10 played a positive role in regulating anthocyanin biosynthesis.【Conclusion】There were significant differences in the total antho- cyanin contents in different phenotypes, and anthocyanin content was closely related to the expression level of the genes related to the anthocyanin biosynthesis. Among the eight candidate genes, FaCHI,FaDFR, FaANS and FaGST showed significantly lower expression levels in the white fruit type than in the red fruit type during fruit ripening, suggesting that these genes might be vital structural genes in the anthocyanin biosynthesis. The expression level of FaMYB10 in the white fruit type was significantly higher than that in the red fruit type. The transient transformation of fruit proved that FaMYB10 was a key positive regulatory gene for the anthocyanin biosynthesis. FaMYB10 gene sequence insertion mutation might result in the loss of intact FaMYB10 protein function and then the loss of anthocyanin accu- mulation in the white octoploid strawberry.