Transcription factor FabHLH148 is involved in the color development of strawberry fruit

Abstract:【Objective】Strawberry is the most widely cultivated small berry fruit in the world due to its unique flavor as well as high nutritional and economic value. Meanwhile, it is a prominent problem to improve the quality of strawberry in cultivation. Coloring is an important evaluation index of fruit quality. Anthocyanin is the main component affecting the color of strawberry fruit. The basic helix-loop-helix (bHLH) transcription factor family as the second largest transcription factor families in plants has been proved to be involved in many processes of plant growth, development, morphogenesis and stress response. Some members of bHLH transcription factors play crucial regulatory roles in plant anthocyanin synthesis. To analyze the function of bHLH transcription factors during strawberry fruit coloring, the FabHLH148 gene was cloned and we used physiological and molecular biology methods to reveal the function of FabHLH148.【Methods】Firstly, according to the reported transcriptome data of strawberry fruits in five development periods (SG, LG, Wt, IR and PR), a gene increasing rapidly with fruit ripening was screened. As it contained a bHLH superfamily conserved domain, it had the highest sequence similarity with diploid strawberry FvbHLH148 (GenBank accession: XM_004295010.2), and the gene was named FabHLH148. The total RNA from strawberry cultivar Bebihoppe fruit was extracted using the plant total RNA extraction kit (Huayueyang, China) according to the manufacturer’s protocol. Firststrand cDNA was synthesized and the reverse transcription was carried out using Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix (YEASEN, China) and then, the full- length CDS sequence of the FabHLH148 gene was obtained by PCR. Secondly, some bioinformatics techniques were used in this research. ExPASy website was referred to analyze the molecular weight, isoelectric point and liposoluble index of encoding protein. The conserved domains of FabHLH148 were predicted using NCBI Conserved Domains. The secondary and tertiary structure of FabHLH148 was analyzed by DNA star software and the online software SWISS-MODEL separately. MEGA 5.1 software was used to construct phylogenetic tree of FabHLH148 homologous proteins. Thirdly, we used RT-qPCR to detect the expression level of FabHLH148 in strawberry including various organs and developmental stages. Its subcellular localization was observed by Agrobacterium mediated transient transformation of tobacco mesophyll cells. Finally, the full-length CDS sequence of FabHLH148 was constructed into pSuper1300 vector by homologous recombination to obtain over- expression vector; the 32-383 bp CDS sequence of FabHLH148 was constructed into pK7GWIWG( Ⅱ)RR by Gateway method to obtain RNAi vector. These vectors were transformed into Agrobacterium. Based on this, the strawberry fruits in the de-green period were infected by the Agrobacterium-mediated transient infection, and the phenotypes were photographed and recorded at 0, 3, 5 and 7 days after injection. The achenes were removed 7 days after injection, and only the injection parts were frozen in liquid nitrogen and stored at -80 ℃. Then, we detected the expression level of FabHLH148 and anthocyanin content in different transgenic strawberry fruits. 【Results】Bioinformatics analysis showed that the whole open reading frame (ORF) of FabHLH148 was 687 bp encoding 255 amino acids. The results of amino acid physicochemical properties analysis showed that putative FabHLH148 protein was C1080H1809N353O312S5, with a molecular weight of 24.89 ku and a theoretical isoelectric point (pI) of 11.56, so it was an alkaline protein. The protein contained 10 negatively charged amino acid residues (Asp + Glu), 42 positively charged amino acid residues (Arg + Lys), with an instability coefficient of 54.49 and average hydrophilicity of -0.636, indicating that the protein was an unstable hydrophobic protein. Conserved domain analysis showed that FabHLH148 contained a typical bHLH superfamily conserved domain. Therefore, FabHLH148 protein was clustered into the bHLH superfamily and shared high identity with amino acid sequence of FvbHLH148 (99.56%). Transient expression in Nicotiana benthamiana showed the green fluorescent signal of Super1300: FabHLH148-GFP was found in the nucleus and cytoplasm of epidermal cells in leaves. Analyses of qRTPCR showed that FabHLH148 was expressed in different organs of strawberry, with the highest relative expression in full red fruit and lowest in achene. It was highly expressed in fruit and reached its peak at full red fruit, which suggested that it played a crucial role in strawberry fruit ripening. Based on Agrobacterium transient infection of strawberry fruit and RT-qPCR, the expression level of FabHLH148 in the overexpression group was significantly higher than that in the control group while the expression level in RNAi group was significantly lower than the control group. The fruit coloration of overexpression group was faster and the anthocyanin content was higher than that from the control group, while the effect was opposite when the expression of FabHLH148 was reduced by RNA interference.【Conclusion】A homolog of FvbHLH148 denoted as FabHLH148 was cloned in cultivated strawberry. FabHLH148 encoded a classic bHLH transcription factor and was located in nucleus and cytoplasm. FabHLH148 was expressed much higher in fruit than in the rest of organs and up- regulated significantly during fruit ripening. Up-regulated or down-regulated expression of FabHLH148 led to a significant induction or reduction of anthocyanin in transient transgenic strawberry fruits. These results suggested that FabHLH148 could play an important role in strawberry fruit coloring.