转录因子FabHLH148参与草莓果实的颜色发育

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

【目的】探究碱性螺旋-环-螺旋(basic helix-loop-helix,bHLH)转录因子在草莓果实成熟过程中的功能。【方法】基于转录组测序和基因组数据库,克隆FabHLH148基因;分析其理化性质、保守结构域、预测编码的蛋白质结构、系统进化树、亚细胞定位等;采用RT-qPCR检测其在草莓中的时空表达水平,并构建过表达(over expression,OE)和RNA干扰(RNAinterference,RNAi)载体,利用农杆菌介导瞬时侵染草莓果实,观察记录表型,检测FabHLH148表达水平,并测定花色素苷含量。【结果】FabHLH148基因CDS区全长687 bp,编码228个氨基酸,预测蛋白分子质量24.89 ku,理论等电点(pI)为11.65;该基因属于bHLH超家族,因其与二倍体草莓FvbHLH148序列相似度最高,所以将其命名为FabHLH148;亚细胞定位显示,FabHLH148主要定位在细胞核。RT-qPCR结果表明,FabHLH148在草莓不同组织部位均有表达,在果实中有较高表达并随果实成熟表达量显著升高。过表达FabHLH148能够促进果实着色以及花色素苷的积累,通过RNAi调低Fab-HLH148的表达则作用结果相反,且FabHLH148在OE组果实中表达水平显著高于对照,RNAi组果实中表达水平显著低于对照(p<0.01)。【结论】转录因子FabHLH148属于bHLH超家族,可促进草莓果实着色。

【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®Ⅲ1st Strand cDNA Synthesis SuperMix(YEASEN,China)and then,the full-length CDS sequence of the Fab-HLH148 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 C_(1080)H_(1809)N_(353)O_(312)S_(5),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:Fab-HLH148-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.Fab-HLH148 encoded a classic bHLH transcription factor and was located in nucleus and cytoplasm.Fab-HLH148 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.