苹果14-3-3基因家族的鉴定与MdGRF13的功能分析

Identification of 14-3-3 gene family and functional analysis of MdGRF13 gene in apple

【目的】通过鉴定和分析苹果14-3-3基因家族,并在苹果愈伤组织中过表达MdGRF13,研究逆境胁迫下其对苹果愈伤组织生长的影响,为探究该家族基因功能提供理论参考。【方法】利用生物信息学方法对苹果14-3-3基因家族进行鉴定和表达分析,构建MdGRF13过表达载体并转化苹果愈伤组织。【结果】共鉴定出36个苹果14-3-3基因家族成员。系统进化分析将该家族成员分为ε类和非ε类,每个亚族基因结构相似,且高度保守。共线性分析发现片段重复是该基因家族扩张的主要原因。基因组织特异性表达分析显示,苹果14-3-3家族基因在茎中多数上调表达。亚细胞定位表明MdGRF13蛋白定位在细胞核、细胞质和细胞膜上。qRT-PCR分析发现,在PEG和NaCl处理下分别有14和13个基因在不同时间点上调表达。过表达型(OE)愈伤组织在PEG和NaCl处理下长势优于野生型(WT)。【结论】鉴定出36个苹果14-3-3基因家族成员,且各亚族成员结构高度保守。过表达MdGRF13增强了苹果愈伤组织的抗旱性和耐盐性。

【Objective】In this study,the physicochemical properties,structure and evolutionary relationships of 14-3-3 proteins in the apple genome,as well as their expression in different tissues and under stress conditions were analyzed.Meanwhile,overexpression of MdGRF13 in apple callus under stress was investigated.The purpose of this study was to provide theoretical references for exploring the functions of 14-3-3 family of genes in apple.【Methods】The 14-3-3 genes of apple were blasted via Arabidopsis and rice protein sequences,and then the conserved domains were searched by SMART and Pfam online websites to remove the apple sequences without conserved domains.ExPASy and SOPMA tools were used to predict the protein's physical and chemical properties and secondary structure,respectively.MEGA7.0 software was used to construct the phylogenetic tree.The gene structures and protein conserved motifs were analyzed by online tools GSDS and MEME,respectively.Tbtools software was used to analyze the synteny among apple,Arabidopsis and rice,and the protein interaction network was analyzed through STRING protein interaction database.The qRT-PCR was used to analyze the differential expression in roots,stems and leaves of the Gala seedlings.The expression of 14-3-3 genes treated with 15%PEG,100μmol·L^(-1) ABA and 200 mmol·L^(-1) NaCl at different time points were measured in Gala plantlet leaves.The MdGRF13 was cloned and transformed into Agrobacterium tumefaciens strain GV3101 cells.The recombinant plasmid pCAMBIA1300-MdGRF13-GFP and the control pCAMBIA1300-GFP were genetically introduced into Nicotiana benthamiana leaves.After the tobaccos were cultured in dark for 2 days,the GFP fluorescence was observed under a laser confocal microscope.Finally,the apple callus was genetically transformed by GV3101,and the transgenic callus were identified by PCR.The fresh weight,MDA content,and the enzyme activities of CAT,POD and SOD of wildtype and overexpression callus were measured after 15 days of PEG and NaCl treatment.【Results】According to the sequence analysis of the apple 14-3-3 genes,the amino acid lengths were 98-1006 aa,the molecular weights were 11221.52-113854.16 Da,and the theoretical isoelectric points ranged from 4.68 to 10.55.The secondary structure of the 14-3-3 proteins was dominated by alpha helix,followed by random coil,beta turn and extended strand.Phylogenetic analysis revealed that 14-3-3 gene family members in apple were divided into ε-group and non-ε-group,as well as conserved motifs showed that each subfamily gene contained motif 3,which was highly conserved.Meanwhile,the analysis of gene structures found that the distribution positions and lengths of exons in different subfamilies had obvious differences.MdGRF11,MdGRF14,MdGRF23 and MdGRF27 had only one intron,MdGRF16 and MdGRF26 had two introns,and MdGRF7 and MdGRF24 had 14 introns.MdGRF24 and MdGRF33 were significantly longer than other 14-3-3 genes.In addition,MdGRF2,MdGRF8,MdGRF13 and MdGRF28 belonged to the intron deletion class,and all sequences only contained different exons.The synteny analysis revealed that 10 syntenic gene pairs were contained in this gene family,and there were 6 gene pairs tandem duplication.The 14-3-3 proteins had obviously interacted with FT2,MdFT1 and XP_008366662.1,which were involved in growth,development and flowering regulation processes.The prediction of subcellular localization indicated that MdGRF13 proteins were localized in the nucleus,cytoplasm and cell membrane.The 14-3-3 genes were expressed in different tissues,and they were obviously up-regulated in stems.With ABA treatment,the expression levels of most genes were downregulated.With PEG treatment,14 genes were up-regulated.With NaCl treatment,the expression levels of MdGRF4,MdGRF5,MdGRF13,MdGRF14 and MdGRF16 were significantly up-regulated compared with the control.The phenotypes of the wild-type and overexpression lines treated with different concentrations of PEG showed that the growth of wild-type(WT)callus was weaker than that of overexpression(OE),while the growth of callus by 2%PEG treatment was better than that by 4%PEG.With NaCl treatment,the growth of transgenic(OE)callus was better than that of wild type(WT),and the growth treated with 0.06 mol·L^(-1) NaCl was better than that with 0.08 mol·L^(-1) NaCl treatment.With 0.08 mol·L^(-1) NaCl treatment,the fresh weight,MDA content,the activities of CAT,POD and SOD of WT callus were not significantly different from those of OE.While with other NaCl and PEG treatments,the fresh weight,the activities of CAT,POD and SOD of WT callus were lower than those of OE,the MDA content of OE was lower than that of WT.【Conclusion】In this study,36 members of apple 14-3-3 family were identified by bioinformatics.Phylogenetic analysis showed that they had high homology with Arabidopsis thaliana,and the members of each subfamily were conserved.Subcellular localization assay showed that MdGRF13 protein was localized in the nucleus,cytoplasm and cell membrane.Overexpression of MdGRF13 enhanced the drought resistance and salt tolerance of apple callus.It provided a theoretical basis for the study on apple 14-3-3 family in response to stress.