| The B-box containing proteins(BBXs)belong to zinc finger transcription factors,which play important roles in photomorphogenesis,flowering regulation and plant hormone transduction of seedlings.In recent years,it was found to be also related to abiotic stress resistance in plants.Previous results of apple transcriptome in this laborabory showed that a MdBBX1 was significantly up-regulated under salt stress.Whether it is related to stress resistance still remains unclear.In this study,the expression of MdBBXl in apple seedlings under different stress conditions was analyzed by qRT-PCR.The anti-stress ability of recombinant MdBBXl was assayed in E.coli.By using homozygous lines of MdBBXl,the mechanism of response to abiotic stresses in plants was further explored.The main results are as follows:(1)Bioinformatic analysis of MdBBXl.Pre-laboratory results showed that the induced expression of MdBBXl in apple leaves was up-regulated about 10 times under salt stress.The registration number of MdBBX1 coding gene in GenBank was LOC103442491.The gene was located on chromosome 1 in apple,and its full length was 1023 bp,including an open reading frame of 1020 bp and encoding a polypeptide chain of 340 amino acid residues.The molecular weight of MdBBXl was 38.139 kDa and the theoretically predicted isoelectric point was 6.01.Homologous sequence comparison and protein clustering analysis showed that MdBBXl contained two B-box domains in the N-terminal and a CCT domain in the C-terminal.It is a kind of typical BBX protein.(2)Subcellular localization and tissue expression pattern analysis of MdBBXl.The fusion of MdBBXl and GFP gene was transformed into tobacco leaves.The results showed that MdBBXl was located in the nucleus of tobacco cells.The subcellular localization of MdBBXl was consistent with the other known MdBBXs in plants.It was also consistent with BBX protein as a transcription factor involved in gene expression regulation.The expression of MdBBX1 in different tissues of apple was analyzed by qRT-PCR.It was found that MdBBX1 can express on different levels in roots,stems,leaves,flowers and fruit organs,of which the highest expression was located in leaves.(3)MdBBXl was induced by various abiotic stresses in apple seedlings.The promoter sequence of MdBBXl was predicted and analyzed by online software PlantCare.It was found that there were many elements related to stress response,such as ABRE element,MBS element,LTR element and P-box element,thus we speculated that the expression of MdBBXl gene responded to abiotic stress.By treating one-year-old apple seedlings under various stresses,including NaCl,PEQ 4℃ and exogenous ABA,we found that the expression of MdBBXl was up-regulated to a certain extent.After the treatment with ABA,the expression of MdBBXl was up-regulated about 120 times in apple roots,while after the treatment with PEG,the expression of MdBBXl was up-regulated about 20 times in apple leaves.The results showed that the expression of MdBBXl was induced by abiotic stress.(4)Recombinant expression of MdBBXl can improve the stress resistance of E.coli.The recombinant E.coli expressing MdBBXl was cultured on solid stress medium containing NaCl,KCl and mannitol respectively.The results showed that the expression of MdBBXl significantly improved the resistance of cells to stress,and the number of colonies was significantly more than that of control group.In liquid medium containing NaCl,KCl and mannitol,the expression of MdBBXl also significantly improved the tolerance of E.coli to stress,and its growth rate was significantly higher than that of the control group,which entered the logarithmic growth phase 4 hours earlier than the control group.Both solid and liquid stress culture showed that MdBBXl could improve the endurance of E.coli under abiotic stress.(5)Overexpression of MdBBX1 in Arabidopsis thaliana improved its tolerance to abiotic stresses.Homozygous lines of overexpressing MdBBX1 in Arabidopsis thaliana were obtained using flower leaching method.Under normal growth conditions,there were no significant differences in seed germination rate and growth status between MdBBXl-overexpressed plants(OE)and wild type plants(WT).However,in the medium containing NaCl,mannitol and exogenous ABA,OE seeds germinated faster and the germination rate was significantly higher than that of WT,and the root length of OE plants was significantly longer than that of WT.After the treatment with NaCl or PEG on Arabidopsis thaliana seedlings for 20 days,OE appeared later leaf wilting and lighter yellowing than WT,and its survival rate was significantly higher than that of WT plants;NBT staining experiment showed that the O2·-accumulation in OE plants was significantly lower than that of WT plants.The results showed that the expression of MdBBXl could improve the abiotic stress resistance of Arabidopsis thaliana.(6)The mechanism that MdBBXl improves the stress resistance of plant is related to ABA signaling pathway and scavenging reactive oxygen species(ROS).To explore the stress resistance mechanism of MdBBX1,we examined the expression of related genes in ABA signaling pathway and ROS clearance pathway after the treatment with stress.After the treatment with 250 mM NaCl solution,the expression of ABA signaling pathway related genes ABA1,ABA2,NCED3,RD29A and ROS scavenging pathway related genes SOD and CAT in WT and OE plants were increased,but the up-regulation multiple of OE plants was more significant than that of WT plants,suggesting that MdBBXl might participate in stress response through ABA signaling pathway and ROS scavenging pathway. |
