Clone And Function Of Ethylene Response Factor ZjERF53 In Jujube

Jujube（Ziziphus jujuba Mill.）is a special economic forest fruit originated in China,which includes fresh edible and dried.Fresh jujube is an important part of jujube industry in China.It is of great significance to study the ripening biology of jujube fruit for the harvest,storage,transportation and sale of fresh jujube.AP2/ERF is a plant-specific transcription factor.Studies have found that AP2/ERF may be involved in the regulation of jujube fruit ripening,but its biological functions are still unclear.This study is based on genome database of Chinese jujube,combined with the published transcriptome data and q PCR analysis,as well as gene clone and subcellular localization technologies.The cDNA library of jujube fruit was constructed by Clontech SMART to screen for the protein interacting with ZjERF53.Also we transformed ZjERF53 into Micro-Tom tomato and the phenotypic changes of overexpressed plants were observed to reveal the biological function of ZjERF53in fruit ripening.The main results are as follows:1.A total of 116 AP2/ERF family genes were identified,of which 14 genes were highly related to fruit ripening.q PCR analysis found that the expression of ZjERF35,ZjERF37,ZjERF53,Zj DREB18,Zj DREB26,and Zj AP2.16 genes in the two jujube cultivars increased significantly with fruit ripening and maintained a high level during the ripening process.2.A ZjERF53 gene with the highest relative expression during fruit ripening was cloned from the full red jujube in‘Fucuimi’.The full length of coding region of ZjERF53 was 1155bp,located at 19497761～19751592 bp in Chr 1,containing an intron and encoding 384amino acids.Through bioinformatics analysis,the theoretical isoelectric point of the protein is 4.85,the relative molecular weight is 42.86 k Da,and the average hydrophilic coefficient of the protein is-0.826.The subcellular localization study of ZjERF53 can be expressed on both the cell membrane and the nucleus.3.The yeast expression vectors pGBKT7-ZjERF53,pGBKT7-ZjERF53-N1,pGBKT7-ZjERF53-N2,pGBKT7-ZjERF53-C1 and pGBKT7-ZjERF53-C2 were constructed and transformed into Y2HGold yeast cells for transcription activation analysis.The results showed that ZjERF53 displayed strong self-activating activity which were found to be related to the carbon-terminal 321 bp of protein sequences.4.Clontech SAMRT technology was applied to constructed a cDNA library of jujube fruit.The results of library quality test showed that the library titer was 7.68×10⁷cfu/m L and the recombination rate was 96%.The cDNA fragments inserted were distributed from250～500 bp.And we finally generated totaling four non-abundant protein sequences by yeast two-hybrid technology using a non-self-activating vector named pGBKT7-ZjERF53-N,which are related to biotic and abiotic stress processes such as disease resistance and high temperature tolerance.5.The plant expression vector pCAMBIA2300-35S:ZjERF53::EGFP was constructed and genetically transformed into Micro-Tom tomato by agrobacterium mediated method.A total of 10 ZjERF53 transgenic and empty vector plants of the T₀generation were obtained,respectively,through PCR positive detection.In conclusion,this study cloned the full length of ZjERF53 encoding region and revealed that ZjERF53 may be involved in biotic and abiotic stress processes such as disease resistance and high temperature tolerance of jujube,and it also can affect fruit ripening and size.Our findings gained the understanding of candidate functions of ZjERF53 in jujube,but also laid a foundation for further investigating the regulation of ERF transcription factors in jujube biological processes.