Selecting Of Key AP2/ERF Transcription Factors During The Development Of Jujube Witches' Broom?Difference Expression Analysis And Genetic Transformation

Jujube Mill.?Rhamnaceae?Ziziphus Mill.)is the main economic tree species and fruit tree species in China.Jujube trees are often infected by phytoplasma of jujube witches'broom.After infection by phytoplasma,it usually causes symptoms such as branching,yellowing of leaves,Hormone metabolism.Jujube witches'room is devastating to the production of jujube trees.Related studies have shown that AP2/ERF transcription factors have a wide variety and diverse functions,which can regulate plant growth and development,hormone metabolism,and participate in the regulation of abiotic stress and biotic stress.In order to study the role of AP2/ERF transcription factors in the pathogenesis of jujube witches'broom,bioinformatics analysis was used to identify the AP2/ERF transcription factor family in jujube,and the AP2/ERF transcription factors were involved in the pathogenesis of jujube witches'broom.Expression analysis selecting genes that may play a role in the pathogenesis of jujube witches'broom,transforming 'K326' tobacco leaves and'Fengmiguan' jujube with different explants(root,hypocotyl,leaf,cotyledon))performing gene function verification.The main results are as follows:1.A total of 145 AP2/ERF transcription factors were identified in the jujube by the plant transcription factor database and the NCBI database,The ERF,AP2,and RAV subfamilies contain 116,23,and 5 members,respectively,and one transcription factor is independent;There are distributions in the 12 chromosomes of jujube;constructing a phylogenetic tree of ERF protein sequences of jujube and Arabidopsis,and dividing the ERF Subfamily into 12 subgroups,namely Group I-X,Group VI-L,GroupXb-L.In addition to the GroupXb-L subgroup,each group contain unequal number of genes.At the same time,the motif analysis of the AP2/ERF proteins were found to have the most conserved motifs in the AP2 subfamily,followed by the ERF subfamily.The RAV subfamily contains the least conserved motifs.2.The 'huizao'jujube branches infected with jujube witches'broom and healthy 'huizao'jujube branches with full buds were grafted into healthy 'huizao' jujube roots,and the RNA in jujube leaves was extracted and analyzed by transcriptome analysis according to the key period of phytoplasma infection process and related symptoms.73 differentially expressed genes(DEGs)were obtained,and 10 genes with up-regulated expression(ZjERF1,ZjERF46,ZjERF18,ZjERF102,ZjERF36,ZjERF49,ZjEFF88,ZjERF38,ZjERF85,ZjERF88)were selected.The diseased jujube branches and healthy jujube branches were grafted into healthy rootstocks,and the spatial and temporal expression analysised of the selected ten genes in the process of phytoplasma infection,It was found that the expression of ZjERFl,ZjERF36,ZjERF38,ZjERF49,ZjERF85 and ZjERF102 genes in phloem were significantly up-regulated during phytoplasma infection,the expression of ZjERFl?ZjERF85 were the most significant,and the expression of ZjERF46 and ZjERF88 were significantly up-regulated in leaves.3.The Agrobacterium-mediated method was used to verify the gene function of ZjERFl,ZjERF85 and ZjERF88 overexpression vectors,the tobacco leaf explants can differentiat into callus after 10 days in selecting culture,can grow into buds after 25 days,the buds can grow to 4-5cm after 40 days,and can grow into plants aftert 60 days.Molecular identification of transgenic tobacco was carried out by PCR.A total of 95 positive plants were obtained,including 18 ZjERF1 transgenic plants,64 ZjERF85 transgenic plants and 13 ZjERF88 transgenic plants.4.The Agrobacterium-mediated method was used to verify the genetic function of different explants(including hypocotyls,roots,cotyledons,and leaves)transformed with ZjERF85 vector,the roots and hypocotyls grow more fast than cotyledons and leaves.The roots and hypocotyls can grow buds in the selecting medium after 15 days,and can grown into plants after 25 days.The leaves are differentiated into callus afterlO days,and can sprout after 30 days,and grow into plants after 60 days;cotyledons callus can be formed after 15 days,the buds are sprouted after about 25 days,and can grow into plants after about 50 days.Molecular identification of transgenic plants was carried out by PCR,and 22 ZjERF85 transgenic positive shoots and 13 intact plants were obtained.