Identification And Function Prediction Of Differential Expressed Jujube NAC Transcription Factors During Jujube Witches’ Broom Pathological Process

Jujube Witches Broom is a kind of jujube disease caused by phytoplasma.At the time of onset,there were phenomena such as plexus branching,yellowing,and flower returning.And then,it got weak and reduction of output,and finally it became to death.This is the most serious jujube disease.At present,in view of the genome sequence of jujube witches’-broom phytoplasma and its pathogenic factors are unclear,therefore our laboratory are planning to find the genes which are affected by the phytoplasma infection expressing confusedly and lead to phenotypes such as plexus,yellowing,lower returning and so on.Some studies have shown that after phytoplasma invades plants,its pathogenic factors can interact with transcription factors of plants,thereby affecting expression of downstream gene.This study started with the plant-specific NAC transcription factor family,identified its members and performed bioinformatics analysis.And then we grafted the diseased jujube branches onto the healthy jujube seedlings,making the phytoplasma to infect the healthy jujube seedlings.Judging by the phenotypic observation of the process of jujube witches’-broom and the detection of the distribution of phytoplasma,we determined the critical period of jujube witches’-broom.Using high-throughput sequencing technology,the transcriptome data of diseased jujube seedlings and healthy seedlings at different stages were analyzed to find NAC transcription factors that were differentially expressed during the jujube disease.Next,we perform full-length cloning and function prediction proceeding from the NAC transcription factors in these differentially expressed transcription factors,and construct over-expression vectors which were then transformed into tobacco and Arabidopsis thaliana to identify their functions.The main results are as follows:(1)Through grafting spread method,the mobidity extent of phytoplasma was detected by PCR in the process of phytoplasma infection of jujube.Genomic RNA was extracted from different stages of phytoplasma infection in jujube for transcriptome sequencing.eighty-four NAC transcription factors were identified according to the data of transcriptome combined with NCBI database.The phylogenetic tree,chromosome localization,domain,and motif were analyzed.We analyzed the expression of NAC transcription factor in different stages of phytoplasma infection.The results showed that all of the 84 NAC transcription factors in jujube contained the conserved NAM domain and were unevenly distributed on 12 chromosomes of jujube.(2)The gene expression heat map analysis of NAC transcription factor genes in jujube trees at different stages of phytoplasma invasion.The expression levels of 36 NAC transcription factor genes were detected in the transcriptome data.After mapping the heat maps,36 Zj NAC transcription factors which expression trends were similar could be divided into 6 groups.The results showed that in addition to the 2 genes of the fifth group,the remaining 34 genes were up-regulated in the late stages of disease.Differentially expressed Zj NAC3,Zj NAC41,Zj NAC49,Zj NAC54,Zj NAC55 and Zj NAC73 transcription factors were selected for RT-PCR identification in accordance with the expression trends of the transcriptome data.The trend of the expression levels of Zj NAC3,Zj NAC54,Zj NAC73 these three genes in RT-PCR validation was similar to that of transcriptome data analysis,and the relative expression levels in the later stages of the three genes changed greatly,indicating that these genes may play a regulatory role in the resistance of jujube to phytoplasma infection.(3)Overexpression vectors of Zj NAC3,Zj NAC54,and Zj NAC73 genes were constructed and transformed into tobacco by Agrobacterium-mediated transformation.7 resistant Zj NAC3 resistant buds,4 resistant Zj NAC54 resistant buds,and 3 Zj NAC73 resistant transgenic lines were obtained.(4)Overexpression vectors of Zj NAC3,Zj NAC54,and Zj NAC73 genes were constructed and transformed into Arabidopsis thaliana by Agrobacterium-mediated transformation.6 resistant Zj NAC3 Arabidopsis thaliana transgenic plants,10 Zj NAC54 transgenic Arabidopsis plants and 10 Zj NAC73 transgenic plants were obtained.The transgenic leaves and the control were treated with 100 m M H2O2.The results showed that the activity of CAT and SOD in the transgenic Arabidopsis thaliana were higher than the control from 0 to 18 days,indicating that these three genes may be the functional gene that plays an important role in biotic stress.Based on the study of the function of NAC transcription factors,we defined its mechanism in the process of jujube disease and made reference for the molecular mechanism of Jujube Witches Broom and prevention and treatment of Jujube Witches Broom.