| Jujube(Ziziphus jujuba Mill.)belong to the rat lee branch(Rhamnaceae)plants,is China's first big dried fruit tree species.In recent years,it has become a hotspot of important economic forest development in our country.With its strong adaptability and resistance,jujube is the pioneer species of fruit trees on the mountain beach and ideal ecological economic forest tree species.Jujube cultivating process often accompanied by the occurrence of pests and diseases,of which phytoplasma is the most serious disease,as a cancer of Chinese jujube.In order to obtain the genome sequence of Phytoplasma from jujube witches broom,and study of the interaction between jujube and Phytoplasma,we referenced the mothed of separation Phytoplasma from references and tried to separation phytoplasma genome DNA from jujube witches broom.At the same time,the transcriptional expression levels of AP2/ERF transcription factor gene in jujube was carried out,and the possible role of ERF genes in the process of phytoplasma infection jujube was analyzed.The main results are as follows:1.We used differential centrifugation method,extracted phytoplasma crude extract of the jujube witches broom,and combined with pulsed field gel electrophoresis to separate the genome sequence of phytoplasma.The results indicated that a clear and bright band appears in 1600~2200 kb.Phytoplasma were detected from target band by PCR and real-time quantitative PCR.2.Through grafting spread method,the mobidity extent of phytoplasma was detected by PCR and DAPI in the process of phytoplasma infection of jujube.Genomic RNA was extracted from different stages of phytoplasma infection in jujube for transcriptome sequencing.Ninty-three AP2/ERF 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 AP2/ERF transcription factor gene in different stages of phytoplasma infection.The results showed that all of the 93 AP2/ERF transcription factors in jujube contained the conserved AP2 domain and were unevenly distributed on 12 chromosomes of jujube.The differential expression of Zj ERF04,Zj ERF20,Zj ERF21,Zj ERF11,Zj ERF18,Zj ERF22,Zj ERF28,Zj ERF62,Zj ERF72,and Zj ERF109 indicated that these genes were up-regulated in the period of jujube phytoplasmainfection.3.The real-time quantitative PCR results showed that the expression levels of Zj ERF04,Zj ERF20,Zj ERF21,Zj ERF11,Zj ERF18,Zj ERF22,Zj ERF28,and Zj ERF62 increased gradually from the beginning of phytoplasma infection to jujube pathogenesis,which indicating that these genes may play a regulatory role in jujube resistance to phytoplasma infection.4.To further validate the roles of target genes on responding to stree,we treated the embryo culture seedlings of jujube with salicylic acid and methyl jasmonate,and the expression levels of Zj ERF04,Zj ERF20,Zj ERF21,Zj ERF11,Zj ERF18,Zj ERF22,Zj ERF28,Zj ERF62,Zj ERF72,and Zj ERF109 were analyzed in the treatment of the jujube seedlings with different time course.The results showed that the expression of the above 10 genes increased after salicylic acid treatment,but only Zj ERF04,Zj ERF20,Zj ERF21,and Zj ERF22 were increased after methyl jasmonate treatment,which indicated that the target genes played important roles in the jujube responds to stress treatments,and the response mechanism may not be similar.5.The overexpression vector of Zj ERF04,Zj ERF11,and Zj ERF18 genes was constructed and transformed into tobacco by Agrobacterium tumefaciens.We obtained twenty,ten,and fiftheen transgenic resistant buds,respectively.6.Zj ERF04,Zj ERF11,and Zj ERF18 genes were transiently expressed in tobacco leaves,and the leaves and control were treated with 100 m M H2O2 respectively.The results show that the activities of CAT and SOD in leaves of Zj ERF04 were higher than those in control at 0~12 h,witch indicated that Zj ERF04 may be a potential functional gene that has an important role in biological stress. |
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