| Jujube(Ziziphus jujuba Mill.)belong to Rhamnaceae plants,which is an important fruit tree in our country.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 study the biological function of jujube WRKY transcription factor(TF)and interaction mechanisms between jujube and Candidatus Phytoplasma ziziphi,We explored and identified the pupative jujube WRKY TF family members by bioinformation.The transcriptional expression levels of WRKY transcription factor genes in jujube was carried out,and the expression changes of them were verified by quantitative real time polymerase chain reaction(q RT-PCR)during the jujube witches' broom(JWB)developing.At the same time,we analyzed the expression of WRKY transcription factor genes after the treatment of salicylic acid(SA)and methyl jasmonate(Me JA).The main results are as follows:1.Through grafting spread method,the genomic RNA was extracted from different stages of phytoplasma infection in jujube for transcriptome sequencing.Sixty-nine WRKY 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 WRKY transcription factor gene in different stages of phytoplasma infection.The results showed that all of the 69 WRKY transcription factors in jujube contained the WRKY conserved domain,which could be divided into three groups : Group I,Group II,Group III,contained 17,41,11 members,respectively.WRKY TFs were unevenly located on different chromosomes,but there no WRKY TF on chromosome 7.2.The expression pattern of 28 jujube WRKY TFs were detected to be different during the JWB developing in jujube leaves,and differently expressed Zj WRKY TFs were mainly detected from 39 to 52 weeks after infecting.Six differently expressed jujube WRKY TFs Zj WRKY5?Zj WRKY8?Zj WRKY42?Zj WRKY52?Zj WRKY61 and Zj WRKY69 were screened from the transcriptomics analyses,and the expression changes of them were verified by quantitative real time polymerase chain reaction(q RT-PCR)during the jujube witches' broom(JWB).The results showed that the expression levels of Zj WRKY8?Zj WRKY52?Zj WRKY61 and Zj WRKY69 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.3.To further validate the roles of target genes on responding to phytoplasma stresses,we treated the embryo culture seedlings of jujube with salicylic acid and methyl jasmonate,and the expression levels of Zj WRKY5?Zj WRKY8?Zj WRKY42?Zj WRKY52?Zj WRKY61 and Zj WRKY69 were analyzed in the treatment of the jujube seedlings with different time course.The results showed that after SA treatment,the expression levels of Zj WRKY42 and Zj WRKY52 were significantly increased.The expression levels of Zj WRKY42 and Zj WRKY61 were significantly up-regulated in response to Me JA 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.4.The overexpression vector of ZjWRKY8,ZjWRKY52,and ZjWRKY61 genes was constructed and transformed into tobacco by Agrobacterium tumefaciens.We obtained fifteen,twenty-eight,sixteen resistant buds,respectively.5.Zj WRKY8,Zj WRKY52,and Zj WRKY61 genes were transiently expressed in tobacco leaves,and the leaves were treated with 0.1 mol·L-1 H2O2.The results showed that the activities of CAT in leaves of Zj WRKY8 and Zj WRKY61 was higher than those in control,and the activities of SOD in leaves of Zj WRKY8 and Zj WRKY61 was higher than those in control at 0-12 h;The result of DAB histochemical staining showed that Zj WRKY8-OE had an increased yellow spots. |
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