Study On The Mediated Role Of Mitochondria In The Process Of PCD Induced By SA In Cucumis Sativus L.

Cucumber?Cucumis sativus L.?is the major vegetable crops in China as a genusCucurbitaceae.It is susceptible to be infected by some pathogen which causes serious loss to cucumber production.As a endogenous signal molecules,salicylic acid?SA?can induce expression of genes to enhance the disease resistance of plants.In this study,we investigated the molecular mechanism of SA inducing cucumber resistance by studying the induction of mitochondria-related genes in cucumber PCD induced by SA.The result of experiment are as follows:?1?Identification of cucumber PCD:treating the cucumber leaves with 10mM SA and 0.2%MeJA and applying Trypan blue stainingare to identify PCD in this study.The results showed that Trypanblue dyed the treated leaves blue,indicating that SA and MeJA treatment produced cell death.At the same time,ROS also detected the production of H₂O₂ and O₂^-.Also,the accumulation of ROS caused the occurrence of plant PCD.?2?The screening and expression analysis of cucumber mitochondria genes:mitochondria are not only the energy center cells but also play important role in the process of the PCD.16 genes related mitochondria were screened via Illumina high-throughput sequencing technology,the primers were designed.These mitochondrial genes were tested and analyzed by RT-PCR and qRT-PCR in SA treatment and downy mildew treatment.The Analysis of RT-PCR results revealed that these genes were expressed in SA and downy mildew treatments.The results of qRT-PCR showed that the expression of genes were similar or opposite.16 genes controlled positively or negatively in the PCD process,and all of them were upregulated during SA treatment,including mitochondrial pyruvate carrier 2?A?,Prohibitin-3?B?,cardiolipin-specific deacylase?F?,uncoupling protein 5?G?,carnitine carrier-like protein?H?,Formate dehydrogenase 1?I?,Gamma aminobutyrate transaminase 1?J?,Mitochondrial import inner membrane translocase subunit?K?,Proline dehydrogenase 2?L?,Pyruvate dehydrogenase E1 component subunit alpha?M?,and Acetylornithine amino-transferase?P?in downy mildew treatment and SA treatment seemingly upregulated.However,3- hydroxyisobutyryl-CoA hydrolase like protein?C?,Chaperonin CPN60-2?D?,Glycine dehydrogenase?E?,Elongation factor Tu?O?and Leucine zipper and EF-hand containing transmembrane protein 1?N?five genes were up-regulated in SA treatment but down-regulated during downy mildew treatment.The 16 genes were analyzed and researched by searching literature.These genes belonged to four functional groups,including involved in mitochondrial membrane transport,cell respiration,protein synthesis,folding and transport,mitochondrial signal transduction.?3?Cloning and analysis of CsPHB3 and CsProDH2 of cucumber:cucumber mitochondrial related genes CsPHB3 and CsProDH2 were cloned and analyzed by bioinformatics.The full-length cDNAs of the CsPHB3 and CsProDH2 genes were obtained.After logging in GenBank,the numbers were XP₀04138106.1 and XP₀04141603.2,respectively.The length of CsPHB3 gene cDNA was 837 bp,which encoded 279 amino acids.The length of CsProDH2 gene cDNA was 1479 bp,which encoded 839 amino acids.NCBI BlastX results showed that CsPHB3 belonged to the SPFH superfamily and CsProDH2 belonged to ProDH superfamily.The sequencing results showed that the sequence homology between it and the one from GenBank was100%.?4?The transformation between Cucumber CsPHB3 and CsProDH2 Gene and Arabidopsis thaliana:To construct the overexpression vector of CsPHB3 and CsProDH2 gene,and transfer the plasmid into the wild type Arabidopsis plant by Agrobacterium tumefaciens-mediated immersion method to obtain transgenic Arabidopsis plant T₀.