The Role Of CsMPC1 And CsE1? Genes In PCD Mediated By Mitochondria Of Cucumis Sativus

Salicylic acid?SA?can induce expression of genes to enhance the disease resistance of plants.Previous research by the study team confirmed that SA can induce hypersensitivity?HR?in cucumber leaves,which is a process of programmed cell death?PCD?.To further explore the mechanism of action of SA,transcriptome analysis of the leaves of Cucumis sativus before and after SA treatment showed 1756 differentially expressed genes.In this study,we significantly selected up-regulated expression of CsMPC1 and CsE1? genes associated with mitochondrial function,and studied their expression characteristics,using overexpression of Arabidopsis thaliana and mutant Arabidopsis thaliana to study gene function in order to reveal genes in mitochondria-mediated PCD.The role lays the foundation.Detailed results are shown below:1.treating the leaves of Cucumis sativus with 10 mM SA and 0.2% MeJA,The relative expression of in the roots,stems,leaves,and the leaves of Cucumis sativus treated by MeJA,as well as Determination of pyruvic acid?PA?content and pyruvate dehy-drogenase?PDH?activity in the leaves of Cucumis sativus treated SA and MeJA.A link between gene levels and physiological levels is achieved.CsMPC1 and CsE1?genes had the higher expression levels in the leaves,and up-regulated expression regulation in MeJA mediated of PCD processes;The change of CsMPC1 Mitochondrial pyruvate carrier gene level caused the change of PA content,and it was found to be negatively correlated.The change of CsE1? Pyruvate dehydrogenase E1 component subunit alpha gene level affected the change of PDH activity,and it was found to be positively correlated.there was no significant change in PDH activity under MeJA and SA treatments.2.Primers were designed based on the open reading region of the mRNA,and the CsMPC1 and CsE1? genes were cloned from cucumber.The length of CsMPC1 gene cDNA was 311 bp,which encoded 103 amino acids.The length of CsE1? gene cDNA was 1199 bp,which encoded 399 amino acids.NCBI BlastX results showed that CsMPC1 belonged to the MPC superfamily and CsE1? belonged to TPP superfamily.3.To construct the overexpression vector of CsMPC1 and CsE1? genes,and transfer the plasmid into the wild type Arabidopsis thaliana by Agrobacterium tumefaciens-mediated immersion method to PCR-positive plants.Using the three-primer method,the MPC1 gene?SALK₀89763.42.10.x?and the E1? gene?SALK₀56967.56.00.x?T-DNA insertion mutant were obtained from Arabidopsis thaliana Biological Resource Center by the three-primer method PCR identification to obtain homozygous plant.4.The overexpression Arabidopsis thaliana?OE-1 and OE#-1?and the mutant Arabidopsis thaliana?mpc1-1 and E1?-1?of MPC1 and E1? were measured the phenotype?physiological indicators and PCD identified.The results were as follows:?1?The mutant Arabidopsis thaliana mpc1-1 was sensitive to ABA and had a drought-resistant function;The overexpression plant OE-1 transported elevated levels of PA,indicating that the PA transporter functions;Mutant plant mpc1-1 pyruvate transport was impeded,mitochondrial function decreased,glycolytic pathway was enhanced,plant growth was inhibited,but PCD phenomenon was not detected.This indicates that the overexpression and mutant Arabidopsis thaliana of MPC1 gene,PA metabolism changes but did not affect the occurrence of plant PCD.When MPC1 gene was deleted,other transport proteins of MPC family were still driving,and MPC1 gene overexpressing plants grew normally without cell deathing.?2?The overexpressing plant OE#-1 had no significant difference in phenotype and PDH activity from wild type,no accumulation of ROS,and did not affect the occurrence of PCD;mutant plant E1?-1 PDH activity was inhibited,organism The aerobic metabolism was hindered,which seriously hinders the cells respiration,and the accumulation of ROS may caused plant PCD,which affects the normal growth and development of the plant.This indicates that the E1? gene may play an important role in causing plant PCD.