The Role Of HEMA1 And PaO Genes In PCD Mediated By Chloroplast Of Cucumis Sativus L.

Programmed cell death(PCD)is ubiquitous in the growth and development of plants and animals,and target cells eventually die under certain developmental and pathological conditions.This is critical for controlling the number of cells,removing unwanted diseased or damaged cells and maintaining cell homeostasis.The PCD process is studied in detail in animal cells,but the study of this process in plants is still infancy.Unlike animals with only mitochondria,plants have two energy-generating organelles: mitochondria and chloroplasts.Chloroplasts are unique energy-transducing organelles in plant cells and produce ROS,all of which indicate that chloroplasts maybe involved in PCD.In the process of plant PCD,the mechanism of action of chloroplast-mediated PCD remains blank.This study investigated the mediated effects of chlorophyll-related genes in the induction of Cucumis sativus PCD by SA.The results are as follows:(1)The differentially expressed genes of SA treated Cucumis sativus leaves obtained by Illumina high-throughput sequencing technology were analyzed.In the results of 1759 DEGs,16 genes related to chlorophyll synthesis and degradation were screened according to the given gene annotation.These 16 genes were analyzed by PCR and qRT-PCR methods.The results showed that 14 genes were up-regulated and 2 genes down-regulated on the basis of 24 h.(2)The RT-PCR and qRT-PCR results of 16 chlorophyll synthesis and degradation related genes were analyzed and compared and the key enzyme genes HEMA1 and PaO in the process of chlorophyll synthesis and degradation were selected.The cloning and bioinformatics analysis of the full-length sequences of the coding regions of these two genes showed that the homology of the gene sequencing results with the reference sequence of GenBank reached 100%.The CsHEMA1 gene is located on chromosome 2 of Cucumis sativus.The open reading frame(ORF)contains 1659 base pairs and encodes 552 amino acids.It contains 3 exons and 2 introns.The molecular weight of the encoded protein is 60895.18 Da,the theoretical isoelectric point(pI)is 8.40,containing a conserved domain;The CsPaO gene is located on chromosome 1 of Cucumis sativus.The open reading frame(ORF)contains 1640 base pairs and encodes 546 amino acids.It contains 7 exons and 7 introns.The molecular weight of the encoded protein is 61039.26 Da,the theoretical isoelectric point(pI)is5.74,containing six conserved domains.By constructing the phylogenetic tree,the Cucumis sativus CsHEMA1 gene has the highest homology with the Cucumis melo CmHEMA1 gene;the Cucumis sativus CsPaO gene has the highest homology with the Cucumis melo CmPaO gene.(3)The HEMA1 and PaO gene overexpression vectors of Cucumis sativus were constructed and transformed into Arabidopsis thaliana.The overexpression and HEMA1,PaO gene mutant Arabidopsis thaliana homozygous strains were screened,physiological indicators and PCD were identified.The results showed that the loss of HEMA1 gene caused symptoms of streaking,yellowing and early death in Arabidopsis thaliana leaves,germination lag and germination rate decreased;chlorophyll and soluble sugar content decreased;Overexpression of CsHEMA1 gene in Arabidopsis thaliana,chlorophyll content increased,Salicylic acid induced cell death was reduced,compared with wild type,antioxidant enzyme activity increased at the same treatment time.PaO gene deletion had no significant effect on chlorophyll content,but chlorophyll a content decreased,chlorophyll b content increased,and soluble sugar content decreased;Overexpression of the CsPaO gene in Arabidopsis thaliana reduced chlorophyll content and decreased the degree of cell death induced by Salicylic acid.Compared with wild type,the antioxidant enzyme activity increased at the same treatment time.