Expression And Functional Analysis Of CsATG8c And CsATG8e Genes In Cucumis Sativus

Autophagy is an evolutionarily conserved catabolic process that exists in eukaryotes.During this process,autophagy degrades damaged proteins and organelles in cells and recycles and reuses intracellular materials.When cells undergo programmed cell death,autophagy plays an active role in degrading dead cell components.Autophagy can not only promote defense-related allergic cell death caused by pathogenic bacteria,but also limit unnecessary cell death and disease transmission.This precise regulation has an important role and significance in plant immune response.Autophagy was first studied in yeast,followed by more detailed research in animals,but research in plants is still in its early stages.Autophagy genes are currently found in plants such as Arabidopsis,soybean,rice and corn.Studies on cucumber autophagy-related genes have not been reported.This study screened and identified 20 members of cucumber autophagy gene family at the genome-wide level.The structural characteristics and phylogenetic relationships of the 20 family members were analyzed.The 20 genes were induced by 2 hormones and The expression under three stress treatments was analyzed.In this study,the characteristics and functions of the genes CsATG8 c and CsATG8 e were selected for further analysis,which provided a theoretical basis for revealing that autophagy participates in all stages of cucumber growth and development and resists external stress.The experimental results are as follows:1.Based on the full mining of bioinformatics methods,a total of 20 putative ATG?autophagy-related genes?genes were identified in the cucumber genome.Much less autophagy-related genes than in rice,tobacco and corn.Unlike the autophagy gene in yeast,which has only a single copy,the cucumber autophagy gene has both a single copy and multiple copies,such as the ATG8 gene.There are 5,9,and 11 copies of ATG8 in tobacco,Arabidopsis,and soybean,respectively.In cucumber,the autophagy gene ATG8 has six copies,respectively?ATG8a-ATG8f?,which is similar to that in tobacco.The structure,conserved motifs,amino acid sequence length,predicted molecular weight and isoelectric point,colinearity,phylogeny,chromosome localization,selection pressure and phylogenetic relationship of these ATG genes were systematically analyzed.2.10 mmol/L SA and 0.2% MeJA were added to the leaves for hormone induction treatment,200 mmol/L NaCl,20% PEG6000,and darkness were used to treat the roots,stems,and leaves of cucumber vegetative organs with salt,drought,and carbon starvation stress.20 autophagy-related genes in cucumber were analyzed by qRT-PCR.qRT-PCR expression results showed that 16 CsATG genes were induced by the hormone SA treatment,and 16 CsATG genes were down-regulated by the hormone MEJA treatment;under carbon starvation stress,20 CsATG genes were induced to express in leaves,and under high salt stress 10 CsATG genes were induced in roots.Under drought stress,16 CsATG genes were induced in roots.It was also found that most of the 20 CsATG genes were expressed in cucumber roots,stems and leaves at 6h of carbon starvation stress,suggesting that it has a potential role in nutritional starvation tolerance.3.Two genes were selected from 20 Cucumis sativus autophagy-related genes as CsATG8 c and CsATG8 e.Cloning technology was used to clone the full-length sequences of the coding regions of these two autophagy genes and analyzed by bioinformatics.The results showed that the Cs ATG8 c gene was located in Cucumis sativus on chromosome 5,the full length of the cDNA is 356 bp,the number of protein amino acids is 118,the molecular weight of the encoded protein is 13689.82 Da,the theoretical pI value is 8.71,the total number of atoms is 1954,the instability coefficient is 35.39,and the fat coefficient is 85.93.The CsATG8 e gene is located on chromosome6 of cucumber.The full-length cDNA is 371 bp,the number of protein amino acids is123,the molecular weight of the encoded protein is 13721.89 Da,the theoretical pI value is 9.33,the total number of atoms is 1961,the instability coefficient is 40.39,and the fat The coefficient is 93.58.NCBI BlastX alignment sequencing results showed that both CsATG8 c and CsATG8 e belong to the UBQ superfamily.4.CsATG8 c and CsATG8 e genes transformed into Arabidopsis thaliana: Cucumis sativus ATG8 c and ATG8 e gene overexpression vectors were constructed,and the plasmid was transferred into wild-type Arabidopsis thaliana: plants using the Agrobacterium-mediated dip method to obtain Cs ATG8 e transgenic Arabidopsis thaliana plants T₁.