| Euphorbia kansui Liou is a perennial herb in Euphorbia,Euphorbiaceae family.The laticifer in E.kansui plants is a special secretory cell that can secrete latex,and it is also the main site for the synthesis and storage of the secondary metabolites.Autophagy is a conserved cellular process in eukaryotes which plays an important role in the recycling of nutrients and cytoplasmic components,adjusting the tolerance to biotic and abiotic stresses.When the cells are subjected to starvation or other stresses,production of double-membrane structures,the autophagosomes,is induced,where organelles and macromolecules and other cytoplasmic components were engulfed,and then they fused with lysosome or vacuole for decomposition and reuse.This process requires a class of unique gene,namely autophagy-related genes(Atg)regulation.Previous studies have found that there is a lysosomal autophagy pathway during the development of E.kansui laticifers.At the same time,autophagy can participate in the development of laticifers and play an important role in maintaining intracellular homeostasis.The promoter is a DNA sequence which is located upstream of the 5’ end of the structural gene,and has the ability to initiate the transcription of activating gene.The structure of eukaryotic promoter was complex,and has many cis-acting elements upstream that can affect the process of plant growth and development.The autophagy-related gene Ek Atg18 a is an important regulatory gene of autophagosome membrane synthesis,but the transcriptional regulation mechanism of this gene lacks systematic study at present.Therefore,based on the transcriptome data of E.kansui,the 5’flanking fragment of Ek Atg18 a gene was successfully cloned by using TAIL-PCR.2219 bp Ek Atg18 a promoter fragment was obtained and the transcription initiation site was determined by 5’ RACE.Combined with the prediction results of bioinformatics analysis,the 5’ deletion analysis plant fusion vector of Ek Atg18 a promoter was constructed and transformed into Arabidopsis thaliana.GUS histochemical staining was performed on T3 generation homozygous transgenic A.thaliana seedlings to preliminarily determine the promoter activity.After application of the abiotic stress and the exogenous signal molecules treatment,quantitative activity analysis of GUS was carried out on homozygous transgenic A.thaliana to explore the response mode of cis-acting elements upstream of Ek Atg18 a promoter.The research results are as follows:1.The 5’ flanking fragment of Ek Atg18 a gene was successfully cloned by TAIL-PCR using E.kansui genomic DNA as template.The transcription initiation site was determined by 5’RACE.After sequence proofreading,the result showed that 2219 bp promoter fragment of Ek Atg18 a was obtained.Bioinformatics results showed that there are many potential cisacting elements upstream of Ek Atg18 a promoter that can respond to abiotic stress and participate in signaling molecule regulation pathway.2.In order to explore the structure,function and corresponding response mode of cis-acting element of Ek Atg18 a promoter,different 5’ deletion plant fusion vectors were constructed,which were Full(2219 bp),P1(1901 bp),P2(1689 bp),P3(1076 bp),P4(900 bp)and P5(572 bp).Then Agrobacterium mediated transformation of A.thaliana,the T3 generation homozygous transgenic A.thaliana plants were identified and harvested.3.GUS histochemical staining was performed on homozygous A.thaliana seedlings transformed with different 5’-deletion Ek Atg18 a promoter vectors.The results showed that the Ek Atg18 a promoter had the activity of initiating and activating the transcription and could effectively induce the expression of GUS.At the same time,the expression of GUS activity was different in different transgenic lines.Quantitative analysis showed the GUS activity expressed the highest in transgenic A.thaliana transformed with P3 promoter fragment,indicating that the P3 promoter fragment had strong activity under untreated conditions.4.The drought and high salt stresses were applied to different lines of homozygous A.thaliana plants.The GUS activity in transgenic plants was detected quantitatively.After drought stress,the GUS activity of Full,P1,P2 and P3 transgenic lines increased significantly compared with the other two groups;while after 200 m M Na Cl treatment,only P5 transgenic line showed a decrease in GUS activity.The results showed that Ek Atg18 a promoter was sensitive to abiotic stress treatment,and the cis-acting elements upstream the promoter could respond to abiotic stress treatment.5.In addition,quantitative analysis of GUS activity showed that there was a cis-acting element between Full and P1 promoter fragment that could respond to Me JA treatment.The cis-acting elements on the promoter fragments of Full,P1 and P2 transgenic lines were more sensitive to 2 m M SA treatment.Meanwhile,the Full,P1,P2 and P3 transgenic lines showed more significantly increase of GUS activity under 200 μM ABA treatment.The results showed that the treatment of Me JA、SA、ABA,and the exogenous signaling molecules resulted in prominent GUS expression in transgenic plants equipping with the corresponding response elements.In conclusion,the 5’-deletion analysis and functional study showed that there were multiple cis-acting elements upstream of Ek Atg18 a promoter which could respond to drought and high salt stress and participate in the SA,ABA and Me JA molecular regulatory pathways.The location and functional response of elements were basically consistent with the bioinformatics prediction results.The results might lay a theoretical foundation for exploring the transcriptional regulation mechanism of Ek Atg18 a and also provided an available endogenous promoter for transgenic E.kansui. |
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