Evaluation Of High Temperature Acclimation And Functional Analysis Of MicroRNA171 Concerning Leaf Chlorosis Induced By Allopolyploidy In Cucumis

Polyploidization enriches plant diversity by giving rise to novel phenotypes and offering plants increased environmental plasticity with duplicated genomes.Polyploids are prevalent in nature,and all angiosperms have undergone one or more polyploidizations in their evolutionary history.Newly synthesized allotetraploid(Cucumis ×hytivus)with distinct young yellow-green leaves stably inherited in the offspring was observed to be potentially tolerant to high temperatures under field condition.To validate and understand the HT tolerance,the alletetrapliod(Cucumis×hytivus S14)and two diploid parents(Cucumis sativus and Cucumis hystrix)were used in experiment under the high temperature(HT)(38℃/30℃)compared with control(CK)conditions(28℃/20℃).The differences in growth,chlorophyll accumulation and photosynthesis between alloterotetraploid and parents were investigated.The transcriptome sequencing analysis of leaves after high temperature treatment was carried out to explore the transcriptional differences between allotetraploid and parents.In addition,the miRNA171 was differently expressed between allotetraploid and diploid parents according to the results of miRNA sequencing in the previous experiments.Hereof,the miRNA171 was cloned and functionally analyzed in Cucumber.The results are as follows:1.Physiological study on the allopolyploids and diploid parents under high temperatureIn this experiment,the high-generation inbred line(S14)of allotetraploid(C.×hytivus)and two diploid parents(C.sativus and C.hystrix)were used as experimental materials in the seedling stage(3-4 true leaves),there were treated with high temperature(38℃/30℃),and control(28℃/20℃).The growth rate,chlorophyll content,photosynthesis and fluorescence parameters were determined.The results showed that the growth rate of C.×hytivus was significantly increased after high temperature treatment,and the high temperature significantly promoted chlorophyll(Chl)accumulates in C.×hytivus,photosynthesis was significantly enhanced as well,and the maximum quantum efficiency value of photosystem II(Fv/Fm)in C.×hytivus was uneffected.In summary,our result demonstrated that C.×hytivus had better photosynthetic acclimation to high temperature in terms of chlorophyll accumulation,photosynthesis compared with its parents.2.Transcriptome analysis between allotetraploid and diploid parent under high temperature treatmentThe same leaves which were used for physiological measurement were sampled.Transcriptome analysis showed that the transcription levels were significantly altered between the allotetraploid and parents after high temperature treatment.The differential genes were mainly enriched into photosynthetic carbon assimilation.The expression analysis indicated that GLTX,CAO,NYC and CHL genes have important roles in promoting chlorophyll accumulation of C.× hytivus under HT.In addition,the additive expression genes were increased and the non-additively expressed genes in C.×hytivus are enriched in different metabolic pathways.Enrichment analysis showed that ELD-CC and TUR genes play important roles in carbon metabolism and removal of peroxides,the ELD-HH and TDR genes are more involved in lipid and energy metabolism.The heat map analysis of SOD,POD,CAT,GR,DHAR,FAD and heat shock protein(HSP)showed that the expression of C.×hytivus gene was positively responded to high temperature treatment.3.Cloning and Functional Analysis of mircroRNA171 in CucumberBased on the differential expression of microRNA171(miR171)between the C.×hytivus and parent.We further cloned four precursors(Csa-miR171a/b/c/d)of Csa-miR171 family genes in the northern Chinese cucumber C.sativus L.‘Beijingjietou’.Sequence alignment and phylogenetic analysis of the four precursors were conducted.Target genes of the mature sequence of Csa-miR171 were predicted and functional analysis were performed in Arabidopsis thaliana by genetic transformation.The results showed that the two mature sequences,Csa-miR171a/b were cleaved from the four Csa-miR171 precursors.Phylogenetic analysis showed that Csa-miR171a/b/c/d were closer to Cme-miR171e/c/a/b in melon than those in Arabidopsis,rice and tomato etc.GRAS transcription factor family was predicted to be the main target gene of Csa-miR171.Overexpression of Csa-miR171a in Arabidopsis significantly affected many phenotypes including leaf morphology and leaf color,blooming time,root,stem,fruit and floral morphogenesis.