Transcriptome Analysis Of Acquire Thermotolerance In Cucurbita Moschata And Identification Of Related Gene Families

Plants can only grow in a fixed location and cannot autonomously escape from hot environments,so they have evolved different adaptive mechanisms to survive under unfavorable conditions.Plants,when exposed to short-term high temperatures,develop the ability to withstand severe temperatures and this phenomenon is often referred to as acquired thermotolerance.As a more heat-tolerant and drought-tolerant plant,Cucurbita moschata has a high basal thermotolerance,and the study of acquire thermotoleranc in pumpkin seedlings can not only understand the molecular mechanism of acquire thermotoleranc,but also provide a theoretical basis for further improving the heat resistance of pumpkin.In this study,24 pumpkin materials were screened for heat tolerance through heat damage index survey,and their high temperature semi-lethal temperature was subsequently determined,and "24" C.moschata experimental materials with high temperature tolerance were selected for subsequent experiments,and the time of heat induction and high temperature stress treatment was determined by measuring peroxidase activity.When "24" C.moschata seedlings reached the two leaves and one heart periods,they were subjected to different heat stress assays in two groups,and the transcriptome was sampled and measured.To compare the differences between two different heat stress treatments based on RNA-seq sequencing results and to analyze the molecular regulatory mechanisms of heat tolerance in C.moschata to explore the key genes of acquired thermotolerance in C.moschata.The HSP70 family of genes was selected by searching and comparing using the C.moschata genome database,and then the gene family was identified using bioinformatics techniques,while its gene expression was analyzed in different patterns.The results of the study are as follows:(1)The 24 pumpkin materials were ranked by heat damage index,and the heatresistant candidates with the lowest heat damage index were: 8,23 and 24.Combined with the results of high temperature semi-lethal temperature determination: 24(55.36°C)> 23(54.14°C)> 8(48.78°C),the most heat-resistant experimental material was identified as "24 " C.moschata.(2)Design of experimental protocol in combination with the change of peroxidase activity with temperature treatment.the acquired heat treatment(with 38°C heat induction),in which two-leaved seedlings were first acclimated at 38°C for 180 min,allowed to recover at 25°C for 5 h,then challenged by severe heat stress at 45°C for 180min;the heat treatment(without 38°C heat induction),in which samples were directly introduced to a temperature of 45°C for 180 min.(3)There were 6193 differentially expressed genes(DEGs)in the acquired heat treatment and 6293 DEGs in the heat treatment.Analysis of the differences between the two heat treatments revealed that without the 38°C heat induction treatment,C.moschata needed to initiate more gene functions in order for the plant to better withstand the heat stress.KEGG pathway enrichment revealed 10 KEGG-enriched pathways specific to acquired heat treatment,with the largest number of genes enriched in the protein processing in the endoplasmic reticulum(csv04141)pathway,which was enriched for a total of HSPs and HSFs,which are mainly associated with endoplasmic reticulum-associated protein degradation.The results of the metabolic pathway visualization analysis of DEGs using Map Man software showed that underwent without heat induction process were significantly down-regulated in the auxins signaling pathway.(4)The Hidden Markov Model files of the HSP70(PF00012)structural domains were downloaded from the Pfam protein family database,and a Hidden Markov Model(HMM)search of the protein sequences of C.moschata was conducted to obtain15 Cm HSP70 genes.Analysis showed that the Cm HSP70 gene family contains a large number of cis-acting elements in response to biotic/abiotic stresses.Expression analysis showed that after the heat induction process at 38°C could effectively induce an increase in the expression of some Cm HSP70 genes,and all except Cm HSP70-10 were involved in the response process to high temperature stress.The molecular regulatory mechanisms of C.moschata leaves under two different heat stress treatments were investigated at the transcriptome level using RNAseq technology,and the results indicate that the heat-induced process not only activates the expression of genes such as HSPs,but also induces better metabolic and cellular regulation to adapt to the subsequent heat stress.These results provide a theoretical basis for better understanding of the mechanisms of acquire thermotoleranc in C.moschata.