Bioinformatics And Expression Analysis Of NAC Transcription Factor Family Under Low Temperature In Cucumis Sativus Var.hardwickii

Cucumis sativus var.hardwickii belongs to the Cucumis family of Cucurbititaceae,and it is recognized as the wild ancestor of cultivated cucumber and considered to be the key species to study the origin and evolution of Cucumis species.It contains the resistant gene(s)against stress and diseases,which makes it to be an important germplasm to broaden the genetic basis of cucumber.Studying the gene regulation mechanism and evolutionary relationship of agronomic traits of hardwickii plays an important role in cucumber genetics and cultivar improvement.The results of the whole genome sequencing of hardwickii helps to identify and study important functional genes and transcription factor families at the genome level.The NAC transcription factor is one of the largest transcription factor families in plants and plays an important role in plant resistance to low temperature stress.However,the current research on the NAC transcription factors of hardwickii under low temperature stress is still very limited.In this study,using hardwickii as a research material was identified the NAC transcription factor of hardwickii,based on the results of genome-wide sequencing.We analyze the sequence characteristics,phylogeny,gene and protein structure,promoter cis-acting elements,protein interaction,replication patterns and evolutionary selection direction.By using existing transcriptome data,the tissue-specific expression of NAC gene was investigated,and fluorescent quantitation technique was used to study the expression of NAC gene in hardwickii under low temperature stress.The research contents and results are as follows:1.Bioinformatic analysis of NAC transcription factors in hardwickiiIn this study,a total of 77 NAC transcription factors were identified based on sequencing hardwickii genome.The NAC transcription factors were highly conserved.The length of sequences varied and unevenly distributed in seven chromosomes.The theoretical isoelectric point(pI)were ranged from 4.54 to 9.10.Most were hydrophilic proteins.Most of NAC transcription factors were located in the cell nucleus and plasma membrane,and are rarely located on organelles such as mitochondria and chloroplasts.Phylogenetic analysis showed that NAC proteins could be classified into six subfamilies,the members of the subfamily had similar genetic structures and conserved motifs,while the third subfamily members showed diversity.The random coils account for the majority in the secondary structure.Promoter element analysis showed that in addition to the basic promoter elements,the HdNAC gene family also has cis-acting elements related to light regulation,meristem and endosperm development,hormonal regulation such as ethylene and gibberellin,and drought and heat stress responses.The results of protein interaction showed that the HdNAC protein not only interacted with CUC2,MYB46 and VND7 proteins involved in the regulation of secondary cell wall formation and apical meristem development,but also had interactions among members such as HdNAC55 and HdNAC26,HdNAC69.The collinearity analysis of the HdNAC gene family is similar to the phylogenetic evolution relationship,and there is a linear replication relationship between the paralogous genes.We identified 9 NTLs of hardwickii,8 NTLs of melons(cucumis melo L.cv.DHL92),8 NTLs of watermelons(Citrullus lanatus subsp.Vulgaris cv.97103),15 NTLs of pumpkins(Cucurbita maxima cv.Rimu).Phylogenetic analysis showed that there was some homology between the NTLs of hardwickii and the other three species of NTLs.2.The evolution relationship of HdNAC transcription factorThe evolutionary analysis of the HdNAC gene family revealed that 14 genes belonged to segmental duplication,7 genes had tandem duplication,and the remaining 56 genes were derived from dispersed duplication or singleton duplication.Seven of the 23 pairs of paralogous genes found in the phylogenetic tree were from segmental duplication,3 pairs belonged to tandem duplication,and the remaining pairs were derived from Dispersed duplication.The Ks values of all replicated gene pairs were significantly larger than 1(except HdNAC06-HdNAC31).The Ka/Ks values of the HdNAC paralogous gene pairs were all lower than 1,suggests that purifying selection played a major role in the evolution of HdNAC transcription factors.3.Analysis of HdNAC gene expressionTissue-specific expression analysis showed that the expression level of NAC transcription factor was highest in roots,followed by female flowers and leaves.Fluorescence quantitative analysis showed that only 3 of the 47 NAC genes detected were down-regulated under low temperature treatment,while other genes were up-regulated at different levels.Among those,HdNAC32 and HdANC47,a pair of segmental duplication genes,showed opposite expression patterns.