Genome-wide Identification Of LEA Gene Family In Rosa Chinensis And The Mining Of Cold-response Dehydrin Genes In R.beggeriana

Late-embryogenesis-abundant protein(LEA)is a class of stressexpressed proteins that play an important role in normal plant growth and protection from abiotic stresses such as drought,salinity,and cold.Dehydrin(DHN)is a highly hydrophilic protein of the D-II family of LEA proteins,which is widely involved in the regulation of abiotic stress related to dehydration in plant cells,especially in cold resistance.Rosa beggeriana is widely distributed in northwest of China and has strong cold resistance.It is a key germplasm for cold-tolerant rose breeding,but its genome has not been sequenced.In this study,LEA gene family were identification and analysis in Rosa chinensis ’Old blush’ which has genome data.Based on the identification results and transcriptome data of low temperature treatment of R.beggeriana,the low temperature stress response gene DHN,subfamily of LEA gene family,of R.beggeriana were explored.Then,by low temperature treatment of R.Chinese ’Old blush’ and R.beggeriana,a comparative analysis of DHN gene expression patterns was carried out to determine the DHN gene related to low temperature stress in R.beggeriana,followed by gene cloning,subcellular localization and Genetically transformed tobacco plants.The detail result is as follows:1.In R.chinensis genome,49 LEA genes were identified and divided into 8 subfamilies,including 6 DHN genes.The amino acid length of family members ranges from 89 to 550 aa,the protein molecular weight ranges from 9211.86 to 59770.3 Da,and the isoelectric point ranges from4.55 to 11.42.Motif and gene structure analysis that DHN was mainly concentrated on Motif 2 and was highly conserved.49 genes are irregularly arranged on 7 chromosomes,among which DHN genes are mainly concentrated on chromosomes No.2 and 6.Analysis of cis-acting elements revealed that the Rc LEAs promoters were involved in various responses to temperature stress,and salt stress.These results demonstrate that LEA genes exhibit differences in the number and location of distributions,which in turn lead to functional differences.According to the tissue-and organ-specific RNA-seq data of R.chinensis ’Old blush’,22 genes were expressed in roots,11 genes were expressed in stems,2genes were expressed in leaves,and 9 genes were expressed in stamens,5genes were expressed in the pistil and ovary.DHN gene was significantly expressed in roots,leaves,stamens and shoots.2.After low temperature treatment,of the number of differentially expressed genes of LEA genes were increased at 4 °C for 6 h and 24 h,especially at 24 h in R.beggeriana.DHN genes were all up-regulated.Then the DHNs gene was compared with the sequences of Arabidopsis,apple and rice,and it was found that the homology was relatively large.When real-time quantitative PCR was performed on 6 DHN genes,it was found that the expressions of DHN5,DHN7,DHN9,and DHN11 in R.chinensis ’Old blush’ and R.beggeriana showed an upward trend.3.The 4 initially screened Rosa beggeriana DHN(Rb DHNs)genes were cloned,and it was found that Rb DHN5 belonged to YKn type,Rb DHN7 and Rb DHN9 belonged to SKn type,and Rb DHN11 belonged to Kn type.Subcellular localization by transient expression of tobacco leaves found that Rb DHN5,Rb DHN7 and Rb DHN9 were localized in the cytoplasm and Rb DHN11 was localized in the nucleus.T1-positive transgenic plants with 4 genes were initially obtained by Agrobacterium infested tobacco,which provided materials for the functional verification of the above-mentioned dehydrin genes.