Genome-wide Identification Of EfPHD Gene Family And Response Of EfPHD15 Gene To Cold Stress In Erianthus Fulvus

Sugarcane(Saccharum spp.)is an important sugar crop in the world,contributing to 80% of the world’s sugar production and serving as a feedstock for ethanol and some natural compounds.Sugarcane is a typical C4 crop,which likes heat and light.However,in recent years,due to the influence of changeable environment and other factors,the temperature of sugarcane planting area has become an important factor restricting the yield and quality of sugarcane.As a close wild species of sugarcane,Erianthus fulvus has strong cold tolerance,so the excellent cold resistance gene source in Erianthus fulvus is used to improve the variety of sugarcane.PHD transcription factor plays an important role in plant growth and development and stress response by regulating the recognition and binding of DNA,protein and RNA.In this study,based on the whole genome database,the EfPHD family gene in Erianthus fulvus was identified and analyzed,from which an excellent candidate gene was screened to respond positively to low temperature conditions.Then,the gene was cloned,the overexpression vector was constructed and genetically transformed into Arabidopsis thaliana,and the transgenic Arabidopsis thaliana was treated with low temperature stress.Then the physiological indexes and phenotypic characters were analyzed.The results are as follows:(1)A total of 76 EfPHD family genes(EfPHD1～EfPHD76)were identified in the whole genome of Erianthus fulvus.Evolutionary analysis showed that these genes were divided into 11 groups,of which group1 contained the most EfPHD family members.These genes were unevenly distributed on 10 chromosomes of Erianthus fulvus.Gene collinearity analysis showed that the expansion of EfPHD family genes was mainly driven by the replication of large chromosome segments,and there was no tandem duplication.Ka/Ks analysis showed that the differentiation time of 8 pairs of large fragment replicate gene pairs was 23.81～108.93 Mya,and the Ka/Ks values were all less than 1,indicating that the PHD family gene pairs were under strong purification selection pressure.EfPHD family gene motif and gene structure analysis showed that the motif of each branch within the same subgroup was more similar.The prediction results of cis-acting elements showed that EfPHD family genes had multiple response elements such as low temperature and drought.The results of multiple sequence alignment showed that EfPHDs had obvious Cys4-His-Cys3 protein sequence characteristics.The interaction network analysis of PHD family proteins of Arabidopsis thaliana showed that EfPHD25,EfPHD63,EfPHD54,EfPHD36,EfPHD10 and EfPHD49 had strong interaction with proteins in Arabidopsis thaliana.The collinearity analysis between Erianthus fulvus and Arabidopsis thaliana,Rice,Sorghum bicolor,Saccharum spontaneum and Maize showed that Erianthus fulvus and Sorghum bicolor were more closely related.The expression of leaves,stems and roots treated under cold stress(0 h,24 h,72 h)was analyzed.Among the 76 EfPHD family genes,37 EfPHD family genes were up-regulated.Ten EfPHD genes with large up-regulation multiples were selected for RT-q PCR verification,and the expression of all 10 genes changed with the extension of low temperature stress time,but all of them responded positively to low temperature environment.Among them,the EfPHD15 gene showed continuous and significant up-regulation expression during low temperature treatment,so it was used as a candidate gene for cold stress expression pattern analysis.(2)The EfPHD15 gene was cloned(Gen Bank No.OK356615)to obtain a 1863 bp nucleotide sequence containing a PHD＿MMD1＿like domain.The physicochemical properties of protein showed that EfPHD15 protein was mainly composed of α-helix,which was a hydrophilic protein with no signal peptide,multiple phosphorylation sites exist,and no transmembrane structure,which belonged to extramembrane proteins.The highest similarity with the sequence of EfPHD15 protein was Sorghum bicolor.(3)The overexpression vector PU1301-PHD was constructed with the total length of 16290 bp The total length of the plasmid was 16290 bp.After transforming Agrobacterium GV3101 receptor cells with PU1301-PHD plasmid,Arabidopsis thaliana was genetically transformed to produce transgenic positive Arabidopsis Thaliana plants.7 Arabidopsis thaliana seeds were collected and seeded.The EfPHD15 gene expression level of Arabidopsis thaliana was detected by RT-q PCR.Strains OE1 and OE6 with significantly higher expression level were selected for cold stress treatment,and the wild type was used as the control.The activity of superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)and the content of proline(Pro)in Arabidopsis thaliana were determined under 4℃ stress.The results showed that the EfPHD15 transgenic Arabidopsis thaliana plants had higher enzyme activity and Pro content than those of wild-type.Further analysis of frost resistance phenotype of Arabidopsis thaliana plants showed that the wild-type plants cannot resume growth,but the transgenic plants could recover growth.Therefore,it is speculated that EfPHD15 gene is positively regulated in the physiological state regulation of Arabidopsis thaliana.In summary,in this study,EfPHD family genes were identified,bioinformatics analysis and cold stress expression analysis,and a high expression EfPHD15 gene was screened out and cloned.After genetic transformation of Arabidopsis thaliana,cold tolerance and cold resistance of EfPHD15 gene were verified.