Analysis And Functional Verification Of Stress-tolerant PUB Gene Family In Cotton (Gossypium Spp.)

Cotton is the most important fiber crop.The study of cotton abiotic stress is one of the important contents of cotton breeding as the cotton growing areas of china started to transfer to the northern salt alkali land,xinjiang,Inner Mongolia and coastal beach.PUBs were U-box-domain-containing ubiquitin ligases and were involved in abiotic stress in various plants.There are less research about PUB proteins and genes in cotton.To explore the PUB in the abiotic stresses the role of cotton,in this study,we identified the PUB families in whole genome of cotton,and analyzed the evolution of them in cotton and the expression profile of them in upland cotton.A couple of GhPUB homologous genes were cloned in upland cotton,the function of the preliminary verification.The main results of this study were as follows:(1)With U-box domain(PF04564)data as a query,we identified 93,96,185 and 208 PUB gene families in four sequenced cotton species G.raimondii(D5),G.arboreum(A2),G.hirsutum(AD1),and G.barbadense(AD2),respectively.The number of exons of PUB gene families rang from 1 to 25,around one third of them had a single exon.The phylogentic tree showed that the PUBs could be clustered into six subfamilies I-V in each cotton species,and the evolutionary relationships of all PUBs in D5,A2 and AD1genome were similar to the relationships in single genome.(2)The homologous analysis showed that 15 segmental duplication and 3 tandem duplication in D5genome,25 segmental duplication and 2 tandem duplication in A2 genome,and 143 pairs homologous genes in intra-genome of A2 and D5.In AD1 genome,14 pairs homologous genes in A-subgenome(A_t),17pairs homologous genes in D-subgenome(D_t),and 105 pairs homologous genes in intra-subgeome of A_t and D_t.These results indicated that one gene of the pair of homologous genes may be lost.(3)The the expression profiles of GhPUB were made using the transcriptome sequencing datas.Results show that intercomparing the expression level of GhPUB genes in root,stem and leaf,only three GhPUB genes were highly expressed in all organizations,and 21 genes expressed were hardly expression in all organizations.The expression levels of 22 GhPUB genes have a significant difference during salt,drought,cold and heat stress.(4)A pair of homologous genes(GhPUB85A and GhPUB45D)were cloned from upland cotton.The expression levels of them reached the highest at about 6 h during drought stress,and reached the highest at near 12 h undring low temperature and salt stress.After GhPUB85A and GhPUB45D were silenced respestively,GhPUB85A and GhPUB45D expression level were significantly decreased and were no difference in the expression level of the same gene in different gene silent plants.(5)The red fluorescent fusion expression vectors were built and transiently expressed in leaves of tobacco to confirm subcellular localization of GhPUB85A and GhPUB45D.GhPUB85A and GhPUB45D were localized in cytomembrane.The over-expression vectors were built and transformed GhPUB85A and GhPUB45D into Arabidopsis,and 6,11 of them are single T-DNA insertion transgentic Arabidopsis lines were obtained respectively.