Bioinformatics Analysis Of The Isopentenyltransferase Gene Family In Wheat And Functional Identification Of TaIPT8 In Drought Tolerance

Wheat is rich in nutrients such as protein,fat and vitamins.It is one of the most important staple crops in the world and feeds a large number of people in the world.In addition,flour can also be used as raw materials for food processing and feed processing.Wheat plays an important role in agricultural production.With the rise of global temperature and the intensification of competition for water resources for different uses,drought has become the main factor affecting wheat yield and growth.Water stress limits the growth and productivity of wheat to a great extent.In order to adapt to environmental changes,plants have evolved sensitive protection systems,in which plant hormones are an important part.The tiny content of plant hormones can also play an important role in transmitting stress-related signals.In many species,CKS has been proved to improve plant drought tolerance by enhancing photorespiration,protecting plant photosynthetic system and delaying leaf senescence.Cytokinins have attracted extensive attention in the study of plant drought resistance.In addition,increasing the expression of cytokinin synthesis gene IPT during water stress can change the source sink relationship and reduce yield loss.However,the interaction between CK and wheat drought resistance are not yet entirely clear.In this paper,the role of cytokinin in wheat drought resistance was analyzed by regulating the expression of the key gene IPT（isopentenyltransferase）of cytokinin synthesis.The main results are as follows:1.Bioinformatics analysis of the isopentenyltransferase gene family in wheat.In the present study,based on the reported IPT gene family members of wheat,barley,Arabidopsis and rice,24 isopentenyltransferase（IPT）genes,which encode rate-limiting enzymes in CK biosynthesis were identified in the wheat genome.Furthermore the genes’chromosome locations,gene structures,protein properties,phylogenetic relationships,motif composition and Cis-Acting elements were characterized.24 TaIPT genes were distributed widely and unevenly on the 15 chromosomes,and no ipt gene was identified on chromosomes 4a,4b,4D,6a,6b and 6D.Among these 24 TaIPTs,TaIPT4-2A,TaIPT4-2B,and TaIPT4-2D contained many exons（11,11,and 10,respectively）;TaIPT1-1A contained one exon,and the remaining 20 TaIPT members lacked introns.A phylogenetic analysis of 24 wheat TaIPTs,nine Arabidopsis AtIPTs,ten rice OsIPTs and seven barley Hv IPTs was performed by generating a phylogenetic tree which classified all IPTs into four major groups.TaIPT4-2A/2B/2D and TaIPT9-7A7/B/7D were closely related to AtIPT2,AtIPT9,OsIPT9,and OsIPT10,which are involved in tRNA prenylation,TaIPT2-1B and TaIPT8-5A/B/D showed the closest relationship with AtIPT3,5,and 7,which predominantly affect the formation of iP-and tZ-types in Arabidopsis.Cis-acting element analysis showed that the promoter region of wheat ipt gene contained a variety of stress response and plant hormone response cis acting elements,such as ABA response element（ABRE）,MeJA response element（TGACG motif,CGTCA motif）,drought inducing element（MBS）and defense and stress response element（TC rich repeats）.2.Expression and functional analysis of TaIPTs.Expression analyses indicated that the ATP/ADP TaIPT genes exhibited tissue-specific expression patterns.TaIPT2,TaIPT7,and TaIPT8 expression was rapidly induced by 0.5-1 h drought treatments,which subsequently decreased to low levels after 2 h drought treatment,as did most other TaIPT genes.We chose TaIPT8,which showed rapid responses to drought stress,for further study.Functional analyses showed that TaIPT8-5a/5b/5d triple mutants exhibited decreased levels of tZ-type CK under normal and drought conditions,After drought treatment,there was no significant difference in water loss rate between mutant plants and WT.The contents of MDA,H₂O₂ and 0₂^-were significantly higher than WT,while the content of proline was significantly lower than WT,which showed low drought tolerance.By contrast,transgenic wheat plants with drought-induced TaIPT8 showed lower wilting degree after drought treatment,and could recover quickly after rehydration,showing enhanced drought resistance.Our study provides a foundation for further investigation of TaIPT genes and provides novel insights into the role of CKs in the drought response of wheat.