Gene Expression And Interaction With F-box Analysis Of Wheat TSK41,TSK43.2 And TSK62

SKP1(S-phase kinase-related protein 1)is the core component of SCF ubiquitin ligase,which directly binds to members of the F-box family.SKP1 proteins mediate protein degradation,thereby regulating many cytological processes,such as the growth and development of plants,hormone signaling and biotic/abiotic stress etc.Unlike protists,some fungi and vertebrates,plants have multiple SKP1 homologues.Understanding the category,quantity,phylogenetic characteristics,and function of SKP1 in plants is essential for dissecting the mechanism of the SCF complex.At present,the knowledge of this family mainly comes from model plants,such as Arabidopsis and rice,and there are few research reports on the SKP1 family in wheat.In this situation,the present study carried out a systematic bioinformatics analysis on the wheat SKP1 family at the proteome level.At the same time,wheat TcLrl5 was used as experimental material to obtain the different types of SKP1.The expression patterns of the genes obtained were clarified with the help of qRT-PCR in response to hormone signals and stresses,and the yeast two-hybrid was used to verify their interactions with F-box proteins.The main results of this study are as follows:1.115 sequences were identified in the wheat proteome,which were encoded by 101 SKP1 genes;SKP1 protein has two conservative domains,SKP1 and SKP1-POZ.These proteins can be divided into four groups according to the composition of the domains;SKP1,SKP1_POZ,1 SKP1+1 SKP1_POZ and 1 SKP1+2 SKP1_POZ.1 SKP1+1 SKP1_POZ accounts for a large percentage.All SKP1 members in wheat are acidic proteins,and most of them are predicted to subcellular localize in the nucleus.Wheat SKP1 genes distributed on each chromosome,and the density on chromosome NO.3 and 5 is relatively higher than that of the others.The results of heat map analysis showed that a few SKP1 genes have higher expression levels in different developmental stages of 5 tissues in wheat,and some of them showed high expression levels in a certain tissue or a specific stage of development.But most of them were not expressed or expressed at low level in 5 tissues at different stages.The expression patterns of SKP1 family genes in wheat varied greatly under drought and/or heat stresses,and some of them responded to the invasion of fungi and bacteria.Prediction results for the protein interaction network showed that SKP1 protein could directly bind to Cullin and F-box proteins.2.Three different types of SKP1 genes:TSK41(containing SKP1_POZ domain),TSK43.2(containing SKP1 domain)and TSK62(containing SKP1 and 1 SKP1 POZ domains)were obtained from wheat line TcLr15.The three proteins are all non-spherical,and composed of random coils,α-helixes and β-sheets.Subcellular localization of three proteins were predicted to be in nucleus.These three TSK genes shared high similarity with wheat related species,such as Hordeum vulgare,Brachypodium distachyon,Triticum urartu,Setaria italica,Zea mays,and Sorghum bicolor,which indicated that they are evolutionarily conservative.3.The expression patterns indicated that the three stress-related hormones ABA,SA and MeJA had little effect on the expression of the three TSK genes.The expression levels of the three TSK genes increased first and then decreased in wheat TcLr15 after treatment by drought and salt,and decreased in TcLr15 under the treatment of oxidative stress.Although the three TSK genes were differentially expressed during the infection process of leaf rust fungus,different virulent leaf rust pathogen strains had little effect on their expression trends.In general,the two stresses of NaCl and H2O2 caused more dramatic changes in the expression of the three TSK genes.4.Yeast two-hybrid prey vectors containing three TSK genes:pGADT7-TSK41,pGADT7-TSK43.2 and pGADT7-TSK62,were constructed in the experiment.And all of them were used to test their interactions with eight yeast bait vectors containing F-box genes(BD-ToPP2-A13,BD-TaSKIP27,BD-TaFBK19,BD-TaFBK50,BD-TaFBK34,BD-TaFBK71,BD-TaFBL14 and BD-TaSKP2A)via Y2H.TSK41 bind to TaFBK 19 and TaFBK34.TSK62 can interact with TaFBK 19,TaFBK34,TaFBK71,TaSKP2A and TaSKIP27.All the other 17 combinations showed negative results in the present experiment.Y2H results presented that SKP1 proteins and F-box proteins had multiple interaction modes in wheat.