Genome-wide Analysis Of Evolution Of CKI Gene Family And Functional Analysis Of Factors In Regulation Of GhCKI Responsive To High-temperature In Cotton

The rapid global climate change has raised serious concerns all over the world.As a consequence of heat stress,male sterility can be widely observed and results in catastrophic loss of cotton yield.It is known high temperature causes abnormal development of male organs which leads to male sterility in cotton.But the molecular regulatory mechanism of high-temperture indiced male sterility is not clear.GhCKI gene was reported to encode a homolog of casein kinase I in cotton,and regulate tapetal programmed cell death and anther dehiscence.The expression of GhCKI was increased at the early stages of anther development under HT.In current research,we identified the cotton CKI gene family and analyzed gene structure,evolutionary history and expression profiles.Besides,we attempted to elucidate the mechanisms of transcriptional regulation of CKI genes in early anther development after HT exposure in Arabidopsis and cotton.The main results of this study were as follows:1.Based on CKI genes in Arabidopsis and rice,homologous analysis revealed 31,30 and 61 CKI members respectively in G.raimondii,G.arboreum and G.hirsutum acc.TM-1 and these CKI genes were classified into two types designated as the type I and the type II.The investigation showed that CKI proteins possessed a highly conserved kinase domain,but differed significantly in the length and primary structure of their N-terminal and C-terminal domains.Furthermore,it was found that the origin of the CKI genes was very ancient,occurring before the ancestral angiosperm WGD event.The evolution of CKI genes was characterized by a history of gene duplications involving multiple evolutionary stages(e.g.an ancient WGD event occurred even earlier in angiosperm evolution and the triplication event).After the common ancient species of Gossypium and cocoa differed,the CKI gene family was involved in the Gossypium whole genome replication events.2.Expression profiling of cotton CKI gene family showed that 22 genes were specifically expressed in anther and 34 genes showed the different expression at the anther development stages(tetrad stage;tapetal degradation stage;and anther dehiscence stage)under HT.It is likely that CKI genes are components of a complex network regulating anther development under NT and HT conditions.We analyzed the gene expression patterns of 13 AtCKLs in Arabidopsis anthers.AtCKL1,AtCKL2,AtCKL5-AtCKL9,AtCKL11,AtCKL12 and AtCKL13 displayed the expression.The expression of AtCKL2 and AtCKL7 during anther development under HT was observed in the tapetum and microspore of anther,and later in the pollen grain,which was similar to the observed expression pattern of GhCKI.3.In order to screen the regulatory factors corresponding to transcriptional regulation of GhCKI genes in anthers under HT,we analyzed the potential cis-acting elements and the responsive transcription factors.The results showed that HT would induce the expression of R2R3-MYB transcription factors(e.g.At MYB4 and At MYB24)at the early stage of anther development and At MYB24 could bind to the AtCKL2 and AtCKL7 promoters and activated them at transcriptional levels.Based on the aforementioned results,we deduced that R2R3-MYB family would target at the promoter regions of AtCKL2 and AtCKL7 and activate these two genes expression at early stage of anther development after HT exposure.4.By comparing the promoter sequences of GhCKI,AtCKL2 and AtCKL7,three progressive 5′deletions of pro GhCKI and together with the whole sequence fused with GUS were proposed to figure out the core regulatory region.The results showed that the minimal region necessary for the expression of GhCKI in the early anther development under HT was confined within the 310-bp fragment.Furtherly,by analyzing the expression of transcription factors during the two different anther development stages(tetrad stage and tapetal degradation stage)of cotton lines “84021”(HT-tolerant)and“H05”(HT-sensitive)under NT and HT conditions,a total of 241 differentially expressed genes were found.No matter at tetrad stage or tapetal degradation stage,the number of differentially expressed genes in “H05” was more than that in “84021”.Besides,these differentially expressed genes mainly belong to WRKY,AP2-EREBP,ARF,and MYB.5.We selected one potential gene for functional study.GhMYB4 encodes a R2R3-MYB transcription factor in cotton.The expression of GhMYB4 was detected in the tapetum and microspores at early anther stages and later in the pollen grain under HT.Moreover,by means of yeast one-hybrid and EMSA,it was verified that GhMYB4 could bind to the promoter sequences of GhCKI.Overexpression of GhMYB4 caused male sterility,suggesting that GhMYB4 may regulate GhCKI gene expression in early anther development and affect the anther development.From the future perspective,this study paves the way to unravel the underlying molecular mechanism of CKI in regulating the anther development and the function of response to HT at stages of anther development.