The Arabidopsis Thaliana Casein Kinase 1 Affects The Regulation Of Cotton Fiber Development By Regulating The Accumulation And Reduction Of Ethylene Biosynthesis

Cotton is the important crop for its fiber in the world.Improving the production of cotton fiber has become one of the main problems faced by our country.It make sense to discover genes and regulation network related to cotton fiber development for cotton genetic improvement and guidance of cotton production.In cotton,ethylene is an important plant hormone regulator during cotton fiber development.The application of an appropriate amount of exogenous ethylene promotes fiber growth,but studies by changing endogenous ethylene to explore fiber development are rarely reported.The Arabidopsis casein kinase（PK1）and its allelic mutant gene PK1^D128N can cause the ethylene content of A.thaliana to decrease or constitutive triple response（CTR）,respectively.In order to create stable genetic material with altered endogenous ethylene content in cotton,we conducted the following studies and derived some molecular regulatory networks by which ethylene affects fiber development.The main findings are as follows:1.PK and PK1^D128Ninteract with GhACS-1 and GhACS2 in vitro and in vivo,respectively.Two target proteins,GhACS-1 and GhACS2,were screened in the upland cotton fiber tissue c DNA library by screening yeast library with BD-PK1 as bait.After cloning the full-length cDNAs of these two genes,the interaction of PK and PK1^D128N with GhACS-1 and GhACS2 in vitro and in vivo was verified by Y2H and BiFC methods,respectively.2.GhACS-1 and GhACS2 genes positively regulate fiber development.GhACS-1 and GhACS2 genes were silenced in upland cotton by VIGS experiment,and1DPA ovules were observed by electron microscope scanning.It was found that the onset of fibers on the silenced cotton ovules was slower than that of the negative control.Cotton fibers with GhACS-1 or GhACS2 genes were significantly shorter than those on control cotton（p-value<0.01）.It indicated that GhACS-1 or GhACS2 may positively regulate fiber growth and development.3.PK1 and PK1^D128Ntransgenic cotton affects the decrease and accumulation of ethylene content during the process of rapid fiber elongation.To investigate whether the heterologously expression of PK1 and PK1^D128N in cotton can affect ethylene biosynthesis.The PK1 and PK1^D128N genes were driven by the fiber-specific promoter RDL1 and transformed into G.hirsutum cv ZM24,respectively.qRT-PCR and semi-quantitative methods were used to detect the expression levels of these two target genes in the respective transgenic cottons at 5,10,15,20 and 25 DPA stages.It was found that PK1and PK1^D128N genes were mainly expressed at 5-15 DPA,that is,the fast fiber elongation stage..Western Blot also detected more protein accumulation in OE-PK1^D128N cotton fibers.The ACC content and ethylene release of cotton fibers at 5 and 15 DPA were detected.In OE-PK1 cotton,ACC and ethylene content decreased compared with ZM24 cotton at 5DPA,while OE-PK1^D128N cotton had extremely high ACC content and ethylene at 5DPA.For the OE-PK1^D128N cotton,although the ACC content was significantly lower than that of ZM24 at15DPA,its ethylene release amount was still higher than that of the control cotton,about 4times that of ZM24.4.Transgenic cotton with PK1 and PK1^D128N genes responds to ethylene biosynthesis inhibitors and promoters.With different concentrations treatment of ACC and AVG in the in vitro experiments,it was found that fiber development of OE-PK1 and ZM24 genotype can be promoted under ACC treatment,while fiber of OE-PK1^D128N cotton ovule was inhibited by ACC,which might be caused by its own accumulation of endogenous ethylene.On the contrary,using the ethylene biosynthesis inhibitor AVG to culture the ovules of the transgenic material in vitro,it was found that the fiber development of OE-PK1 and ZM24 cotton ovules was more inhibited under AVG treatment due to their low endogenous ethylene release levels,while the excess ethylene released by OE-PK1^D128N was inhibited by AVG,and the ethylene release was reduced to a suitable concentration,resulting in a promoting effect on fiber development of OE-PK1^D128N.These results indicated that PK1 and PK1^D128N did affect the release of endogenous ethylene from cotton fibers.5.Functional analysis of genes related to fiber development and responsed to changes of endogenous ethylene by RNA-SeqTo explore genes that respond to ethylene changes in cotton fibers after the endogenous ethylene content was decreased or increased,RNA-Seq was performed using the 15DPA fiber.DEG analysis found that 4319 genes were differentially expressed after ethylene content was down-regulated,of which 1621 and 2698 genes were up-and down-regulated in OE-PK1,compared with ZM24,respectively.GO enrichment analysis found that these genes were associated with plant secondary cell wall biosynthesis,peroxidase activity and xylem development;while in OE-PK1^D128N,there are 1698 up-regulated and 4698 down-regulated genes,respectively,and these genes are mainly enriched in microtubule development,kinesin,rhamnogalacturonic acid I side chain metabolic processes,cellulose synthase,arabinose biosynthesis.etc.Through Venn diagram analysis,we found 791（or 2841）genes that are sensitive to low（or high）concentration of ethylene and insensitive to high（or low）concentration of ethylene,these genes are mainly enriched in"flavonoid biosynthesis"”and“plant hormone signal transduction”and“phenylpropanoid biosynthesis”and“fructose and mannose metabolism”pathways.In addition expression patterns of 237 and 230 genes were positively or negatively correlated with ethylene concentration,respectively.Furthe KEGG analysis showed that positively correlated genes were mainly enriched in cellulose synthase activity and secondary cell wall biogenesis of seed trichome differentiation,while negatively related genes were mainly enriched in"keratin biosynthesis","heme binding"and"oxidoreductase activity"pathways.