The Study Of GhWRI1 Structure And Functions

As one of the major nutritional components in the plant,the vegetable oil is of great importance for human beings.The relationship between WRINKLED1(WRI1)and seeds oil content of cotton(Gossypium hirsutum L.)was explored by our team.By up-regulating GhWRI1,oil content of seeds was decreased in cotton,which conflicted with the functions of WRI1 in Arabidopsis thaliana reported.To identify the major role of Gh WRI1 on transcriptional regulation in cotton and the effects of GhWRI1 domains on the subcellular localization,transactivation and binding cis-acting element ability,the content of oil and soluble sugar in different tissues of over-expression GhWRI1 transgenic lines was measured at first.And then,the domains of GhWRI1 were predicted to obtain different GhWRI1 truncated variants by 5? and 3? terminal deleting technique and PCR site-directed mutagenesis.In addition,a set of expression vectors were constructed for tobacco agrobacterium tumefaciens mediated transient expression system,Dual-Glo Luciferase Assay System and yeast two-hybrid system to assess the effects of GhWRI1 domains on it's function.The key results are as followed:1.The repartitions of carbon in the cotton leaf,ovule and fiber were regulated by over-expression GhWRI1To identify the role of Gh WRI1 in cotton,the highly over expressed GhWRI1 transgenic lines were confirmed by Quantitative Real-Time PCR,and then the oil content and soluble sugar content in T3 generation leaf,27 DPA ovule and 27 DPA fiber were measured.The results showed that: the oil content of ovule and fiber of transgenic lines decreased by 4.93%~10.46% and 19.95%~42.53% compared to the wildtype respectively;the soluble sugar contents increased by 10.44%~26.02% and 31.21%~69.54% compared to the wildtype respectively.A greater increase in total oil content of leaves,from 22.45% to 22.94%,was observed in two of the preselected transgenic lines,while the levels of soluble sugar contents decreased by 14.80%~22.24%.These results indicated that the carbon flow in leaf,ovule and fiber were changed and repartitioned by upregulating the expression of GhWRI1 in cotton.2.Domains and nuclear signal localization of GhWRI1 amino acid sequence were predicated and analyzedTo figure out the domains and nuclear location signal(NLS)in GhWRI1 amino acid sequence,a set of in silico analyses were performed by NCBI(Conserved Domain Search),NLStradamus and PSORT prediction algorithms.It turned out that GhWRI1 has a “PKAKRARK” NLS,which was in the N-terminal 48~55 amino acid residues,and 7 domains including TauE superfamily,Oxidored_q4 superfamily,67 superfamily,DUF3446 superfamily,up AP2,down AP2 and EpsG superfamily.The AP2 domain was highly conserved among the transcription factor WRI of all kinds of species.3.Domains in GhWRI1 have certain effect on nuclear translocationTo explore the impacts of GhWRI1 domains on it`s nuclear translocation,GhWRI1 truncated variants were obtained by 5?and 3?terminal deleting technique,firstly.And then a GFP protein was respectively fused to the N-terminal and C-terminus of GhWRI1 and GhWRI1 truncated variants.Subsequently,the subcellular localizations were detected by tobacco agrobacterium tumefaciens-mediated transient expression system.It turned out that nuclear location signal existed in 48~76 amino acid residues(GhWRI148-76)in the N-terminal of GhWRI1 protein and played a pivotal role in GhWRI1 nuclear translocation.In addition,the 7 domains in GhWRI1 had certain influence on GhWRI1 protein nuclear translocation.Otherwise,the nuclear translocation of GhWRI1 truncated variants was partly blocked by EpsG superfamily.That is to say,EpsG superfamily will effect the stability of GhWRI1.4.A 55 amino acid domain at C-terminus of Gh WRI1 is necessary for transactivationTo explore the effects of GhWRI1 domains on transactivation,GhWRI1 truncated variants were detected by a GAL4-based two-hybrid system.No transactivation activity was observed for GhWRI1 truncated variants,which has a deletion of the C-terminal 306~439 amino acids or only one AP2 domain existed.Different transactivation activities were observed for other truncated variants containing C-terminal 306~439 amino acids.The 7 domains and NLS have certain effects on GhWRI1 transactivation activity,and a 55 amino acid domain at the C–terminus of GhWRI1 is sufficient for transactivation.5.Cys at 315 th and 339 th amino acid of Gh WRI1(GhWRI1C315,GhWRI1C339)are not essential for Gh WRI1 transactivation activityTo explore the essential amino acid in GhWRI1 transactivation,Multiple Sequence Alignment cluster analysis of transactivation domain of GhWRI1 orthologs proteins searched by the NCBI was conducted,and the results indicated that there were two relatively conservative Cys at 315 th and 339 th amino acid.To further identify the potential role of Cys amino acid residues in GhWRI1 transactivation,GhWRI1 C-terminal 315 th and 339 th Cys amino acids were substituted respectively by alanine(A)(GhWRI1C315A,GhWRI1C339A).Stong transactivation were observed at GhWRI1 point mutants.Results showed that 315 th and 339 th Cys were not essential for GhWRI1 transactivation activity.6?The two AP2 domains and EpsG superfamily domain are essential for GhWRI1 to recognize and combine target cis-acting elementsTo research the effects of GhWRI1 domains on binding cis-acting elements of upstream sequence of target genes,Gh WRI1 truncated variants were constructed for Dual-Glo Luciferase Assay System and transient expression system in tobacco.The result showed that the ratio of LUC/Ren of GhWRI1 truncated variants with both two AP2 and EpsG superfamily domains was higher than others.On the contrary,the recognizion and combination between GhWRI1 and cis-acting elements of upstream sequence of target genes were declined when none AP2 or unabridged EpsG superfamily or only one AP2 domain existed.That is to say,both the two AP2 domains and EpsG superfamily were requisite for GhWRI1 to recognize and combine cis-acting elements.