Conservative And Regulation Analysis Of The Promoter Of Self-incompatibility Gene BnSCR1 And BnSRK1 In Brassica Napus

Self-incompatibility(SI)is a natural phenomenon to prevent self-fertilization and promote out-crossing in flowering plants.Previous studies indicated that the self-incompatibility recognition results from the interaction between SP11/SCR(S-locus protein 11 or S-locus Cys-rich)expressed in stamen and SRK(S-locus receptor kinase)expressed in stigma,and the following Bn SRK mediated signal transduction.This study is focusing on the conservative signal pathway of BnSCR1-BnSRK1-BnARC1 in A.thaliana,and cis-element and trans-acting factors of BnSCR1 and BnSRK1 in the self-incompatibility line “W-3”.Our study will be helpful for dissecting the self-incompatibility mechanism and the self-incompatibility signal pathway in Brassica.The main results are as following:1.The two overexpress vectors,pORE_O4-BnSCR1+BnSRK1 and pORE_O4-BnSCR1+BnSRK1+BnARC1,were constructed and introduced in the A.thaliana Col-0 and C24.21 BnSCR1+BnSRK1 transgenic Col-0 plants and 13 BnSCR1+BnSRK1 transgenic C24 plants were obtained.Meanwhile,12 BnSCR1+BnSRK1+BnARC1 transgenic Col-0 plants and 9 BnSCR1+BnSRK1+BnARC1 transgenic C24 plants were obtained.BnSCR1,BnSRK1 and BnARC1 were expressed in all the transgenic plants.All the transgenic plants were self-compatibility by the investigation of pod length,the pod numbers per pod and pollination experiments.We speculated that the self-incompatibility signal pathway of BnSCR1-BnSRK1-BnARC1 of Brassica don’t work in the A.thaliana Col-0 and C24.2.2 kb promoter of BnSRK1 gene was cloned.Promoter analysis showed that PR1-GUS(-1809 to-1 bp)and PR2-GUS(-1314 to-1 bp)could drive GUS gene expression in stigma.The futher promoter delete analysis indicated that the key regulatory region of promoter of BnSRK1 gene is located at-1314 bp to-1000 bp.504 bp promoter of Bn SCR-1300(class-II)and 416 bp promoter of Bn SCR-6(class-II)were cloned.Promoter analysis showed that 60P-GUS(-504 to-1 bp)and 15P-GUS(-504 to-1 bp)could drive GUS gene expression in tapetum from stage 9 to stage 11.3.A transcription factor BnPHL2α which could interact with 684 bp promoter of BnSCR1 was obtained by Y1 H.Two rounds of truncation analysis indicated that BnPHL2α could interact with the 16 bp promoter sequence “TTTAATTTCTTGCATA” of the BnSCR1.Subcellular localization results indicated that BnPHL2α is a nuclear gene.The BnPHL2α has no transcriptional activity by detection with the AH109 yeast strain.And then,The transcriptional activation/repression structure analysis of BnPHL2α showed that two conserved region(MYB CC domain: 136-185 aa,MYB CC DNA binding domain:37-90 aa)may be the repression region of this protein.We further demonstrated that the P342 promoter activity was repressed by BnPHL2α with DLR system.A series of mutation experiments about the P342 promoter region(from-310 to-295 bp)sequence “TTTAATTTCTTGCATA” indicated that the repression sequence of the P342 activity regulation is “TTTAATTTCTTGCATA”(from-310 to-295 bp of the BnSCR1 prompter).We also verified that BnPHL2α protein can interacte with the “TTTAATTTCTTGCATA” sequence by EMSA.Using the CRISPR technique,BnPHL2α gene has been knocked out and four T0 chimera transgenic lines was obtained.