Cloning And Analysis Of Self-incompatibility Genes BoPUB3L And BoGSTL21 In Brassica Oleracea L.

Self-incompatibility(SI)is a genetic mechanism that effectively restricts self-fall and promotes heterosis in plants during long-term evolution.Brassica plants exhibit sporophyte self-incompatibility(SSI).In recent years,although people have made significant progress in studying the SI signaling mechanism in Brassica plants represented by Brassica oleracea,the research on the isolation and identification of downstream signaling elements of S-locus receptor kinase(SRK)is relatively slow.ARC1(ARM repeat containing 1)is a U-box protein with E3 ubiquitinated ligase activity.In the cabbage self-incompatibility signaling pathway,ARC1 acts as a downstream target protein of SRK to promote self-incompatibility.However,it was found that antisense inhibition of Arabidopsis lyrata and Brassica napus ARC1 gene expression,and overexpression of Exo70A1 gene in Brassica napus can only partially break self-incompatibility.This indicates that whether ARC1 is the only downstream substrate of SRK is questionable,or there may be other signal elements and signaling pathways other than ARC1-Exo70A1 involved in regulating SRK downstream signaling.Based on this,this study used two highly self-incompatible and incompatible Brassica oleracea "A4" and "F1" as materials,and analyzed the differential expression of the stigma transcriptome after self-pollination and cross-pollination to screen for self-incompatibility.Related genes,amplify the complete c DNA sequence,and study its function by bioinformatics analysis,tissue-specific expression analysis,promoter activity analysis,prokaryotic expression,subcellular localization,q RT-PCR,and yeast two-hybrid.The main research results obtained are as follows:1.Cloning and analysis of SRK interaction protein encoding gene BoPUB3 L in Brasscia oleraceaUsing stigma transcriptome data analysis after cabbage pollination,a plant ubiquitin protein family member gene BoPUB3 L that was differentially expressed after cabbage self-pollination was obtained.The CDS of BoPUB3 L gene was 2214 bp in total length,encoded 737 amino acids,theoretical isoelectric point was 6.04,molecular weight was 81.27 k Da,done not contain signal peptide and transmembrane domain,contained 5 arm repeat ARM domains and 1 U-box domain.The BoPUB3 L gene promoter contained various cis-acting elements such as stress response,hormone response,metabolic regulation,and endosperm development.BoPUB3 L protein was located in the nucleus.The tissue quantification of BoPUB3 L gene showed that it was expressed in the roots,leaves,flowers and pods of cabbage,and the expression level in the stigma was higher than other tissues,which was basically consistent with the GUS staining results.Through yeast two-hybrid experiments,it was found that BoPUB3 L protein interacts with the intracellular domain of SRK.Using gene over-expressioned technology to transform the cabbage BoPUB3 L gene into Colombian(Columbia,Col)Arabidopsis thaliana,it was found that the overexpressing Arabidopsis plants can grow normally,but the growth was weak.The above research results had laid the foundation for further study on the function of BoPUB3 L gene,which was of great significance for the in-depth understanding of the mechanism of self-incompatibility.2.Cloning and expression analysis of self-incompatibility related gene BoGSTL21 in Brasscia oleraceaFrom the stigma transcriptome data of self-pollination of Brassica oleracea from 0to 60 min,a glutathione-s-transferase gene BoGSTL21,which was up-regulated and induced by self-pollination,was successfully screened.BoGSTL21 had an open reading frame of 900 bp,encoded 299 amino acids,had a theoretical isoelectric point of 8.49,done not contain signal peptides and transmembrane regions,contained GST-N and GST-C domains,and was a member of the GST-Lambda family.The BoGSTL21 gene promoter contained various cis-acting elements such as light response,auxin response,low temperature and drought response.The tissue quantification results of BoGSTL21 gene were basically consistent with the GUS staining results.They were expressed in different tissues,and were highly expressed in the mature stigma during the flower development stage.Fluorescence quantitative PCR results showed that the expression trend of BoGSTL21 gene after self-pollination was up-regulation and then down-regulation,and there was little change after cross-pollination,which was consistent with the results of transcriptome analysis.Through yeast two-hybrid,it was found that BoGSTL21 protein interacted with pollen development-related protein BoFAB1 C,auxin-related protein BoPATL2,and aldolase-type TIM barrel family protein BoF9N12＿9.The BoGSTL21 gene was successfully induced and expressed in E.coli,and the purified protein size was 34 k Da.The results of this study preliminarily proved that glutathione-S-transferase was likely to participate in the self-pollination process of SI cabbage.It was speculated that BoGSTL21 might be a new protein involved in the SI reaction process,which provided new content for further study on the self-incompatibility of Brasscia oleracea.