Molecular Mechanism Study Of PlNCEDs Regulating Seed Dormancy In Paeonia Lactiflora Pall.

Paeonia lactiflora Pall.is a traditional Chinese famous flower,with high ornamental value and strong cold resistance,and is an excellent material for landscape application.P.lactiflora has formed a unique dormancy characteristic.In the breeding process,the germination rate is often reduced because the dormancy is not lifted or not completely lifted.If the target genes that can be used for genetic engineering breeding are screened out,the speed of molecular directional breeding can be accelerated to a great extent,thus effectively improving the promotion of P.lactiflora breeding and the maintenance of germplasm resources.Studies have shown that abscisic acid(ABA)is the main plant hormone affecting the seed dormancy release of P.lactiflora.However,the function and regulatory mechanism of ABA biosynthesis pathway in the seed dormancy release of P.lactiflora are still unclear.In this study,the key rate-limiting enzyme gene NCED in the ABA biosynthetic pathway was used as the research object.Through transcriptome data and transcriptional level determination results,Pl NCED1 and Pl NCED2 with significant differential expression during the seed dormancy release of P.lactiflora were screened.Their roles in ABA biosynthesis and seed germination were clarified by homologous and heterologous genetic transformation.Based on the constructed yeast hybrid c DNA library,combined with yeast hybridization,yeast and plant in vivo verification techniques,the target factor(Pl PIF)and protein(Pl XTH)interacting with Pl NCED1 and Pl NCED2 genes were screened and identified,and preliminary analysis on Pl PIF and Pl XTH functions.The regulatory mechanism of ABA biosynthesis pathway affecting the seed dormancy release of P.lactiflora was preliminarily explored,which laid a theoretical foundation for further revealing the molecular mechanism of the seed dormancy release of P.lactiflora.The main results are as follows :1.To clarify the sequences of Pl NCED1 and Pl NCED2 genes and their roles in plant cells.The results showed that the CDS sequence of Pl NCED1 gene is 1518 bp,encoding 505 amino acids.The 3’ UTR sequence is 142 bp and the promoter sequence is amplified to 732 bp.The CDS sequence of Pl NCED2 gene is 1326 bp,encoding 441 amino acids.The 3’ UTR sequence is 300 bp and the promoter sequence is amplified to 1855 bp.Pl NCED1 and Pl NCED2 are stable,non-transmembrane,and hydrophilic non-secretory proteins.Protein conserved domain analysis showed that both Pl NCED1 and Pl NCED2 had the typical RPE65 conserved domain of NCED family,which are closely related to Paeonia ostii and Vitis vinifera.The subcellular localization of protein showed that Pl NCED1 and Pl NCED2 were located in the nucleus and cytoplasm,respectively.2.The functions of Pl NCED1 and Pl NCED2 genes in ABA biosynthesis and seed germination were verified.The results showed that the expression trend of Pl NCED1 and Pl NCED2 genes was roughly the same as the change trend of endogenous ABA content during the seed dormancy release of P.lactiflora,and the overall trend was downward.The results of endogenous ABA content determination in the P.lactiflora transgenic callus showed that Pl NCED1 and Pl NCED2 genes were positively correlated with ABA content accumulation.The seed germination rate statistics of Arabidopsis thaliana transgenic line and functional complementation line showed that Pl NCED1 and Pl NCED2 genes were positively correlated with seed dormancy.3.The yeast hybrid c DNA library of P.lactiflora seed dormancy release critical periods was constructed,and target proteins interacting with Pl NCED1 and Pl NCED2 were screened and verified.The results showed that the library availability was ensured by the verification of library capacity,titer,average insert fragment length and recombination rate.Meanwhile,the Pl NCED1 and Pl NCED2 bait proteins constructed in this study have no self-activating activity and toxicity,and can be used for yeast library two-hybrid screening.A total of 7 positive clones with mediating seed dormancy and germination were obtained by yeast two-hybrid screening.Combined with bimolecular fluorescence complementation(Bi FC)test results,its were clear that Pl NCED1 and Pl NCED2 did interact with Pl XTH protein.Pl NCED1 and Pl XTH interacted in the nucleus and cytomembrane,Pl NCED2 and Pl XTH interacted in the cytoplasm.The expression of Pl XTH gene was negatively correlated with the seed dormancy release of P.lactiflora.4.The upstream transcription factors regulating the expression of Pl NCED1 and Pl NCED2 genes were screened and verified.The results showed that the Pl NCED1 and Pl NCED2 genes promoter sequences can drive the activity of β-glucuronidase(GUS)gene expression,and the promoter activity is the strongest when the promoter sequences are 661 bp and 1023 bp,respectively.The optimal Aureobasidin A(Ab A)inhibition concentrations of yeast bait strain constructed by the segmentation of Pl NCED1 was 400.The optimal Ab A inhibition concentration of yeast bait strains constructed by Pl NCED2 gene segmentations were 300 and 200 ng/m L,respectively.The results of yeast one-hybrid screening library showed that 8 positive clones were obtained,which could regulate plant metabolism,light signal,jasmonic acid,auxin and salicylic acid signal.Combined with dual-luciferase reporter assay(Dual-LUC)and GUS activity test result,it was clear that the transcription factor Pl PIF could directly and positively regulate the activity of Pl NCED1 promoter.Meanwhile,the expression trend of Pl PIF gene and Pl NCED1 gene were roughly the same.The results of endogenous ABA content determination in the P.lactiflora transgenic seeds showed that Pl PIF gene was positively correlated with ABA content accumulation.In summary,this study clarified that Pl NCED1 and Pl NCED2 genes can promote endogenous ABA synthesis and inhibite seed germination.The interactions between Pl XTH with Pl NCED1 and Pl NCED2 at the protein level,and the regulatory relationship between Pl PIF and Pl NCED1 at the transcriptional and ABA accumulation level were preliminarily proved.These results laid a foundation for systematically elucidating the regulatory mechanism of ABA synthesis metabolic pathway involved in the seed dormancy release of P.lactiflora.