| With a high ornamental value,Paeonia lactiflora Pall.is used as a material for garden landscaping and cut flower.It also has a high medicinal value.However,the special dormancy characteristics of hypocotyl and epicotyl in P.lactiflora seed results in a low germination rate,which will seriously affect the breeding application and the selection of new hybrid varieties.Studies have shown that abscisic acid(ABA)is an important plant hormone that can regulate seed dormancy and germination.At present,the understandings of dormancy release process,especially on molecular aspect,is not systematic and the specific role of ABA in this process is not clear in P.lactiflora.In this study,by analyzing the differentially expressed genes during seed dormancy release process in P.lactiflora,and combining with the determination of ABA content,the Pl CYP707 As gene encoding a key enzyme for ABA catabolic metabolism was screened.Combined with various experimental methods,their functions were analyzed when participated in the ABA decomposition pathway and seed dormancy release.The results can enrich the relevant theories of Pl CYP707 As regulating seed dormancy,and provide a scientific basis for further exploration and clarification of the double dormancy release process in P.lactiflora.The main results in this study are as follows:1.Three stages were divided during the dormancy release process: seed imbibition,hypocotyl dormancy release and epicotyl dormancy release.Seed imbibition was the beginning of dormancy release;the hypocotyl dormancy release was the basis for the whole dormancy release;the epicotyl dormancy release process was the most complex process.A total of 7139 differentially expressed genes were obtained during seed dormancy release process.During seed imbibition process,a total of 1278 genes were differentially expressed,of which 582 genes were up-regulated and 696 genes were down-regulated;during hypocotyl dormancy release,a total of 1809 genes were differentially expressed,of which 836 genes were up-regulated and 973 genes were down-regulated;during the epicotyl dormancy release process,there were 3049 differential expressed genes,of which 2203 genes were up-regulated and 846 genes were down-regulated.2.Plant hormones regulate seed dormancy release.During the process of P.lactiflora seed dormancy release,a total of 91 genes related to plant hormone metabolism and signal transduction were differentially expressed,involving a total of 8 main plant hormones,including abscisic acid,gibberellin,ethylene,auxin,cytokinin,brassinolide,jasmonic acid and salicylic acid.Among them,more differentially expressed genes were related to abscisic acid,auxin and ethylene metabolism or signal transduction,and less related to other five plant hormones metabolism or signal transduction.These genes can be used as candidate genes on plants hormones regulating dormancy release of P.lactiflora seeds in the future.3.Endogenous ABA of were measured in the dormancy release process and in different seed parts.It showed that the ABA content basically presented a downward trend during the dormancy release process,especially in hypocotyl dormancy release process.The hypocotyl dormancy release is also the essential process to the whole dormancy release.Therefore,the decrease in ABA content was the main factor affecting the dormancy release in P.lactiflora seeds.As for the mature seeds,the ABA content in seed coat was the highest,about 10 times than that of in the embryo and endosperm.Combined with the characteristics of P.lactiflora,it is believed that even ABA content in the embryo and endosperm were low,it was the main reason affecting the seed dormancy release.4.Using P.lactiflora callus as the receptor material,the conditions of Agrobacterium-mediated genetic transformation were preliminarily studied.By testing the callus sensitivity to antibiotics,kanamycin and cefotaxime concentrations were determined to be 125 mg·L-1 and 300 mg·L-1,respectively.The transformation rate was calculated according to the GUS gene transformation.Three main factors were studied,including the concentration of the bacterial solution,the time of infection and co-culture time.It showed that the most suitable parameter selection was OD600=0.6,infection for 25 min and co-culture for 3 d.5.The CDS sequences of Pl CYP707A1 and Pl CYP707A2 have been obtained in our previous research.In this study,the CDS sequence of Pl CYP707A3 was cloned,with a length of 1404 bp and encoding 467 amino acids.Sequence alignment found that they were significantly different.Analysis of the protein conserved domains showed that they all contained the cytochrome P450 monooxygenase domain,which was closer to grape and rose.The subcellular localization of the protein showed that they were localized to the cytoplasm and cell membrane.Pl CYP707A1,Pl CYP707A2 and Pl CYP707A3 showed different expression patterns in different seed parts or different dormancy release stages.The synergistic up-regulation of expression during seed imbibition and hypocotyl dormancy release is essential for the decline in ABA content.Pl CYP707A2 played a role in epicotyl dormancy release.Through transgenic experiments in P.lactiflora callus and Arabidopsis thaliana,it was proved that Pl CYP707 As played a certain role in ABA catabolism and seed germination.Therefore,Pl CYP707 As promoted the dormancy release of P.lactiflora seeds by regulating ABA content.In summary,this study systematically divided the dormant release stages of P.lactiflora seeds and analyzed the differentially expressed genes in each stage.Combined with the determination of endogenous ABA content,it was determined that Pl CYP707 As may play a role in the dormancy release process of seeds.The function of Pl CYP707 As was studied by various experiments,which filled the gap in the gene research related to ABA in P.lactiflora.Systematic research from gene differential expression,physiological changes to phenotypic changes has been basically realized,which plays an important role in improving the understanding of the double dormancy release process and lays an important foundation for a deep understanding of molecular mechanisms regulating this process in P.lactiflora. |
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