The Regulatory Mechanism Of MsPHYs On Dormancy Release In Magnolia Sieboldii Seeds

Magnolia Sieboldii K.Koch is one of the ornamental plants of Magnolia in Magnoliaceae,which has important economic and scientific value.However,due to the morphological and physiological dormancy characteristics of Magnolia sieboldii seeds,it is difficult to germinate and sprout.Based on the previous experiments of our research group,it was found that the germination process of seeds induced by stratification at low temperature accelerated after illumination.A large number of studies have proved that light plays a vital role in seed germination.In order to explore the relationship between light and seed germination more comprehensively,the light-sensitive pigment genes(MsPHYs)of Magnolia sieboldii were screened and their gene functions were predicted based on the genomic data of Magnolia sieboldii seeds.The effects of different wavelength light treatments such as darkness,red light,far-red light,white light,red light and then far-red light on the expression pattern of MsPHYs during seed stratification and germination were investigated.The recombinant expression vector was constructed to realize the genetic transformation of MsPHYA and MsPHYB1 in wild-type and mutant Arabidopsis thaliana in Colombia,and to verify the gene function.This study provides a theoretical basis for the involvement of photosensitive pigments in dormancy release of Magnolia sieboldii seeds.The main results are as follows:1.According to the comparison between the genome database of Magnolia sieboldii and BLASTP,five PHY genes of Magnolia sieboldii and other species were screened,and the phylogenetic tree was constructed.It was found that five MsPHYs genes MsPHYA,MsPHYB1,MsPHYB2,MsPHYC and MsPHYE were clustered into PHYA,PHYB,PHYC and PHYE subfamilies,respectively,with typical structures of photosensitive pigments.The five MsPHYs genes were nuclear genes,encoding 1130～ 1151 amino acids.The protein belonged to acidic protein,with low hydrophilicity and no signal peptide,and showed preference in chromosome distribution.2.The expression level of MsPHYs gene in Magnolia sieboldii seeds increased in a ‘M’ shape after 0 ～ 40 days under different wavelength light conditions.Among them,red light is more conducive to the expression of MsPHYs,especially MsPHYA.The change trend of Ms GA20ox6 was similar to that of MsPHYs,showing a ‘M’-shaped increase,while the relative expression of Ms NCED3 was different from that of MsPHYs,showing a downward trend.Therefore,in the process of seed stratification,red light may be an important condition for regulating photosensitive pigment protein to participate in dormancy release of Magnolia sieboldii seeds,in which MsPHYA may play an important role and participate in hormone synthesis and metabolic pathways.3.MsPYHB1 gene was successfully cloned.The eukaryotic expression vectors p RI101-EGFP-MsPHYA and p RI101-EGFP-MsPHYB1 were successfully constructed by single enzyme digestion.The recombinant expression vector was transformed into wild type and mutant Arabidopsis thaliana of model plant Colombia by inflorescence infection,and the transgenic seeds of T3 generation were obtained.Compared with the radicle length of MsPHYA-△Over,MsPHYB1-△Over,MsPHYA-△Homo,phya mutant and Col-0 T3 generation seeds,the radicle length of Arabidopsis thaliana was higher under red light.The overexpression of MsPHYA gene promoted the early completion of radicle and cotyledon growth,and had germination advantages.MsPHYA can restore the late germination phenotype of phya mutant in Arabidopsis thaliana,and MsPHYB1 phenotype was not significantly different from wild type.Therefore,it is speculated that MsPHYA is an important protein in response to red light to relieve seed dormancy.