Effects Of Different Hormone Treatments On Dormancy Of Magnolia Sieboldii K. Koch Seeds And Functional Analysis Of ABA Receptor MsPYR1 Gene

Magnolia sieboldii K.Koch?Ms?is a small deciduous arbor of Magnoliaceae with ornamental and medicinal values.Its seeds are characterized by deep dormancy and difficult to breed.Abscisic acid?ABA?is a plant hormone that can positively regulate seed dormancy.The ABA receptor PYR/PYL/RCARs gene family plays an important role as the initial component of the ABA signaling pathway,and is of great significance to uncover the mechanism of seed dormancy release.In this study,the most representative MsPYR1 gene among the five AtPYRL homologous genes?MsPYR/PYLs/RCAR,MsPYLs?in Magnolia sieboldii was selected based on the transcriptome data of Magnolia sieboldii,and the gene was used as the research object of Magnolia sieboldii.Cloning,real-time quantitative PCR,Western Blot?WB?,subcellular localization,genetic transformation and other technologies to perform a preliminary analysis of its function,lay the foundation for later verification of its function,the main results are as follows:?1?During the stratification process after GA₃ treatment?4??,the GA₃ content in the seeds increased and the seed germination rate increased;during the stratification process after ABA treatment?4??,the ABA content in the seeds increased and the seed germination rate decreased.By measuring the water content of the seeds during the stratification process,the water absorption curve of Magnolia sieboldii seeds was drawn,and the five key time points of dry seeds,stratification at 0 d,16 d,40 d,and 56 d were determined for the later stage.Analysis of various tests.?2?Five MsPYL genes—MsPYR1,MsPYL2,MsPYL4,MsPYL8,and MsPYL12 of Magnolia sieboldii were screened from the transcriptome data of Magnolia sieboldii.Among them,MsPYR1 and MsPYL12 were more highly expressed in dormant seeds of Magnolia sieboldii than other members.After ABA treatment?4??,the germination rate of stratified seeds decreased,and members of the MsPYLs gene family showed three different expression patterns.The expression of some genes is up-regulated: MsPYR1,MsPYL2,the degree of up-regulation is also different,of which MsPYR1 is the most up-regulated;the expression of some genes is not affected: MsPYL12;some genes are suppressed:MsPYL4,MsPYL8.After GA₃ treatment?4??,the germination rate of stratified seeds increased,and the expression of MsPYLs gene family members was down-regulated as a whole.In summary,the ABA receptor,MsPYR1,which has a higher expression level in dormant seeds and is most sensitive to ABA during dormancy release,was selected.?3?The MsPYR1 gene was cloned,and bioinformatics analysis showed that the MsPYR1 gene sequence was 618 bp in length,encoding 204 amino acids.The relative molecular weight of the protein encoded by MsPYR1 is 22.661 k D.It has a domain of PYR/PYL/RACR protein family,and its encoded amino acid sequence is highly conserved in the evolution of different species.It is closest to AtPYR1,AtPYL1,AtPYL2,and AtPYL3 in Arabidopsis thaliana.There may be commonality in the regulation.?4?The expression analysis results of MsPYR1 in different Magnolia sieboldii tissues found that MsPYR1 was highly expressed in tissues other than buds.Through different concentrations of ABA treatment,it was found that within the set concentration gradient range,with the increase of ABA treatment concentration,the germination rate of seeds decreased,and the expression of MsPYR1 gradually increased.Analysis of the expression patterns of ABA signaling pathway-related genes under FL,ABA,and CK treatments found that the seed germination rate under ABA treatment decreased,and the expression levels of MsABI3 and MsPYR1 increased first and then decreased,while the expression levels of MsABI1 were relatively Lower.The germination rate of seeds increased under the treatment of ABA synthesis inhibitor FL,and the expression levels of genes related to ABA signaling pathway decreased.Through WBexperiments,it was found that the expression levels of MsPYR1 were consistent with the protein translation level during the stratification of seeds treated with ABA,FL and CK.?5?The MsPYR1 protein is localized in the cytoplasm and nucleus.In order to study the biological function of MsPYR1,the pMDC32-MsPYR1 plant overexpression vector was constructed,and the Arabidopsis mutant pyr1pyl1pyl2pyl4 was genetically transformed to obtain T₀ generation seeds,and resistant plants were obtained through preliminary screening of corresponding antibiotics.