Study On Germination Conditions And Dormancy Mechanism Of Chinese Medicinal Material Polygonatum Sibiricum Seeds

The tubers of three plants of Liliaceae,Polygonalum sibiricum Red.,P.cyrtonema Hua.and P.kingianum Coll.Et Hemsl.,are used as Traditional Chinese Medicine polygonate rhizome for replenish yin,moisten the lungs and pulmonary tuberculosis with coughing and hemoptysis,and also for good use.For a long time,the wild plants are the main source of polygonate rhizome and large amounts of Polygonatum were consumed in China,these resulted in exhaustion of plant resources of these plants.Artificial cultivation of Polygonatum began to develop in the last 5 years,asexual propagation using rhizome and seed propagation are two methods in practice.There are two problems in cultivation practice,one is asexual reproduction with difficult seedling emergence and time inconsistency in emergence,the other is the seed has the dormancy characteristic and seedling time is long.Therefore,the purpose of this study is to explore the temperature stratification conditions of three plants seed germination,as well as to study the expression of specific genes during seed germination to elucidate the mechanism of seed dormancy release.In this study,based on the analysis of the basic biology characteristics of three plant seeds,P.sibiricum,P.cyrtonema and P.kingianum,temperature stratification was employed to explore the optimal germination conditions under natural environment.The results showed that three seeds have similar characteristics with undifferentiated rodlike,Embryo also called linear embryo as Baskin described.For the temperature stratification,30/20?,25/15?,and 20/10?,12:12h daily alternating thermoperiods were set to clarify the germination habits.The results showed that high temperature is most suitable for three plant seed germination,P.kingianum and P.sibiricum at 30/20?,while P.cyrtonema 20/10?.Meanwhile,P.kingianum and P.cyrtonema could emergence without and additional low temperature stratification,while P.cyrtonema seed need a low temperature process to emergence after the high temperature germination.In addition,a single-molecule real-time(SMRT)sequencing by the so-called Isoform sequencing(Iso-seq)from Pacific Biosciences(PacBio)was used for generate a complete and full-length transcriptome of P.sibiricum seed germination and dormancy release process and these transcript isoforms used for the reference sequence for comparative transcriptome studies.A total of 292,791 circular consensus sequences(CCS)was obtained and then these CCS were classified into 239,376 full length reads and 230,162 full-length non-chimeric reads(Flnc).A further analysis was performed using isoform-level clustering and 132,557 consensus reads were obtained.In this study,RNA-seq strategy yielding approximately 717 million RNA-seq reads to correct the Iso-seq and 87,251 unigenes were produced.A total of 25,998 DEGs were identified among the twelve samples.We also analysis 14 ABA and GA signal transduction related transcription factors compared with the Arabidopsis genome.RT-qPCR were used to verify the expression of the 15 genes sequenced in the transcriptome.The products expressed by these genes are all related to ABA and GA anabolism.Among them,the genes encoding ZEP and AAO3,the key enzymes for ABA synthesis,began to gradually down-regulate expression after the bulb stage was increased at the early stage of germination.The expression of coding genes of signaling pathway receptors PYL4/8 and regulatory proteins ABI5 and ABF also showed the same trend.The coding genes of inhibitor PP2C and inhibitor CYP707A showed the opposite trend.The gene expression of GA pathway-related proteins is generally opposite to that of ABA.The expression levels of genes encoding key enzymes GAMT2,receptor protein GID1,regulatory proteins GASA3/14,and CIGR2 gradually decrease at the early stage of germination and begin to increase after the bulb stage.However,the expression trend of the gene encoding the synthase GA3ox and regulatory protein GASA6 is different,probably because GA3 inside the seeds starts to accumulate at the bulb stage,which promotes the seeds to release dormancy.Through the expression of these genes,it can be seen that ABA and GA are antagonistic,and the ratio change during germination is related to seed dormancy and germination state.