Screening Of Differentially Expressed Proteins And Content Analysis Of Related Metabolites During Paris Polyphylla Var. Chinensis Seed Germination

Paris polyphylla var.chinensis is a rare plant of the genus Paris L.in the Liliaceae family,and is one of the official source plants of the Chinese herbal medicine Chonglou which is the raw material of various Chinese patent medicines such as Yunnan Baiyao and Gongxuening.However,the Paris polyphylla var.chinensis seeds have to go through a long period of dormancy.It will germinate after two winters and one summer under natural conditions,which aggravates the conflict between the supply and demand of Chonglou in the Chinese herbal medicine market.In order to explore the characteristics and molecular mechanism of Paris polyphylla var.chinensis seeds during germination,paraffin section technology was used to analyze the structure of Paris polyphylla var.chinensis seeds under different physiological states and the proteins of seeds were analyzed using sequential window acquisition of all theoretical mass spectra（SWATH）during the key period of germination.We initially screened out the differentially expressed proteins（DEPs）in this process,and quantitatively analyzed the DEPs in some crucial metabolic pathways from the gene level.At the same time,the content changes of related metabolites were analyzed by high performance liquid chromatography and ELISA in the process.The main results are as follows:（1）The Paris polyphylla var.chinensis seeds at different stages under stratification at 22℃ were used as samples,and the same period seeds at 4℃ were used as the control for morphological observation and paraffin section analysis.The results showed that although the Paris polyphylla var.chinensis seeds were still in the spherical embryo stage at the 20th day,their endosperm had begun to be decomposed at 22℃.On the 40th day,the germination rate reached 64.28%,and the seed developed into the short cylindrical embryo.On the 50th day,the epicotyl grew significantly,and the seed embryo developed into the torpedo shape.On the80th day,the cotyledon had formed and the radicle was about 1.5 cm.Under the condition of4℃,there was no obvious change in the external morphology of Paris polyphylla var.chinensis seeds during the whole culture period,and the seed embryos were always in the stage of spherical embryos.Based on the morphological observation and germination rate analysis,we believe that the 10th day before the germination,early state of the germination,20th day after the germination and the state when the length of the radicle breaking through the seed coat reached 1.5 cm are the critical nodes during Paris polyphylla var.chinensis seeds germination.（2）Paris polyphylla var.chinensis seeds at the 10th day before the germination,early state of the germination,20th day after the germination and the state when the length of the radicle breaking through the seed coat reached 1.5 cm were used as samples.SWATH was performed on the mixed samples of peptides.A total of 11092 peptides were obtained,which matched 1787 proteins.After screening,749 DEPs were obtained,which were annotated in some databases.The functional categories with a large proportion of DEPs in the COG database are post-translational modification,carbohydrate transport and metabolism,etc..The most DEPs were annotated in.the molecular functional categories of binding and catalytic activity of GO database.A total of 103 pathways were obtained in the KEGG database,including carbohydrate metabolism,biosynthesis of secondary metabolites,etc.（3）This study focused on analyzing the change characteristics of related proteins in four metabolic pathways:MAPK signal transduction pathway,glycolysis pathway,ABC transporter pathway and pentose phosphate pathway.Under the 22℃ culture conditions the results showed that only CAT in the MAPK signal transduction pathway was significantly up-regulated at both the protein and gene levels;the metabolic activity of the glycolytic pathway was comparable in the two culture groups.The metabolic intensity of pentose phosphate pathway in the 22℃ culture group was higher than that in the 4℃ culture group.The DEPs identified in the ABC transporter family included ABCC14 and ABCC2,and the ABC transporters played an important role in the initiation stage of Paris polyphylla var.chinensis seed germination.（4）Using high performance liquid chromatography to determine the contents of polyphyllin I,II,VI and VII in the Paris polyphylla var.chinensis seeds at different stages under the conditions of 22℃ and 4℃.The results showed that the Paris polyphylla var.chinensis seeds cultivated at 22℃ contained polyphyllin II,VI and VII,while the seeds only contained polyphyllin II and VII at 4℃.Under the condition of 22℃,the total polyphyllin content in the seeds was lower than that of the seeds cultured at 4℃ before the 50th day,and higher than that of the seeds cultured at 4℃ after 50th day.The results showed that the content of total polyphyllin in Paris polyphylla var.chinensis seeds can promote seed germination when the content is low,and inhibit the seed germination when the content is high.（5）The contents of ABA,IAA,ZT,and GA₃of the Paris polyphylla var.chinensis seed in the above-mentioned period were determined at the same time.The results showed that under the culturing condition of 22℃,the changes of ABA,ZT,and GA₃showed a unimodal curve,and they all reached the highest value on the 40th day.While the IAA showed a bimodal curve with the highest value on 20th day.Under the culturing condition of 4℃,ABA,IAA,GA₃reached the highest value on the 20th day.In addition,when the seeds began to germinate,the contents of the four plant hormones in the seeds at 22℃ were higher than those at 4℃.From the perspective of the change rule,ZT has a promoting effect on the germination and development of Paris polyphylla var.chinensis seeds.