| Saposhnikovia divaricata(Trucz.)Schischk.is a perennial herb of the genus Saposhnikovia of Umbelliferae,which has the effects of dispelling wind,relieving external symptoms,preventing spasms,overcoming dampness and relieving pain,and has high medicinal and dietary value.In recent years,the resources of wild Saposhnikovia divaricata have been decreasing year by year,and artificial planting Saposhnikovia divaricata has become a development trend.Seed propagation is often used in Saposhnikovia divaricata cultivation and production.The dormant nature of Saposhnikovia divaricata seeds formed in the long-term natural system evolution has limited the process of Saposhnikovia divaricata propagation,cultivation and breeding.Based on the analysis of the biological characteristics of Saposhnikovia divaricata seeds,this study adopted the low-temperature stratification test to explore the optimal germination conditions of Saposhnikovia divaricata seeds,used physiological and biochemical methods and enzyme-linked adsorption immunoassay(ELISA),combined with the development characteristics of the seed germination process,and used high-throughput sequencing technology to sequence Saposhnikovia divaricata seeds with low-temperature stratification at different times,and conducted bioinformatics analysis on the significantly differentially expressed SdSAUR gene.The subcellular localization vector was constructed by homologous recombination method and the expression position of SdSAUR was observed by laser confocal microscope.The mechanism of dormancy release of Saposhnikovia divaricata seeds was discussed at molecular level,and the gene expression changes during dormancy release were studied,which the main results are as follows:1.The strong water permeability of seed coat is not the main reason for its dormancy.When the seeds mature,the embryo is not fully developed and there is morphological dormancy.The extraction experiment of endogenous inhibitor showed that the separated phase of seed extract of Saposhnikovia divaricata may be significantly inhibit the germination of Chinese cabbage seeds and the growth of young roots,and the germination inhibitor was the main reason for the dormancy of Saposhnikovia divaricata seeds.The extract was detected by HPLC-MS,which was found that there were organic acids,alkaloids,aldehydes,coumarins,flavonoids,amides,phenols and glycosides in the seeds,and most of them were water-soluble substances.2.The results of low temperature stratification showed that 4℃ low temperature sand layer was an effective method to break the dormancy and promote the germination of Saposhnikovia divaricata seeds.When the 4℃ low temperature sand layer reaches the 4th week,the seeds of Saposhnikovia divaricata begin to break dormancy,the germination rate was 80.6%,and the germination rate reaches 88.0% at the 7th week.3.The results of the study on the content of stored substances and enzyme activity showed that the soluble sugar content increased first and then decreased during the stratification process,and reached the highest at the 4 weeks.The starch content in the seeds decreased by 43.0%compared with the control group after 7 weeks.The soluble protein content showed a decreasing trend,and dropped to the lowest level after 7 weeks,which was 17.5 μg/g.The activities of amylase,protease and glucose-6-phosphate dehydrogenase gradually increased,and reached1,100.0 μg/g·min,128.7 U/g and 52.6 U/g after 7 weeks.These results showed that the stored substances in seeds were gradually decomposed and utilized by key enzymes.4.The study on the content of endogenous hormones showed that the content of GA and IAA increased during the treatment of 4℃ sand layer,and the content of GA increased from1.28 ng/g·FW increased to 1.77 ng/g·FW after 7 weeks of stratification,IAA content increased from 1.72 ng/g·FW to 6.44 ng/g·FW,ABA content showed a downward trend,and decreased to28.10 ng/g·FW at 7 weeks,GA/ABA ratio also showed an overall upward trend.5.The RNA-seq analysis was carried out on the seeds of Saposhnikovia divaricata in three stages of dormancy(CK),dormancy release(S)and germination(G)using high-throughput sequencing technology,and 166,235 Transcripts and 100,690 unigenes were annotated.The results showed that compared with CK group,13,998 and 16,817 up-regulated genes and 9237 and 14,054 down-regulated genes were obtained in seeds of S group and G group,respectively.Compared with group G seeds,12,859 up-regulated genes and 11,145 down-regulated genes were identified in group S seeds.The analysis of differential gene expression showed that the difference of gene expression in seeds during germination was the largest.The differential gene KEGG signal pathway is mainly concentrated in plant hormone biosynthesis and signal transduction,carbohydrate metabolism and energy metabolism.Through q RT-RCR verification of 12 DEGs related to hormone synthesis and metabolism,it was found that during the dormancy and germination of Saposhnikovia divaricata seeds,the expression of GA and IAA synthesis genes was up-regulated,the expression of ABA synthesis genes was down-regulated,and the expression of decomposition genes was down-regulated.6.The results of bioinformatics and subcellular localization showed that the total length of SdSAUR32 open reading frame(ORF)was 372 bp,encoding 123 amino acids.The total length of SdSAUR23 open reading frame(ORF)is 288 bp,encoding 95 amino acids.The phylogenetic evolution tree was constructed by sequence alignment and MEGA7.0 software with the neighborhood method.It was found that the protein encoded by SdSAUR32 and SdSAUR23 genes had the closest genetic relationship with Daucus carota.The subcellular localization expression vector p C2300-GFP-SdSAUR was constructed by homologous recombination method.After transient expression in tobacco,both SdSAUR32 and SdSAUR23 were located in the nucleus by laser confocal scanning microscopy.These results are helpful to artificially control the germination of Saposhnikovia divaricata seeds,shorten the dormancy time of seeds,which has important theoretical and practical significance for the cultivation and molecular plant breeding of Saposhnikovia divaricata. |
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