| Paeonia lactiflora is a herbaceous flower in the family Paeoniaceae that is grown in China. Its seeds display both hypocotyl and epicotyl dormancy. Incomplete removal of dormancy during seed reproduction leads to a decrease in germination rate. Most research on P. lactiflora has concentrated on physical aspects of the plant, and few studies have collected molecular data.In this study, we used two different germination stages of peony seeds, The male parent was "Fen Yulou", and the female parent was "Fen Yunu". We used two different seed developmental stages (0 and 40 days, we called PDB and PDA) as material. We used the Illumina HiSeqTM2000/MiSeqTM sequencer to sequence the P. lactiflora seed transcriptome. This project aims to elucidate the molecular mechanism of seed hypocotyl dormancy and germination of Paeonia lactiflora, and provide a reliable basis for future research. The main test results are as follows:1. PDB and PDA Paeonia lactiflora of RNA sequencing in this part got 120,181,964 unigene sequence,58,107,876 unigene sequences obtained from the seeds of PDB RNA,6,274,088 unigene sequences obtained from the seeds of PDA RNA. A total of 123,577 contigs were detected after splicing the sequence, and about 13360 of the contig were> 2000 bases.2. After blast comparison, A total of 34,284 Unigenes sequence alignment to Nr, Nt, SwissProt, PFAM, GO, KOG, KEGG database, accounting for 50.37% of all Unigenes total. Using KEGG Pathway to obtain annotation informations, including seed plant hormone signal transduction (ko04075) path information. Genes compared with the Nr, SwissProt, KEGG, and the Cluster of Orthologous Groups databases predicted the encoded protein box (CDS) of 30,803 unigenes. There were 8,203 gene-predicted proteins with more than 300 (26.6%) amino acids and 8,203 gene-predicted proteins with more than 1,000 (2.42%) amino acids.3. The PDB samples had 834 upregulated and 960 downregulated genes relative to those in the PDA samples. Select the plant hormone signal transduction pathways (ko04075) as the main object of study. It involves gibberellin, abscisic acid, auxin, cytokinins, ethylene and other hormone regulatory pathway, Plant germination and growth has a great relationship with hormone regulation, so we lock this path as the research object. Four plant hormone signal transduction unigenes were chosen for qRT-PCR analysis to confirm differences in expression levels between accessions found in the FPKM analysis. These unigenes were GAI1, ARF, and BRI1, which were upregulated in seeds, and JAZ, which was downregulated. The qRT-PCR data confirmed the expression patterns of these unigenes determined by the FPKM analysis.4. This study successfully amplified GAI1 gene ORF sequences from cDNA of Paeonia lactiflora seeds. Analysis of GAI1 gene obtained molecular weight is 60.44kDa, the theoretical isoelectric point of 4.93. Predict it is a hydrophobic protein and it is not a transmembrane protein and it belong to non-secreted proteins.5. The study also verified GAI1 protein expression in E. coli. The results show that the gene could significantly expressed in E. coli. It is provides a theoretical and practical basis for future research, and laid a solid foundation for the future of transgenic trials. |
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