| Betula platyphylla is the one of the main tree species in the Northeast. As the "Eighth Five-Year" key species, there are many advantages and economic value, so its research rapidly and achieved good results, but birch research is still not very deep, especially in the flower developmental mechanism still remain in the initial stage. In order to better undersand the mechanism of flowering, in this study, suppression subtractive hybridization technique had been used to constructe the forwords and reverse subtractive library of Betula platyphylla male and female flowers. Anaslysis the the functional of these differential gene fragments and classified. Followed by semi-quantitative PCR and RT-PCR to detection and analysis of the gene expression during development. The main findings are as follows:1. In the forwords SSH library (bhhx),723 ESTs were sequenced successful, the minimum read length 227bp, the average read length 550.2bp. High-quality ESTs (length greater than 100bp) 701. ESTs sequences using phrap software to assemble. Get a total of 339 meaningful sequences. In the reverse SSH library (blihx), there are 815 ESTs, the minimum sequencing length 156bp, the average sequencing length 550bp. High-quality ESTs 668. Assembly of splicing, a total of 256 meaningful sequences.2. The BLAST showed the homology between the specific gene library screening ESTs and GenBank sequences, while a preliminary analysis of biological information followed by KEGG metabolic pathway database and the COG functional classification. The biological function of the obtained ESTs can be divided into 16 categories:Translation, ribosomal structure and biogenesis; Carbohydrate transport and metabolism; Posttranslational modification, protein turnover, chaperones; Amino acid transport and metabolism; Lipid transport and metabolism; Energy production and metabolism; Signal transduction and metabolisms; Secondary metabolite biosynthesis, transport and catabolism; Coenzyme transport and metabolism; Transcription; Chromatin structure and dynamics; Cytoskeleton; Inorganic ion transport and metabolism; Replication recombination and repair; Cell cycle control, cell division; Cell wall/membrane/ envelope biogenesis. Betula platyphylla inflorescence development process involved in metabolic pathways are: glycolysis/gluconeogenesis; pentose phosphate pathway; fructose and mannose metabolism; phenylalanine metabolism; phenylalanine, tyrosine and tryptophan biosynthesis; glyoxylate and dicarboxylate metabolism; carbon fixation in photosynthetic organisms; methane metabolism; phenylpropanoid biosynthesis; pentose and glucuronate interconversion; ascorbate and aldarate metabolism; oxidative phosphorylation; starch and sucrose metabolism; nucleotide sugars metabolism and so on. 3. Screening of some ESTs associated with flower development, such as the MADS-box gene, AP2 gene, ARF, peroxidase gene, ribulose bisphosphate carboxylase gene, S-adenosylmethionine gene, cytochrome P450, plant receptor protein kinases and aging-related genes. Functional annotation of these genes will make the future research work carried out smoothly.4. Selection of four differentially expressed genes for Realtime-PCR detection. The results showed that RBC gene expression in the female flowers is higher than in the expression in the male. The expression in leaves is much larger than female flowers in the expression.SAMS genes in this period of male and female flower development expression no significant differences. In generally, the expression in the female flowers is slightly higher, but did not reach significant level. SAMS expression in leaves, significantly less than the previous expression in the female flowers, and then was gradually increased in the late basically flat in the female flower. POD1 and POD2 gene expression in the male flowers is significantly higher than the expression in female flowers. Also, the four differential expressed genes show their characteristics and trends in the female flowers and male flowers. In leaves, POD1 expression in the early is much higher than the expression in the female flowers, followed by lower expression in the period. The expression of POD2 is higher than the expression of female flowers except the medium-term. |
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