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Grafting is a horticultural techniques, the survival process contains cell dedifferentiation, redifferentiation major theoretical issues. Pecan grafting is a recognized problem, this study in order to explore the molecular mechanism and regulation mechanism of this precious species grafting, using cDNA-AFLP technology and quantitative RT-PCR technology Pecan Grafting gene screening, separation and biological information. analysis, results showed that: 1. improved CTAB grafted samples extracted total RNA quality, completeness, no impurities for cDNA-AFLP analysis and fluorescence quantitative RT-PCR analysis. By SMARTTM PCR cDNA Synthesis Kit reverse transcription obtained eight of experimental material double-stranded cDNA, good integrity, meet the test requirements. 3. With the identification of the 4 bases, and 6 bases in the restriction endonuclease TaqI, ASEI digest the cDNA was obtained fragment length meet the technical requirements. Restriction fragments and artificial pre-expansion connector, considerable concentration of 200 ~ 1000bp fragment of copy. Selection of 100 pairs of selective amplification primers selective amplification. Improved selective amplification procedure, using a high temperature (65 ° C) annealing combined cycle cooling methods, to obtain a high concentration of specific product. 6% polyacrylamide gel electrophoresis and silver staining differential display of selective amplification product, results were obtained over 300 differentially expressed fragments. After Gel Extraction, purification and secondary cloning differential fragments obtained over 100. 66 total measured back sequence, Blast than sequence, removing repetitive sequence 17, 49 other TDFs were homolo-gous to genes with results obtained. 6 selected 12 genes for quantitative RT-PCR validation analysis, the results show, which eight clones translational initiations for factor eIF-4A, 3'-5'-exoribonuclease / RNA binding (AT2G47220), No. 18 unknown genes, isolate K1 / E32 K1 glycoprotein, putative to auxin response factor 1 water channel (PIP1B) of Glycine max catalase (cat4), 65 unknown genes and expression of cDNA-AFLP, a clone of the 25th unknown genes and expression of cDNA-AFLP consistent , while the remaining three clones 17 unknown genes,, beta family the G-protein the cdc20 of protein (CDC20.2) of expression cDNA-AFLP expression results. 75% in this experiment the expression profiles of the cDNA-AFLP been verified. 49 TDFs 20 encoding known functional protein, 8 encoded unknown proteins, and the rest 21 and unannotated genomic sequence similarity, or non-homologous sequences in the nucleotide databases. Functional analysis of the 20 known genes, which belong to the nine categories of material transport, metabolism and energy, cell cycle, signal transduction, etc., pecan grafting process in complex metabolic changes. Obtained differentially expressed genes, the preliminary draw pecan grafting regulation is diagram of modern cell biology grafting theory is confirmed from the molecular level. This is the first of the cDNA-AFLP technique and quantitative RT-PCR analysis technology used in pecan genes differentially expressed, and to improve them, successfully separated into Pecan Grafting related genes. Gene mapping, cloning, and gene expression characteristics of the study provides a theoretical basis for the the fragment sequence produced probe or converted into the corresponding molecular markers based on these differences, Grafting.
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