Establishment Of CDNA-AFLP System And Analysis Differential Gene For Dimocarpus Longan Lour. Cv Sijimi Flower Bud And Leaf Bud

Longan(Dimocarpus longan Lour.)is a member of the family Sapindaceae, which is an important and distinctive subtropical orchardfruit in China and additionally the mainly raised fruit tree in Fujian Province.This research, using flower bud and leaf bud and the technique of cDNA-AFLP, separates gene segment which is related to flowering of Dimocarpus longan Lour. cv Sijimi. Through clone sequencing and BLAST analysis, three gene segments possibly relevant to Longan flowering were obtained. It was still one subject to clone the full length of the gene and extent the research to site- and time- expression which was helpful to disclose the mechanism of relevant gene in the process of Longan flowering. The main experiment results were as follwes:1 Establishing the isolated technology system adaptable for total RNA of Longan (Dimocarpus longan Lour. cv Sijimi)flower bud and leaf budAccording to the characteristic of the tissue of Longan being rich in polysaccharide, tannins, pigments and phenols, utilizing improved SDS approach and CTAB approach, extraction and purification of the RNA of flower bud and leaf bud of Longan were conducted in this study. The results suggested that the improved SDS approach could be used to extract RNA and the extracted RNA had two lines of 28 and 18s clearly and completely without degradation, which meaned that the RNA extracted by improved SDS approach had higher purity and satisfied the requirements of successive molecular biology research. The isolated technology system adaptable for total RNA in Dimocarpus longan Lour. cv Sijimi floral and leaf bud established in this study was that: 1.00 g flower bud or leaf bud of Dimocarpus longan Lour. cv Sijimi with 5.5mL extraction buffer solution and extending the settling time from 3 h to 10 h could result in high quality RNA2 Optimizing the reaction system of cDNA-AFLP and separating the differential gene connecting to flowering process.The key elements of cDNA-AFLP reaction system was probed and analysis system of cDNA-AFLP with good repetitiveness was set up. It showed that it was possible to divideds-cDNA completely by taking 0.9μL from EcoRI and MseI in the 40μL system separately, and the overnight generation from the corresponding joint could be used for pre-amplification. In the reaction system of 25μL PCR, it was better done by diluting the pre-amplification 1200 times with the density of Mg2+ at 1.6 mmol/L and the density of dNTP at 0.2 mmol/L.Blastn and blastx study by comparing 32 orders of TDF with good predicting capacity to the data in GenBank database was carried out. The results was as below: for 27 out of 32, by search the genes were found, mRNA or cDNA with higher similarity. For the rest 5, there were no results, and they were inferred as unknown genes. Analysis showed that 3 TDFs out of 27 possibly was related to Dimocarpus longan Lour. cv Sijimi flowering as following: the gene of S-adenosyl-L-methionine synthetase amplified from E7M7, the gene of cytochromeP450 amplified from E7M4, and the gene of flavanone 3-hydroxylase amplified from M7E6.3 Applying RT-PCR technique to separate 3'end of SAM gene sequenceBy selecting one of the 3 gene sequences-SAM which was possibly related to Dimocarpus longan Lour. cv Sijimi flowering, designing primers and utilizing RT-PCR technique to expand 3'end sequence of SAM, testing its sequence and putting them together, the 3'end of SAM at the amount of 1252bp was attained. The nucleotide sequence comparison with the SAM gene of Populus trichocarpa showed that identity was all higher than 96%.