Studies On The Differential Expression And The Expression Of Genetic Regulation Of Different Ploidy Watermelon Under Low Temperature Tolerance

Watermelon is the main fruits in summer, which is considered to be rich in nutritional value. Because of watermelon is a kind of thermophilic and heat-resistant crop, so it is sensitive to the low temperature environment. Although using greenhouse and plastic film mulching technology to meet the watermelon growth needed for appropriate temperature in the facility cultivation, but also be persecuted by the low temperature. Up to now, the cold resistance of watermelon germplasm resources are very scare, urgently needs to solve the problem. In terms of physiological and biochemical studies have found that the polyploidy watermelon have strong cold to tolerance, while the research at molecular level is less reported.The purpose of this study is to in-depth investigate the gene expression differences before and after low temperature stress in different ploidy watermelon combining the molecular marker technology, making homologous diploid and triploid of watermelon of female precocious Jingxin as materials. Using MSAP technology, c DNA-AFLP technique and quantitative PCR analysis of different ploidy watermelon genens differentially expressed before and after low temperature stress. All the results of this study were as follows:1. MSAP technology was carried out to analyze watermelon genomic DNA before and after cold stress. A total of 3 534 locus were amplied after using 28 MSAP primer combinations, each primer amplified 126 bands on the average. The rates of double stranded DNA internal methylation of CCGG site were highest in different ploidy watermelon, investigating that this methylation type is the main form of watermelon materials. The diversity of full methylated, hemimethylated and total methylated were all not notable of different ploidy watermelon before cold stress. After low temperature stress, the ratios of hemimethylated in diploid and diploid cold were 12.8%, 9.3%; the ratios of hemimethylated in triploid and triploid cold were 15.3%, 10.0%; the ratios of hemimethylated in tetraploid and tetraploid cold were 14.8%, 8.3%. With U test analysis, the diploid hemimethylated was not significant, but the triploid was significant and extremely significant in tetraploid, which are positively correlated with the ploidy of watermelons. Compared with before cold stress, there were a few of adjustment in DNA methylation pattern in different ploidy watermelon respectively. The ratios of demethylation in 3 different ploidy watermelon under cold were 18.4%, 17.7%, 17.8%; the ratios of hypermethylation were 18.4%, 17.0%, 16.0%. Illustrating that it is very different between different ploidy watermelon in epigenetic mechanisms. Took 13 different bands with watermelon genome database, identified 7 bands as watermelon genome sequence, through the BLASTX analysis found that these genes were related to plant growth and development, environment stress response, signal transduction and so on.2. c DNA-AFLP technique was used to analyze the differential gene expression. After being screened with 26 c DNA-AFLP primer combinations, 1 267 bands were amplied: the ratios of up-regulated expression in 3 different ploidy watermelon were 48.2%, 51.3%, 58.6%, which are positively correlated with the ploidy of watermelons; the ratios of down-regulated expression were 51.8%, 48.7%, 41.4%, which are negative correlated with the ploidy of watermelons. BLASTX analysis showed that 38 bands were homologous to functional genes, including hypothetical protein(25.9%), energy and metabolism protein(31.5%), material transportation protein(5.6%), signal transduction protein(3.7%), DNA modification(1.9%), stress protein(1.9%) and unknown genes(29.6%).3. Using fluorescence quantitative PCR detection up-regulated expression showed that, after low temperature, the relative expression amount of Acetyl-Co A carboxylase carboxyl transferase subunit beta, Zinc finger protein and NADH dehydrogenase subunit 4 were 3.56, 1.45 and 16.68 times of the contrast material, respectively. The results illustrating that low temperature affect watermelon some resistance proteins or other substances, regulate the expression of genes, so that against persecution of low temperature.