| Tobacco (Nicotiana tabacum) is an important economic crops and model organisms. With the development of agriculture production and improvement of people's living standard, the yield and quality of tobacco leaves are put forward higher request. But in the long-term tobacco-nurturing, genetic background of tobacco is narrower, and tobacco growing long-term dependent on imported varieties. Molecular marker is an important means to assist breeding, and have important applications in the construction of genetic map and trait gene positioning.This paper adopts amplified fragment length polymorphism, methylation-sensitive amplified polymorphism and gene cloning technology to study epigenetic information such as methylation level, pattern and sites and methylation-related gene sequences.The results are as follows:1.Polymorphism analysis based on gel electrophoresis profiles for AFLP of tobacco genomic DNABy using eleven primer combinations from Mseâ… primers and EcoRâ… primers, AFLP and PAGE (polyacrylamide gel electrophoresis) were performed on genomic DNA of tobacco samples taken from Yunyan 85, NC89, K326, Yunyan 87 taken from Ya'an, Xingwen and Yaojia of Sichuan in 2009.The electrophoresis map show generally poor polymorphism. Then we use the same primer combination amplify genomic DNA of tabacco sample Yunyan 87 which is taken from Guangyuan, Luzhou and Yibin of Sichuan in 2009 and 2010.Electrophoresis map of amplification products show that 242 Clearly visible and repeatable DNA bands are amplified by eleven pairs of primers.The results of statistics show that the year when samples is fetched have more influence on polymorphism compared with geographical conditions.2. polymorphism for methylation patterns of the tobacco genome DNATo establish the methylation-sensitive amplified polymorphism analysis system suitable for tabacco genome, this paper use EcoRâ… and Hpaâ…¡/Mspâ… to digest genomic DNA.This paper detect six tobacco samples genomic DNA methylation sites in the whole genome level,the six tobacco sample are taken from Yunyan 85, NC89, K326, Yunyan 87 taken from Ya'an, Xingwen and Yaojia of Sichuan.Methylation sites statistics were summerized by comparing MSAP patterns of six tobacco samples.The results show that Location of samples growed have more influence on polymorphism compared with sampling time.In this study, the lowest proportion of methylated sites is from 29.72% up to 43.37%,in accordance with the reported study which show that the MSAP methylation detection rate of plant genome DNA is 4.7%-45%.The results enrich the methylation research of tobacco genome,and provides a reference for future epigenetic studies of tobacco.3.DNA methylation fragments sequences in tobacco genomeSome tobacco DNA fragment of genome methylation sites are recycled and cloned. Through further sequence alignment, tobacco genome DNA, chitinase gene, nitrate reductase gene,photosynthetic system precursor gene, chloroplast DNA, mitochondrial DNA, ornithine decarboxylase gene, ribulose-1,5-bisphosphate carboxylase/oxygenase gene and other homologous sequences and promoter sequences are identified.The results enrich the diversity of detected DNA methylation sites in higher plants genome.On the basis of the definition of CpG island, this paper makes further analysis for the distribution characteristics of CpG dinucleotides on tobacco methylated DNA fragments.The results show that tobacco genome CpG two nucleotide concentrated area is potential sites of DNA methylation cytosine modified4. Methylation-related genes sequence of tobacco DNA genomeThe results of clone and analysis of nitrate reductase, ornithine decarboxylase and ribulose decarboxylase three gene sequences are as follows:(1)by comparing the gene sequence for different samples with corresponding gene mRNA sequence which is found on GENBANK, the result indicate that the changed degree for different samples exon similarity in ribulose-1,5-bisphosphate carboxylase/oxygenase gene sequence are the bigger than other genes.Presumably, ribulose-1,5-bisphosphate carboxylase/oxygenase gene of samples mutated during evolution because of the gene is too long.(2) the analysis of intron sequence revealed that base changed degree among four local air-dried tobacco samples is bigger than that of baked-tobacco samples. Presumably, this is related to the close genetic relationship among the baked-tobacco samples; (3) some methylated fragments in gene distibute in gene intron sequences by comparing and analyzing gene structure and methylated fragments in gene of different samples.There are much biological evolution information in Intron region,and DNA methylation with a hereditary characteristic is a part of the epigenetic study.Therefore,there should be a relationship between intron and biological evolution.But it needs futher experimental study. |
PDF Full Text Request