MS-RDA的改进及在白背飞虱的应用研究

The Improvement of MS-RDA and Its Application in Sogatella furcifera (Horvath)

甲基化敏感性代表性差异分析方法 (MS-RDA)适用于特异、低丰度基因组DNA甲基化的筛查与鉴定,已大量应用于哺乳动物的疾病发生和植物发育机理方面的研究,但昆虫中尚未发现该方法的应用。从DNA提取、酶切、扩增子制备、消减杂交等方面对该方法进行了改进,并在水稻重要害虫白背飞虱上得到成功应用,获得了4条与雌、雄性别相关联的特异性甲基化差异条带,其中有3条序列已被GenBank收录,登陆号分别为JX847621、JX624161、JX472453,经BLAST比对,有3条未发现同源性序列,属于功能未知的新基因;同时还获得了2条与长短翅型相关联的特异性甲基化差异条带,也已被GenBank收录,登陆号分别为JX514031、JX472454,同源性分析发现与多种动物的18S核糖体和28S核糖体RNA基因序列高度相似,因此有关核糖体基因的甲基化可能对白背飞虱的翅型分化起到一定的调控作用。

Methylation-sensitive representational difference analysis （MS-RDA） is suitable for screening and identification of specific and low abundances genomic DNA methylation. It has been applied in many researches on mammal disease occurrence and plant developmental mechanism, but no application was reported in insects. In this study, we developed a MS-RDA method for insects, which was improved from DNA extraction, restriction enzyme digestion, amplicon preparation, and competitive hybridization. The method was further applied successfully in Sogatella furcifera （Horvath）, an important rice pest in China. Four differentially methylated DNA sequences related with female and male were obtained. Three of them had been accepted by GenBank, the accession numbers were JX847621, JX624161, JX472453, respectively. Three of them were not found homologous sequences by BLAST and should be belonging to the new genes with functions unknown. Meanwhile, two differentially methylated DNA sequences related with long and short wing were obtained. Their accession numbers of GenBank were JX514031, JX472454, respectively. They were found highly similar with the 18S ribosomal or 28S ribosomal RNA gene sequences of many kinds of animals by homology analysis, therefore ribosomal RNA gene methyla- tion maybe play some roles on wing dimorphism of S. furcifera.