Total RNA Extraction Of Cucurbita And Amplification Of Antioxidant Enzyme Genes

RNA extraction is the first step in the study of gene isolation and expression. It is verydifficult in some plants which contain phenolics and polysaccharids and secondary metabolites toextract RNA. To create a protocol of RNA isolation from Cucurbita rich in polysaccharides andpolyphenols, five methods for RNA extraction were assessed for their ability to recover ahigh-quality RNA. A cetyltrimethylammonium bromide (CTAB)-based RNA extraction protocolwas developed.Time for isolation of RNA using this protocol was just2h. This protocol used an equalvolume of isopropanol to precipitate RNA for20min at-20℃and a high concentration of NaCl(1.0-2.5M) and sodium acetate to effectively remove the polysaccharides. It was successful toextract total RNA from leaves and pulp of Cucurbita pepo and Cucurbita moschata using thisprotocol.This method clearly showed28S and18s ribosomal RNA bands and produced RNA withhigh yield (210-250μg/g fresh weight) and high quality (A260/280ratio2.08-2.15; A260/230ratio2.0-2.12) which indicated that the RNA was of high purity and without polyphenol,polysaccharide and protein contamination.It was verified that the preservation method of RNA was good that the RNA precipitate wasimmersed in ethanol and then stored at-80℃refrigerator, by which the quality of RNA wasunchanged after6month.It was established that a reliable method for designing degenerate primers. It was obtainedthat the degenerate primers of the genes of Cu/Zn-SOD, Mn-SOD, catalase and glutathioneperoxidase by searching out the same protein sequences of15species of same genus and thecorresponding cDNA sequence, then using CLC Main Workbench6to design, through sequencealignment and combing with the principles of primer designing.The gene fragments of the Cu/Zn-SOD, Mn-SOD of Cucurbita pepo and Cucurbitamoschata leaves and pulp were amplified out and the gene fragments of catalase and glutathioneperoxidase of leaves were also amplified out.The cDNA sequences of Cu/Zn-SOD of Cucurbita pepo and Cucurbita moschata leavesand pulp were sequenced，and the accession of the cDNA sequence of Cu/Zn-SOD of Cucurbitapepo pulp was KF192818in GenBank. It was verified that the same gene exhibited differentialexpression in different parts of a plant.