Cloning And Identification Of Several Rice 3'-UTR Sequences

The existence of exogenous gene in Genetically Modified(GM)crops has sparked public's concerns about GM food and biosafety,the application of plant-derived genes especially the endogenous gene is the best way to eliminate public 's concerns.Furthermore,the most of GM regulatory elements which are now widely used are foreign patent will be a huge risk for our application of GM rice.We must have our own patent for cultivating GM varieties with independent intellectual property rights.Therefore,mining and using the plant's own genes and regulatory elements is the key to promoting the development and application of our GM crops.In this study,based on the constitutive expression system of rice and bioinformatics database of rice named "Rice Genome Annotation Project"(http://rice.plantbiology.msu.edu/expression.shtml),we obtained the chip of gene expression profile of the wild Nipponbare.After running data statistics analysis by excel,we screened preliminarily several constitutive expression genes as candidate genes.Analyzing the expression level of each candidate genes in stem,leaf,spike and other tissues via RT-PCR,we further screened seven constitutive genes with high expression level as target genes.Then we searched the 3'-UTR sequence of those target genes by NCBI,cloned the 3'-UTR DNA fragments by PCR and constructed the plant expression vectors driven by 35s and ubiquitin promoter of GUS gene to connect these seven 3'-UTR segments.For positive control,we used pCX35S-GUS-Tnos and pCXUbi-GUS-Tnos.We did preliminary validation of 3'_UTR of target genes via transient expression in tobacco.We obtained GM rice plants by Agrobacterium-mediated transformation.After doing GUS histochemical staining analysis on root,stem,leaf,spike and seeds of T1 transgenic rice,we found strong expression of GUS protein in root,stem,leaf,spike and seeds of transgenic rice which terminator is 3'-UTR.Comparing GUS protease activity of root,stem,leaf,spike and seeds of transgenic rice with Tons,we found that expression level in each part of 3'-UTR PL7 is higher than Tons.We further verified 3'-UTR of target genes in regulation of gene expression.