Cloning And Functional Verification Of Vascular Bundle-specific Promoters In Rice

Promoter is an important regulator at transcriptional level.In recent years,tissue-specific expression promoters have gradually become a research hotspot with in-depth studies on plant genetic engineering.By using tissue-specific promoters,we can avoid encountering some of the negative effects when using constitutive promoters to drive gene expression.Rice is one of the important food crops,as a model plant,the whole genome of which was sequenced.The purpose of our study was to find and clone the vascular bundle-specific promoters from T-DNA mutants of rice by staining specific tissue with GUS,and to characterize these cloned promoters.The experssion of target genes was usually controlled by tissue-specific promoters in rice.This study is benefited for genetic engineering by exploring the vascular bundle-specific promoters in rice.The main contents and results of this study were as follows:1.111 strains were screened out from T-DNA mutants(enhancer trap system)by GUS staining in Nipponbare.And the offsprings of 72 were stained stably in vascular bundle sheath.2.The T-DNA locations of 17 strains were detected by using hiTAIL-PCR and studied upon sequence alignment on NCBI Web.3.After analysis of the DNA sequences with Ensembl Plants,seven insertion sites were found within the structural gene regions,and they had the same direction as the inserted GUS gene.According to the sequeces of structural genes,their upstream sequences of promoters were botained from NCBI database.In addition,Plant CARE analysis of promoter sequences revealed that there were many cis-acting elements in each sequence.4.Seven promoters were successfully cloned,and their expression vectors were constructed,loading with cloned promoters and the GUS reporter genes.5.Rice and tobacco,as the transgenic acceptors were used to verify seven expression vectors of promoters by GUS staining.And the results showed that the promoter of S23 P,coming from the gene of Os02g0752800,had the high activity of constitute promoters in transgenic tobacco and rice.Whereas,the promoter of S10P,from gene of Os01g0699100,regulated the GUS expression in vascular bundle.