Cloning And Cis-acting Regulatory Element Signal Analysis Of The Promoter Regions Of Two Multiple-tillering Genes, MT1 And MT2, In Japonica Rice (Oryza Sativa L.)

Rice is a leading cereal crop and staple food for over half of the world population. Tillering in rice is one of the most important agronomic traits that determine grain yield. Tillering influences the number of spikes which has effects on the yield per unit directly. Cloning and functional analysis of genes controlling rice tillering is the theoretical basis of the yield improving via molecular techeniques. Eukaryote gene expression regulation is one of the hotspots in molecular biology field. The procedure, time and position of gene expression are regulated in various levels by different factors, which decide the expression level and the spatio-temporal order. As an important regulatory element on transcription level, promoter is precisely controlled in the spatio-temporal gene expression of plants. Therefore, isolation, identification and functional investigation of promoter have become a very pop research orientation.In this study, two rice mutants, mt1-1 and mt1-2, characteristic of strong-tillering, were found in the progeny of Zhonghua 11 (Oryza sativa L.subsp.japonica) treated with 0.1 %EMS. MT1 was located in the long arm of rice chromosome 1 firstly, and then based the fine mapping, it was located on a BAC contig, AP003376, with the region being limited to about 30kb including only one ORF(Open Reading Frame) which was considered as the candidate gene of MT1. And the complementary test was employed to identify it as MT1 gene.Additionally, through BLASTp (NCBI), a new gene was found to be highly homologous to MT1. So the gene was tentatively designated as MT2 with its BAC contig as AP003141. The result of ClustalW (http://www.ebi.ac.uk/clustalw/) suggested that the AA homology parameter was up to 63% between MT1 and MT2. Therefore, we focused our interest on the promoter region of these two genes to find something in common or different by analyzing promoter regions.PCR and enzyme digestion were employed to clone the promoter regions of MT1 and MT2. Based on the cloning, we constructed 5'region deletion- GUS expression fused vectors which were introduced into Nipponbare via Agrobacterium tumefacien mediated transformation method.GUS staining results showed that, as to MT1 promoter, GUS expressed in young tissues (leaves, nodes and roots) of all constructed vectors with blue displayed and weaker expression was detected after heading. Weak blue could also be observed in young glumes and branches. Except for the same results to MT1 promoter, GUS expression controlled by MT2 promoter also could be detected in branches of mature seeds.GUS quantitive detection was applied to measure GUS activity. We compared GUS activities in young tissues (leaves, nodes and roots) of the constructs with the full-length promoters. As to MT1, GUS activity in node was the highest followed by that in leaf, and the activity in root was the lowest, while the order of MT2 should be changed as leaf, node and root, from high to low. GUS activities in nodes were also compared among constructs. The result of MT1 was pC1301/MT1P-GUS>pC1301/MT1psmaâ… -GUS >pC1301/MT1pkpnâ… -GUS>pC1301/MT1p-GUS, while the order of MT2 was pC1301/MT2P-GUS>pC1301/MT2psacâ… -GUS >pC1301/MT2pkpnâ… -GUS>pC1301/MT2p-GUS. And these results were estimated to be related to the up-stream sequences which always played important roles in gene expression. In accordance with PLACE results, we analyzed the functions of cis-acting regulatory elements controlled shooting and differences between them in detail. They were predicted to be functional here, and their numbers were of vital importance for gene expression degree. Meanwhile, total RNA of Nanjing 6 was extracted and specific primers were designed for RT-PCR. The results demonstrated that, gene expression of MT1 was detected in mature leaves, stem, node and spike, which was consistent to the results of GUS staining. As to MT2, the higher expression was detected in node and spike while lower expression was detected in stem and leaf. Additionally, the difference was displayed between the results of RT-PCR and these of GUS quantitative, and the accountable reasons were also discussed here.