Preliminary Studies On The Recognition Sequence And Downstream Targets Of The Gene Which Confers Submergence Tolerance In Rice

Like all crops, rice plants of course require water to grow. But you can not have too much of a good thing:when excessive water results in prolonged submergence, the effects on rice production can be devastating. Rice is an indispensable staple food, especially in the large areas of Asia, Latin America and Africa that are characterized by a semi-tropical climate with alternating rainy and dry seasons. Production in regions where rice cultivation is subjected to stresses such as the seasonal flooding that occurs during the monsoon season, particularly the lowlands of south, southeast and east Asia. Floods during and immediately following the rainy season can submerge young rice plants for several days, resulting in wilting and even death.Tolerance to complete submergence is mainly conferred by the Sub1A,Snokell and Snokel2 gene, which encode ethylene-response-factor (ERF) type transcription factors. They act downstream of ethylene and modulate gibberellin-mediated shoot growth, participating in two opposite adaptive strategies to submergence, i.e., elongation (escape) and inhibition of elongation (quiescence).This research mainly includes two parts:serial deletion analysis of SublA promoter and function analysis of Snokell and Snokel2 gene.SublA promoter deletion analysis:Analysis of SublA Promoter sequence through PLACE database revealed three important motif, Anaerol consensus site, GCC core site and EIN3 binding site. In order to obtain fragments of the SublA promoter containing different motif, Promega Erase - a - base kit was used to make a serial deletion of the SublA promoter. Plant expression vectors for analyzing temporal and spatial expression patterns of the SublA-1 gene were constructed in which 12 SublA promoter fragments of different size was used to drive the GUS reporter gene. These vectors have been transformed into rice cultivar Nipponbare. Analysis of GUS staining pattern of these transgenic rice plants under submergence will help to find out the important motif related submergence tolerance in the SublA promoter. Snokell and Snokel2 gene function analysis:To identify the direct responsive genes to SKI and SK2, we constructed two glucocorticoid-inducible gene expression vectors that contain the chimaeric transcription factor GVG driven by the Actinl promoter. When induced with glucocoriticoid hormones such as DEX, GVG is transferred to the nucleus where it will specifically initiate the transcription of the full-length SK1、SK2 cloned downstream of GVG recognition sites, and the direct responsive genes could be found by microarray analysis.To study the mechanism of the transcriptional factor SKI and SK2 in vivo, we constructed two plant expression vectors:ActinI promoter:SK1/SK2.SublA promoter:GUS vectors and glucocorticoid-inducible gene expression vectors were transformed into mature embryo-derived calli from a submergence-intolerant rice cultivar Nipponbare through Agrobacterium-medicated transformation method. Actinl promoter:SK1/SK2 vectors were transformed into d18 (GA synthesis mutants, GSOR300185) and Shiokari (WT, GSOR300192), with an empty vector controls. After two rounds of hygromycin selection and regeneration, hygromycin-resistant plants were obtained, which lay a foundation for the identification of the SublA promoter、SK1、SK2 binding sites in whole genome and the downstream target genes.GST-SKI and GST-SK2 fusion proteins were expressed in E.coli and purified via the GST affinity chromatography. These purified proteins provide the prerequisite for further studies that aim to find out the binding sites of SK1 and SK2 through random binding site selection (RBSS) and electrophoretic mobility shift assay (EMSA).