Constructing And Function Analysis Of 5'-Deletion Mutations In The Promoter Region Of The GT Gene Induced By Both MeJA And SA

Glycosyltransferase (GTs) are enzymes that attach a sugar molecule to a specific acceptor molecule, thereby creating a glycosidic bond. These enzymes are found in most living organisms. In plants, GTs produces important molecules including cell-wall polysaccharides, glycoproteins, and many different types of small molecules that have sugars attached to them. These molecules mediate a wide range of functions, from structure and storage to specific signaling. For understanding the functions and role of GTs, It is essentral to determine the negulatry elements of GTs gene promoter.In this study, the -1150~0 bp of 5'-upstream regulation region (just upstream ORF) of a novel GT gene cloned by our laboratory, whose expression is dually induced by MeJA and SA, was used to determine its regulatory elements. Several putative cis-acting elements, including TATA-box and some special manipulative elements, were found in the promoter sequence by method of PLACE analysis. Five 5'- progressive deletion mutations in the GT gene promoter region were linked to theβ-glucuronidase (GUS) coding region instead of the CaMV 35S promoter in pCAMBIA1301 vector respectively, thereby 5 recombinants, pGTPA, pGTPB, pGTPC, pGTPD and pGTPE, were constructed. With the help of Ti-plasmid of Agrobacterium tumefaciens, pCAMBIA1301 vector and pGTPA~ pGTPE plasmids were transferred into Nicotiana tobacum cv. Wisconsin 38 leaf discs respectively and the transgenic tobacco plants were finally obtained. Furthermore, the objective genes in transgenic tobacco genomes were checked by the method of PCR amplification and the GUS activities of the basal expression or treated with salicylic acid (SA) and methyl jasmonate (MeJA) in the transgenic plants and control were measured by histochemical staining and fluorometric analysis. The main results ware as follows:1. GUS activities ware detected in the leaves, leaf sheath of the transgenic plants with the five delection fragments and in the roots with four delection fragments of GT gene promoter except -220~0 bp fragments. It was showed that all of the five deletion mutations of the promoter could be transcribed but four of them, GTPA, GTPB, GTPC and GTPD, were transcribed with characteristic of spatial and temporal differeces. It was deduced that the sequence which controlled the spatial- and temporal- expression of the gene in root lay in -468~-220 bp.2. The leaf GUS activity of the transgenic plants with -524~0 bp fragment was the strongest, and the leaf GUS activity of the transgenic plants with the -468~0 bp was the second. Activity of the transgenic plants with the -1150~0 bp was the same as that with -800~0bp fragments, but both were much less than those with -524~0 bp and -468~0bp fragments. The activity of the transgenic plants with -220~0 bp fragment was the weakest. The results suggested that the sequence lain in -800~-524 bp of the promoter could restrain the expression, and there might be elements in -468~-220 bp, which could induce the gene expression.The leaf GUS activity of the transgenic plants with -524~0 bp fragment and -468~0 bp ware 4 and 7 times higher than that of the plants transformed with pCAMBIA1301 respectively. It was indicated that there might have an element within the regon of -524~0 bp or -468~0 bp of the promoter, which could basically enchance the initiation of the transcription downstream.3. GUS assay was performed after the transgenic plants were treated with SA and MeJA. The results showed that the gus gene expressions in each group of transgenic plants with same deletion mutation of promoter fluctuated greatly. GUS activity of the leaves treated with MeJA or SA in the transgenic plant A10 with -1150~0 bp fragment were 4 or 7 times higher than that of the leaves untreaed, respectively, it was suggested that there might be a MeJA- or SA- element within the -1150~-524 bp of the promoter. GUS activity of the leaves treated with MeJA in the transgenic plant with -564~0 bp fragment were a little less than that untreaded, which appeared that MeJA treatment had some negative effect on the transcription initiation of the -564~0 bp fragment promoter. The detail analysis of those functional sequences for the expression would redound to elucidating the transcription mechanism of this promoter as well as the inducing and antagonizing interactions between SA and MeJA.