Cloning Of The Root-specific Expression Genes In Rice (Orvza Sativa L.) And Analysis Of Their Promoters

Gene promoters direct gene expression in spatial, temporal patterns. There have been intensive efforts to study the molecular structure of promoters, including site, types and numbers of cis-elements scattering over the promoter, and the proteins that interact with cis-elemetns. Plant roots are importance of nutritions and water absorption, storage, transportation. Root-specific promoters are important to develop root-specifically driving gene expression for nutritions and water absorption.We found seven genes that highly expressed only in root tissues of rice (Oryza sativa L.) by rice microarrays analysis. RT-PCR and qRT-PCR analysis revealed that the genes of Os03g01700and Os02g37190were exclusively expressed at high levels in rice root. According to annotation in TIGRE, Os03g01700locating on chromosome3(431723-430210bp) with1419nucleotides in genomic sequence length encode a citrate transporter, which has two exons and an intron. Os02g37190locating on chromosome2(22472507-22471989bp) with519nucleotides in genomic sequence length is an unknown gene without intron.To confirm the root-specific expression patterns of the two genes, the3500bp promoter upstream regions from ATG of Os03g01700and Os02g37190were isolated by PCR, respectively. The activity of the corresponding root-specific promoters was quantitatively analysed in transgenic rice plants using the GUSPlus reporter. Deletion from the5'end of Os03g01700and Os02g37190were then investigated. The Os03g01700promoter was also used for root-directed overexpression of a low-affinity phosphate transporter gene OsPT2. The results are summarized as follows:1. RT-PCR analysis for different tissues revealed that the two genes were expressed only in root. The qRT-PCR analysis further confirmed a root specificity pattern of the genes. These results demonstrated that Os03g01700and Os02g37190were exclusively expressed at high levels in root tissues throughout the developmental stages in comparison with OsCcl, OsActl, and OsUbil, previously characterized as strong constitutive genes.2. To determine the root-specific expressions of the two genes, we used the modified β-glucuronidase (GUSPlus) reporter gene fused to3500bp upstream region of Os03g01700and Os'02g37190genes respectively and introduced into rice to determine histochemical localization. GUS staining shows that the Os02g37190and Os03g01700promoters were found to be active in primary and secondary roots, but not in root hairs and root cap. Horizontal sections of epoxy resin-embedded root demonstrated that the GUS staining in the epidermis (Ep), cortex (Co), endodermis (En), phloem (Ph), xylem (X) and lateral root primordium cells. No GUS staining were detected in root-shoot base, leaf, stem, seed, glume; pistil and stamen. Overall, the results indicate that the Os02g37190and Os03g01700promoters are root-specific promoters that are highly active in rice roots.3. Studies of GUSPlus expression that is controlled by deletion derivatives of the Os02g37190or Os03g01700promoters in transgenic rice were conducted. Ten subclones of Os02g37190and Os03g01700promoter deletion fragments into pBI101.3were developed, including3p-312GUSPlus,3p-561GUSPlus,3p-1077GUSPlus,3p-1475GUSPlus,3p-2013GUSPlus,2p-293GUSPlus,2p-560GUSPlus,2p-1023GUSPlus,2p-1521GUSPlus and2p-2004GUSPlus. The GUS activities were tested by transient expression assays in callus and in transgenic rice plants. GUSPlus expression that is controlled by deletion derivatives of the Os03g01700promoter in transgenic rice were detected GUS activities.3p-2013bp:GUS and3P-1077bp:GUS showed the root-specifically activityes. These suggested that other cis-acting elements apart from RSEs may be present in-2013bp～1475bp and-1077bp～-561bp of Os03g01700promoter sequences. GUSPlus expression that is controlled by deletion derivatives of the Os02g37190promoter in transgenic rice were undetected GUS activities. The results implied the transcription start site (TSS) of Os02g37190may be properly positioned in upstream of2004bp and other cis-acting elements apart from RSEs in upstream of2004bp may be responsible for the root-specific expression.4. The construction consisting of the transactivator gene GAL4-VP16controlled by the root-specific promoter Os03g01700and OsPT2controlled by the upstream activation sequence (UAS; cis-element to GAL4) was used in this study to transform rice (Oryza sativa L.). Analysis revealed OsPT2controlled by the root-specific promoter Os03g01700was expressed only in root tissues of transgenic rice. Under Pi-sufficient conditions (10mg Pi L-1), the Pi concentration in the shoots of the transgenic plants root-specific expressing OsPT2was increased approximately two to three fold, but was approximately similar in roots compared with wild-type plants. These results indicate that root-directed OsPT2overexpression increases Pi uptake and translocation of Pi from roots to shoots, resulting in the accumulation of excess shoot Pi under abundant Pi conditions.