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| Promoter Functional Analysis of RBCS11 Gene Participate in the Abiotic Stress Responsible in Wheat (Triticum aestivum) |
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Abstract Photosynthesis-associated nuclear genes (PhANGs) are able to respond to multiple environmental signals which include light, drought, sugar and abscisic acid (ABA). In previous studies, several cis-elements related to light regulation have been identified in promoters of PhANGs. However, systematic research on cis-elements of PhANGs which are response to abiotic stress, such as drought and ABA, has not been conducted yet. Here, we performed a series of investigation that mainly aimed at the cis-element that located on ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene (RBCS) promoter and roles in abiotic stress response. Firstly, a 1 839 bp upstream sequence of the TaRBCS11 gene was isolated from the genomic DNA of wheat (Triticum aestivum). Sequence structure analysis showed that the transcriptional start site was located at 59 bp upstream of initiation codon (ATG). Then, the sequence was submitted to the PlantCARE database and the analysis result revealed that the sequence contained large numbers of light-related cis-elements such as BoxⅠ, G-Box, I-box and GATA-motif, and dehydration, salt and ABA response-related cis-elements such as MYB binding site (MBS), CCAAT-box, ethylene-responsive element (ERE) and gibberellin-responsive element (GARE-motif). Based on these results, the promoter regions of TaRBCS11, TaRBCS13, TaRBCS10 and TaRBCS14 that from the same subfamily had been isolated from wheat genome. The alignment indicated that these 4 promoter sequences shared highly identity ranged from 50.9% to 74.17%. Many light-responsive motifs such as MNF1, GATA-motif, I-box, G-Box and Sp1 were also observed at the relatively same position in the proximal region (-200 bp~ATG) of these promoters. In order to obtain the promoter deletion fragment, five forward primers (F1, F2, F3, F4 and F5) and one reverse primers (R1) were designed according to the distribution of the structure and expression element of the TaRBCS11 promoter sequence. PCR amplification was conducted and 5 deleted fragments in length of 1 656, 1 258, 866, 533 and 277 bp were obtained, respectively. Subsequently, the 5 different length of deletion fragments of TaRBCS11 promoter were fused with the β-glucuronidase gene (GUS) and Agrobacterium tumefaciens-mediated transient expression assay were conducted. The histochemical staining of GUS showed that the leaves with 35S and TaRBCS promoter were stained blue which indicated that all promoters possessed activity to drive GUS. Meanwhile, quantitative analysis of GUS activity showed that GUS activities decreased gradually as the length of TaRBCS promoters decreased from P1656 to P277 in the leaves. The result also indicated that light induction could enhance GUS activities. Furthermore, the deletion of light responsive element such as ATCT-motif, Box I, Box 4, G-Box, and chs-CMA2b could reduce GUS activities. Assay of tobacco (Nicotiana tabacum) leaves which harboured the complete promoter in the fusion construct indicated that P1656 was negatively regulated by salt, ABA, dark and especially drought. The 5' deletion analysis of TaRBCS promoter confirmed that a region between -804 and -474 bp included the drought and salt-response region, and ABA-response elements might be located in the region between -1 597 and -1 198 bp. Taken together, these results identified some potentially important regulatory element in the TaRBCS11 promoter, and confirmed that the cis-regulatory region that responsible for repression by drought, salt, ABA and light. The results provide important information for further study on the mechanism of PhANGs expression regulation.
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Received: 26 January 2016
Published: 20 May 2016
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