| Promoter plays a key role in the regulation of gene expression. It is important to achieve temporal and tissue-specific expression of the foreign gene(s) through promoter control. Some constitutive promoters such as the cauliflower mosaic virus (CaMV) 35S promoter and maize ubiquitin are widely used in the current commercial genetic engineering products, and usually result in high and constitutive expression of the genes in host plants. However, constitutive expression of the foreign gene(s) could be harmful to the growth and development of plants because overexpression can lead to metabolic stress. Therefore, application of tissue-specific promoter may increase the safety of genetically modified crops and has an important significance to expression efficiency of target gene. Several seed-specific promoters have been cloned from rape, but few promoters with vegetative organs specificity have been well characterized. In this study, a promoter was cloned from the upstream of the stem-specific expression gene SH4 in rapeseed, and integrated with the GFP reporter gene. A preliminary functional analysis of the pSH4 was made after transformed into Arabidopsis thaliana. The results would provide a valuable promoter for genetic engineering of rapeseed. The results were obtained as follows:1. By screening tissue-specific gene from Arabidopsis thaliana, 6 stem-specific genes were selected as candidates.2. From the rapeseed material Zhongshuang No.9 (in full bloom), RNA was extracted from 8 organs (root, stem, leaf, flower and so on) and quantitatively analysed for the 6 candidate genes. The candidate gene SH4 was shown to express in a stem-specific way.3. According to the known genome sequence (GenBank: AC189510.1) of B.rapa c.v.pekinensis, a 1779bp pSH4 or the 5'flanking sequence of SH4 was isolated. Bioinformatic analysis shows that pSH4 has a variety of cis-acting elements, including the stem tissue-specific elements CCA1ATLHCB1, DOFCOREZM, the hormones-inducable elements ASF1MOTIFCAMV, LTRECOREATCOR15 , physical induced elements GT1CORE,GT1CONSENSUS.4. To characterize the expression features of pSH4, plant expression vectors pBI121-pSH4-GFP was successfully constructed and used to transform Arabidopsis thaliana through the Agrobacterium-mediated method and with the pBI121-GFP as control. We observed the expression of GFP in T1 progeny, indicating that pSH4 is a vascular tissue-specific promoter. This study involved in the isolation and signal scan analysis of pSH4, verification of its tissue-specificity. All these data will give valuable materials and theoretical guidance to study and applification of pSH4 promoter.Genetically modified organisms (GMO) detection is essential for biosafety management but lack of positive control, which become a challenge and a bottleneck of GMO detection. In order to provide a general positive control for GMO screening, plasmid pBI121-ELEMENTS was constructed and confirmed. The pBI121-ELEMENTS contained 11 target elements including promoters (nos, CaMV 35S, FMV35S), terminators (NOS 3', T-35S, g7 3', E9 3') and selective markers (bar, NPT II, hpt II and pmi).The plasmid sequence was submitted to GeneBank and Accession number HM047294 received. These elements covered 91.5% of the worldwide approved transgenic events of major crops including soybean, oilseed rape, maize, cotton and rice. They also covered 100% of transgenic events that China approved. The positive plasmid was used to detect seven related elements and the results showed its effectiveness for GMO screening. |
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