Cloning And Functional Analysis Of A Multiple Stress-inducible Lehsp23.8 Promoter

The production of heat shock proteins (HSPs) is a universal response to high temperature exposure. The synthesis of HSPs is regulated by transcriptional activation and translational selection of heat shock transcripts.The expression of hsps is regulated mainly at the transcriptional level. This regulatory control occurs through the interaction between the"heat shock factors (HSF) and the cis-acting"heat shock elements"(HSEs). In this paper, an approximately 1.9 kb of Lehsp23.8 5'-flanking sequence was isolated from tomato genome. By using theβ-glucuronidase (GUS) reporter gene system, the developmental and tissue specific expression of the gus gene controlled by the Lehsp23.8 promoter was characterized in transgenic tomato plants. The heat-induced expression profiles of the Lehsp23.8::GUS were defined in transgenic tomato fruits at different developmental stages. Obvious GUS activities under cold, exogenous ABA and heavy metal (Cd2+, Cu2+, Pb2+ or Zn2+) stress conditions were also detected. Furthermore, series of the mutagenesis Lehsp23.8 promoter-gus expression vectors(pK1915, pK1327, pK871, pK565, pK255, pK∞AT14, pK△AT15, pK△AT34, pK△AT12 and pK*HSE)were constructed. In order to further define the functional cis elements in the Lehsp23.8 promoter, quantitative GUS assays were performed under multiple-stress conditions in transgenic tobacco plants.The main results were shown as follows:1. The 5'-flanking sequence of Lehsp23.8 was isolated from tomato genome. The Lehsp23.8 promoter (AB239774) was 1915 bp in length with several typical sequences, such as TATA box and CAAT box. A total of seven HSEs and several AT-rich regions were found in the sequence. Additionally, there are some transcription factor binding motifs related to stress response, such as ABA-responsive element [ABRE], C-repeat_DRE and activating protein binding sites [AP-1]. Electrophoresis mobility shift assay (EMSA) analysis showed that the purified HsfA2 protein bound specifically to HSEs of the Lehsp23.8 promoter in vitro, and bound strongly to the proximal five HSEs than to the distal HSE6.The fusion construction of Lehsp23.8::GUS was introduced into tomato. Southern blot results showed that the chimeric gus gene was integrated into the tomato genome with a single copy or two copies. At normal temperature, most of the tissues showed no GUS activity. The developmental and tissue specific expression of the gus gene controlled by the Lehsp23.8 promoter was characterized in transgenic tomato plants. Strong GUS staining was detected in the roots, leaves, flowers, fruits and germinated seeds after heat shock. The heat-induced GUS activity was different in the floral tissues at various developmental stages. Fluorometric GUS assay showed that the heat-induced GUS activity was higher in the pericarp than in the placenta, and it was the lowest in the locular gel. The heat-shock induction of the Lehsp23.8 promoter depended on different stages of fruit development. The optimal heat-shock temperatures leading to the maximal GUS activity in the pericarp of green, breaker, pink and red fruits were 42°C, 36°C, 39°C and 39°C, respectively. The heat-induced GUS activity in tomato fruits increased gradually within 48 h of treatment and weakened during tomato fruit ripening. Obvious GUS activities under cold, exogenous ABA and heavy metal (Cd2+, Cu2+, Pb2+ or Zn2+) stress conditions were also detected. The maximal GUS activity in the leaves under cold and exogenous ABA conditions was about one fourth as much as that after severe heat shock. These results show that the Lehsp23.8 promoter is characterized as strongly heat-inducible and multiple-stress responsive.2. The expression vectors (pK1915, pK1327, pK871, pK565 and pK255) were constructed carrying the gus gene driven by the 5'deletion products of the Lehsp23.8 promoter. And the corresponding transgenic tobacco plants were generated. The GUS activity under stress conditions, such as heat, cold, exogenous ABA and heavy metal (Pb2+), was characterized in different transgenic tobacco lines.High levels of heat-induced GUS staining were detected in the leaves, shoots, roots and different developmental flowers of the pK1915 transgenic tobacco plants. The result of fluorometric GUS assays showed that the heat-induced GUS activity of the 565 bp promoter was the strongest, while that of the 255 bp promoter was the lowest. The heat-induced GUS activity mediated by the 1915 bp, 1327 bp or 871 bp promoter was similar, and it was lower than that of the 565 bp promoter. Deletion analysis showed that the five HSEs within the 255 bp upstream of the Lehsp23.8 gene were necessary for the heat-inducible activity of the promoter. The AT-rich regions between -255 bp and -565 bp in the Lehsp23.8 promoter served as enhancers for the heat-induced expression of the chimeric gene. And the two HSEs between -871 bp and -1915 bp were not necessary for enhancing the Lehsp23.8 promoter activity.The chimeric gene with the 565 bp promoter still can be induced by cold, heavy metal and exogenous ABA. The putative elements C-repeat_DRE, AP-1 and ABRE in the Lehsp23.8 promoter were not involved in the stress response to cold, heavy metal and exogenous ABA. The proximal HSEs and AT-rich regions may, to some extent, contribute to the multiple-stress responsive character of the Lehsp23.8 promoter.3. To analyze in detail the role of proximal HSEs and AT-rich regions in the Lehsp23.8 promoter under multiple stresses, the expression vectors (pK∞AT14, pK△AT15, pK△AT34, pK△AT12 and pK*HSE) were constructed carrying the gus gene driven by the mutagenesis Lehsp23.8 promoter. And the corresponding transgenic tobacco plants were generated. The GUS activity under stress conditions, such as heat, cold, exogenous ABA and heavy metal (Pb2+), was characterized in different transgenic tobacco lines.Construct pK∞AT14 containing repeated AT-rich1-4 region showed higher GUS expression level than construct pK871, but lower than construct pK565. The result showed that the 331 bp sequences between -565 and -235 bp had enhancer activity. However, it restrained the GUS expression when the distance becomes farther from the core promoter elements. The deletion of internal region AT-rich1-2 or AT-rich3-4 significantly affected the heat-induced GUS expression, which suggested that AT-rich1-2 and AT-rich3-4 had important enhancer activity. Promoter harboring the mutagenesis HSEs lacked the heat-inducible activity. In addition, repeating AT-rich1-4 region decreased the transgenic silence ratio while deletion of the internal AT-rich1-2, AT-rich3-4 or AT-rich1-5 resulted in improved transgenic silence ratio.The chimeric gene with construct pK∞AT14 or pK△AT34 still can be induced by cold stress, while that with construct pK△AT12, pK△AT15 or pK*HSE can't. The result showed that AT-rich1-2 and HSEs played an important role in the chimeric gene expression under cold stress, while AT-rich3-4 didn't do.The chimeric gene with construct pK∞AT14, pK△AT12 or pK△AT34 can be induced by exogenous ABA and heavy metal stresses. However, construct pK△AT15 and construct pK*HSE lacked the chimeric gene expression in the transgenic tobacco plants under exogenous ABA and heavy metal stresses. It might have the similar pathway in the response to exogenous ABA and heavy metal stresses of the Lehsp23.8 promoter.The innovations in this dissertation were shown as follows:1. It was the first time to clone the multiple stress-inducible Lehsp23.8 promoter from tomato. By using the gus reporter gene system, the developmental and tissue specific expression of the gus gene controlled by the Lehsp23.8 promoter was characterized in transgenic tomato plants. The results also proved the multiple-stress responsive character of the Lehsp23.8 promoter.2. It was the first time using the gus reporter gene system to analysize the heat-induced expression profiles of the HSP in tomato fruits. The heat-induced expression profiles of the Lehsp23.8::GUS were defined in detail in transgenic tomato fruits. We proved that the heat-shock induction of the Lehsp23.8 promoter depended on the stages of fruit development.3. It was the first time to define the functional cis elements in the Lehsp23.8 promoter. Our results proved that AT-rich regions both serve as important enhancers in the expression of Lehsp23.8 and can influence the transgenic silence ratio. It was the first time to prove that AT-rich regions and HSEs interact with each other in the expression of Lehsp23.8 under low temperature stress condition.