The Expression Regulation Of Terpene Synthase Gene TPS10 In Zea Mays

As a staple crop and industrial raw materials, maize has an important role in national economy. Herbivore is one of the key factors to maize yield loss. The existing results showed that the maize terpene synthetases (TPSs) played a very important role in indirect defense against herbivores. When attacked by lepidopteran larvae, maize leaves emit a complex blend of volatiles mainly composed of sesquiterpenes to attract the natural enemies of herbivores. Maize has been established as a model system to study the indirect plant defense against herbivores during the past two decades, however, the molecular components controlling the complex transcriptional reprogramming in this process are still obscure.The promoter of TPS10 was the main research object in the study. The purpose was to elucidate the expression regulation mechanism of TPS10 gene. The results of quantitative real-time PCR (qPCR) showed that TPS10 gene was inducible in leaf by herbivore and MeJA, and its expression level affected by photosynthesis. The ris-elements in the 1727 bp TPS10 promoter were predicted in the PLACE database. There were many cis-elements in the promoter which related to plant defense, such as GCC-box, G-box, W-box. The results of GUS histochemical staining and enzyme activity analysis in transgenic Arabidopsis showed that the region from-300 to-200 of the TPS10 promoter was the promoter function sequence which had the herbivore-induced activity, GCC-box in the region was a key element to the TPS10 expression. Meanwhile, the sequencing results of 189 varieties of maize inbred lines revealed that the GCC-box was conservative in TPS10 promoter. A high-throughput screening of an Arabidopsis transcription factor library using the region from-300 to-200 of the TPS10 promoter as the bait identified seven AP2/ERF family transcription factors. Among their 17 close homologs in maize, EREB58 was the only gene responsive to herbivory, with a spatiotemporal expression pattern highly similar to that of TPS10. Subcellular localization analysis showed that EREB58 protein was localized in the nucleus. Yeast-one-hybrid (Y1H) assay and electrophoretic mobility shift assay (EMSA) showed that EREB58 specifically bound the GCC-box on the promoter of TPS10. The transient expression assays in Arabidopsis leaf protoplasts showed that EREB58 had transcriptional activation, it could bind to the GCC-box and positively regulates TPS10. Transgenic maize plants overexpressing EREB58 constitutivfy over-accumulated TPS10 transcript and also (E)-β-farnesene and (E)-a-bergamotene, two major sesquiterpenes produced by TPS10. In contrast, jasmonate induction of TPS10 and its volatiles were abolished in EREB58-RNAi transgenic lines. These results indicated that the transcriptional up-regulation of EREB58 was both necessary and sufficient for TPS10 induction and TPS10 volatile production in maize. In addition, we had also carried on the preliminary study on the promoter of TPS6. The results showed the region from-100 to-1 of the TPS6 promoter had the promoter activity. We would do detailed research on this region in the next step.All of the results in this study demonstrated the expression regulation mechanism of TPS10 gene that EREB58 bound to the GCC-box on the promoter of TPS10 and positively regulated TPS10 expression and TPS10 volatile production. It was great significance to parse the molecular regulation network of maize defense system and cultivate the insect-resistant maize varieties. Meanwhile, it provided a new method to study the molecular regulation network of defense system in other plant.