Functional Characterization Of Terpene Synthase In Rice And Its Effect On Brown Planthopper

Among the three types of secondary metabolites of plants,which terpenoids are the most abundant.Terpenoids not only attract pollinators and seed carriers,but also act as antimicrobial agents against pathogens,and attract pest parasitoids or natural enemies of insects to indirectly defense against tissue damage caused by herbivores.The precursor intermediates of terpenoids including isopentenyl diphosphate and dimethylallyl diphosphate,which are synthesized by mevalonate pathway in the cytoplasm and 2-C-methyl-D-erythritol-4-phosphate pathway in plant plastids.The condensation of isopentenyl diphosphate and dimethylallyl diphosphate into farnesyl diphosphate and geranyl diphosphate.Then,terpene synthetase with GPP and FPP as substrates to produce many kinds of terpenoids.Terpene synthetase is a key enzyme in the synthesis of terpenoids.By regulating the expression of terpene synthetase,the release of terpenoids in plants can be affected,and thus regulate the defense response of plants.As one of the three important food crops in China,rice is an important staple food for China's population.Its genome sequencing has been successfully completed.There are many terpene synthetase genes in rice genome.Therefore,terpene synthetase genes were cloned and identified by prokaryotic expression,enzyme assay,subcellular localization,Spatial and temporal expression,and laboratory experiment.The main results are as follows:1.Prokaryotic expression of terpene synthetaseIn this study,six terpene synthetase genes,OsTPS1,OsTPS10,OsTPS13,OsTPS21,OsTPS30 and OsTPS35 were successfully cloned.The recombinant protein was obtained in vitro by prokaryotic expression.The enzyme assays was detected by solid-phase microextraction(SPME)combined with gas chromatography-mass spectrometry(GC-MS).OsTPS1 with GPP as substrate to produce monoterpene compounds,The main product is linalool,which has the activity of monoterpene synthetase;OsTPS10 with FPP as substrate to produce sesquiterpene compounds,The main product is ?-Cadinene,which has the activity of sesquiterpene synthetase;OsTPS35 not only with GPP as substrate to produce monoterpenes,the main product is terpinolene,but also with FPP as substrate toproduce sesquiterpenes,the main product is1,2,3,4,4a,7-hexahydro-1,6-dimethyl-4-(1-methylethyl)-Naphthalene,Thus,OsTPS35 has both monoterpene synthetase activity and sesquiterpene synthetase activity.2.Expression characteristics of terpene synthetaseThe expression of terpene synthetase gene has obvious tissue difference.The expression of six terpene synthetase genes,OsTPS1,OsTPS10,OsTPS13,OsTPS21,OsTPS30 and OsTPS35,detected by real-time quantitative PCR.The results showed that the expression of OsTPS1 was higher in leaf sheath and root at tillering stage;the expression of OsTPS10 in the leaves of the booting stage and the booting stage was higher,and the expression of OsTPS13 in roots at seedling stage,tillering stage and booting stage was higher.OsTPS21 was expressed in seedling,tillering and booting stages,but was relatively high in seedling leaves.The expression of OsTPS30 was the highest in the leaves of the seedling stage;and OsTPS35 expression was higher in leaf sheath at tillering stage and booting stage.3.Subcellular localization of the terpene synthetase geneOsTPS1,OsTPS10,OsTPS21,OsTPS30 and OsTPS35 were detected by subcellular localization.The results showed that they were all localized to the plastids.4.Laboratory behavioral experimentThe experiment of H-type olfactory apparatus showed that brown planthopper(Nilaparvata lugens)significantly selected tps10 overexpression plants,but had no significant selection for tps10 and tps35 mutant plants with gene knock-out.The volatiles of the mutant and overexpression plants were detected by GC-MS.The results showed that there was a significant difference in the tps10 overexpression plants before the feeding of the brown planthopper.There was no significant difference in the volatile content of the tps10 and tps35 mutant plants with knock-out.After the feeding of the brown planthopper,nine volatiles in the tps10 mutant plants with knock-out were found to be significantly lower than those of wild-type plants.