Functional Identification Of Rice Limonene Synthase

Terpenoids are the most diverse species of secondary metabolites in plant,some of which are induced in response to biotic or abiotic stresses.Terpenoids play important role in direct and indirect defense of plants,and therefore have aroused people's wide concern.As rice is one of the main foods in the world,rice diseases and insect pests are important factors affecting the yield of rice.The use of terpenoids for green sustainable defense protection in rice is the future direction for rice diseases and pests control.In previous study,microarray data of rice seedlings inoculated with brown planthopper,showed TPS19 and TPS20,are up-regulated by brown planthoppers.And by in vitro enzyme experiments early,TPS19 and TPS20 were verified to be limonene synthase genes.In this study,we wanted to carry out the biological function analysis of rice lemonene synthase gene TPS19 and TPS20 in rice.This work provides a theoretical reference for cultivating new type of insect-resistant and pest-resistant rice.The main results are as follows:1.Over-expression vector of TPS19 and CRISPR-cas9 vectors of TPS19 and TPS20 were transformed into rice variety ZH11.By PCR and Southern blot,the stably integrated and highly expressed transgenic plants,as well as knockout mutant plants were obtained and chosen for further study.2.The volatiles of over-expressed TPS19 and TPS20 plants were collected by headspace adsorption and the components were analysed by GC-MS:limonene is the major product.Subcellular localization results showed that both TPS19 and TPS20 were localized in plastids,indicating that both TPS19 and TPS20 are monoterpene synthase.The above results demonstrate that both TPS19 and TPS20 function as a limonene synthase in rice.3.The H-tube test and electrical penetration graph of rice brown planthopper were carried out on the T₁ generation of TPS19 and TPS20 over-expression plants.The results showed that over-expressed transgenic rice had no significant effect on the host selection and feeding behavior of brown planthopper.4.The over-expressed TPS19 and TPS20 T₁ generations were tested for the resistance ability against Rhizoctonia solani Kuhn in vitro.The results showed that the over-expressed transgenic rice are more resistance against the Rhizoctonia solani Kuhn to a certain extent,but it was greatly affected by environmental conditions.5.TPS19 and TPS20 have high similarity in both CDS and amino acid sequences,but their tissue expression patterns are different:expression of TPS19 is mainly in the three-leaf stage seedlings,stems,panicle,and palea/lemma,while expression of TPS20was mainly expressed in the three-leaf stage seedlings,shoot,stems,palea/lemma,and spikelets.Based on the GUS staining of the promoter,it was found that both TPS19 and TPS20 were mainly expressed in rice sheath and leaf.6.A yeast one-hybrid vector was constructed,screening of the TPS19 promoter plasmid library and found the transcription factors LOC_Os01g39310,LOC_Os06g41010,LOC_Os07g35870 and LOC_Os09g12770.