Functional Identification Of Rice Homoterpene Synthase And Development Of Transgenic Rice With High Emission Of Homoterpene

Plant have evolved an indirect defense method to respond to pests by releaseing volatile organic compounds(VOC)to attract pests' natural enemies in their long-term "struggle" with herbivores since they cannot move.Since plants cannot move,s,an indirect defense method has evolved to deal with pests by releasing volatile organic compounds(VOC)to attract natural enemies of pests.Homoterpenes are the most widely distributed volatiles in plants.Homoterpenes play an important role in plant protection for that they can be released from the leaves injured by pests and attract the natural enemies of pests.This study is centered on these two homoterpenes and is mainly divided into three parts.In the first part,four exogenous genes(At CYP82G1*,PlCYP,PlTPS3 and Pl TPS4)in the metabolic pathway of homoterpene were transferred into rice individually or in pairs according to the TPS+CYP combination.Since the synthesis of homoterpene is mainly limited by the supply of their precursor of terpenes,and the content of terpenes is determined by the content of terpenes direct precursors(GPP,FPP,GGPP).So we transferred PlTPS4 gene fused with the chloroplast targeting signal peptide(ctp)or the mitochondrial targeting signal peptide(mtp)at the 5' terminus of coding sequence into rice,and obtained the transgenic rice which PlTPS4 located in chloroplasts with a high content of GPP and GGPP or in mitochondria with high FPP content,respectively.Then,Homozygous lines with high expression of the target gene were selected for volatile collection and analysis,and the attraction to the parasitic wasp Cotesia chilonis and its repellent effect on the brown planthopper were investigated.In the second part,a homoterpene synthesis gene OsCYP92C21(LOC?Os03g44740)which was upregulated by alamethicin(ALA),jasmonic acid,brown planthopper(BPH)and Chilo suppressalis(SSB)was newly identified in rice.Its function was determined by the identification of the reaction products with the substrate after expression in yeast,the analysis of the volatiles of OsCYP92C21 knockout rice mutants induced by ALA,and the analysis of volatiles of hybrid polymerized transgenic rice which both overexpression of OsCYP92C21 and PlTPS4.The third part is to find the potential transcriptional factors that regulate the expression of OsCYP92C21 gene with the techniques of yeast one-hybrid and DNA-pulldown by analyzing the promoter of OsCYP92C21 gene.The main results of this research are as follows:1)Aiming at four exogenous genes,eight plant overexpression vectors PTGH3-ctp*-AtCYP82G1*,PTGH3-rbcs-At CYP82G1*,PTGH3-AtCYP82G1*,PTGH3-PlTPS3,PTGH3-PlTPS4,PTGH3-ctp*-PlTPS4 and PTGH3-mtp-PlTPS4 were constructed and transferred into rice.Through Southernblot detection and q RT-PCR detection,the homozygous lines(ctp*-At CYP82G1*-4,24,rbcs-At CYP82G1*-11,13,AtCYP82G1*-6,7,PlCYP-8,9,Pl TPS3-2,19,PlTPS4-9,22,ctp*-PlTPS4-10,20,mtpPlTPS4-5,9)of single copy with high expression of target gene were obtained in progeny,respectively.2)There was no significant change in the content of volatiles related to homoterpene metabolism pathway in wild type rice plants ZH11,ctp*-At CYP82G1*,rbcs-At CYP82G1*,At CYP82G1* and Pl CYP transgenic rice.PlTPS3 transgenic rice not only increased linalool emission,but also detected a small amount of DMNT.The emissions of linalool and DMNT were increased from Pl TPS4,ctp*-PlTPS4 and mtpPl TPS4 transgenic rice plants.The emission of DMNT was highest when PlTPS4 was expressed in mitochondria,and the emission of linalool was highest when PlTPS4 was expressed in chloroplasts.Interestingly,a very small amount of TMTT was detected in ctp*-Pl TPS4-10.3)Four plant expression vectors,p YLTAC380DTH-PlCYP-PlTPS3,pYLTAC380DTH-PlCYP-Pl TPS4,p YLTAC380DTH-At CYP82G1*-PlTPS3 and pYLTAC380DTH-At CYP82G1*PlTPS4,were constructed and transferred into rice.Two lines with high expression of target genes were obtained by q RT-PCR detection,and the volatiles of these lines were detected by GC-MS.The emissions of DMNT and TMTT from transgenetic rice were increased.4)DMNT and TMTT can be detected in the volatiles of ZH11 with treatment of ALA,JA,BPH and SSB.It was confirmed that there was a homoterpene biosynthesis pathway in rice and can be induced by ALA,JA,BPH and SSB.5)Six CYP genes(OsCYP71AK2,OsCYP71C16,OsCYP89B1,OsCYP92A9,OsCYP92A11,OsCYP92C21)were selected as candidate biosynthesis genes from the comparative microarray analysis between the resistant rice variety Rathu Heenati(Oryza sativa L.ssp.indica)(RH)and the susceptible rice variety Taichung Native 1(Oryza sativa L.ssp.indica)(TN1)infested by the brown planthopper(BPH)and the comparative transcriptome analysis of the rice cultivar ZH11(Oryza sativa L.ssp.Japonica)before and after alamethicin-treatment.Among them,only the recombinant protein OsCYP92C21(LOC?Os03g44740)expressed in yeast could convert(E)-nerolidol and(E,E)-geranyllinalool into DMNT and TMTT,respectively.6)Compared with wild-type rice plants,the CRISPR/cas9 knockout homozygous mutants of OsCYP92C21 gene could not produce DMNT and TMTT with the treatment of ALA,indicating that OsCYP92C21 gene is indispensable in the synthesis of homoterpene in rice.7)There was no significant difference in the composition and content of volatiles between OsCYP92C21 overexpression plants and ZH11,indicating that the content of nerolidol and geranyllinalool(the precursors of DMNT and TMTT)may be very low,which could not provide enough substrate for the enzyme expressed by OsCYP92C21.High emission of homoterpene was required a high level of expression of the corresponding terpene synthase.On this basis,the hybridized polymerized rice PlTPS4-9/OsCYP92C21-4,Pl TPS4-22/OsCYP92C21-4,mtp-PlTPS4-5/OsCYP92C21-4,mtpPl TPS4-9/OsCYP92C21-4,ctp*-PlTPS4-10/Os-CYP92C21-4 and ctp*-PlTPS4-20/OsCYP92C21-4 were constructed.The volatiles of polymerized rice were collected and compared with wild type and single gene transgenic rice plants.The results showed that the emissions of DMNT and TMTT from doubl-gene polymerized rice plants were significantly higher than that of PlTPS4 single gene transgenic rice.The mtp-Pl TPS4-9/OsCYP92C21-4 line had the highest emission of DMNT(943 ± 22 ng plant-1day-1),which was 73–157 folds those of single-gene transgenic lines PlTPS4-9 and PlTPS4-22.The ctp*-PlTPS4-10/OsCYP92C21-4 line had the highest TMTT emission,which reached 179 ± 48 ng plant-1day-1 and was 179 folds that of ctp*-Pl TPS4-10 line.8)Y-type olfactometer test showed that there was no significant difference in the number of wasps attracted by OsCYP92C21-4 plant and the wild type ZH11 plant.The transgenic plants PlTPS4-9,mtp-PlTPS4-9 and ctp*-PlTPS4-20 attracted significantly more C.chilonis female wasps than the wild type ZH11 plants.The double-gene introgression lines PlTPS4-9/OsCYP92C21-4,mtp-PlTPS4-9/OsCYP92C21-4 and ctp*-PlTPS4-20/OsCYP92C21-4 were extremely more attractive to C.chilonis female wasps than the wild type ZH11 plants.9)Seven transcriptional factors that may be involved in regulating the expression of OsCYP92C21 gene were found by yeast one-hybrid and DNA-pulldown techniques.On this basis,we can obtain transgenic rice with high emissions of DMNT and TMTT,and provide theoretical and technical support for the future use of plant volatiles such as DMNT and TMTT to form crop-pest-natural enemy three levels of nutrition in the field to control pests.