Cloning And Functional Characterization Of Chilo Suppressalis And Nilaparvata Lugens Inducible Promoters Of Rice

During the long-term coevolution of plants and herbivorous insects,a variety of complex but delicate defense reactions have been gradually formed to resist insect damage.Among them,jasmonic acid(JA)signal pathway and green leaf volatiles(GLVs)are the two most popular plant defense responses.Allene oxide synthase(AOS)and hydroperoxide lyase(HPL)catalyze the production of JA and GLVs on the same substrate,respectively.AOS and HPL show high specificity in response to different insect damage,which affects the production of JA or GLVs,and then affects the plant choice of defense response.However,no research has been reported on the regulated mechanism of AOS and HPL to insect damage.Therefore,this study intends to clone the promoters of rice AOS and HPL,and explore the response patterns of the two promoters to sucking herbivore brown planthopper(Nilaparvata lugens St?l,BPH)and chewing herbivore striped stem borer(Chilo suppressalis Walker,SSB),combined with the 5' truncated assays,GUS activity analysis,and histochemical GUS staining to identify functional regions in Os AOS and Os HPL promoters that respond to the damage of BPH and SSB.Our results will provide new evidences to reveal the internal reason of rice specific defense response to different mouthparts insect.Furthermore,The cloned functional fragments in this study could be used to drive the insect-resistant genes to explore the application potential of these promoters in breeding insect-resistant transgenic crops.This study achieved the following research results:1.Os HPL2 is highly expressed by the infestation of SSB,but its expression cannot be induced by wounding,BPH infestation or phytohormones(jasmonic acid,salicylic acid,abscisic acid).The isolated full-length promoter of Os HPL2 is located at the translation initiation site ATG(+1)to upstream 2009 bp.The full-length promoter(PHPL2)driven GUS reporter gene was then cloned into expression vector(DX2181)(PHPL2)and transformed into Zhonghua 11,the GUS activity quantitative assay,histochemical GUS staining analysis and q RT-PCR were performed to detect the induced activity of promoter,the results showed that after infestation by SSB,PHPL2 has increased expression activity in the sheath,but no induction of the PHPL2 expression was detected by BPH infestation and wounding.In order to identify the damage response region of SSB in PHPL2,PHPL2 was gradually deleted from the 5' and transformed into Zhonghua 11,the results showed that the regions of-1452 to-1213,-903 to-624,and-376 to-176 contained positive regulatory elements response to SSB damage,of which-903 to-624 region showed the strongest inductive and activated ability in the Os HPL2 promoter.Subsequently,the three positive regions and one,two or three-903 to-624 region repeated sequences were respectively fused and linked with min 35S(P2R123-min 35 S,P2R2-min 35 S,P2DR2-min 35 S,P2TR2-min 35S).The whold promoter(P2)and above constructed promoters were respectively used to drive the expression of cry1 C,and transformed into Zhonghua11.Finally,the P2,P2R123-min 35 S and P2TR2-min 35 S promoters showed the highest expression of Cry1 C protein and SSB larval mortalities.P2,P2TR2-min 35 S and P2R123-min 35 S transformed rices also have the characteristics of normal seed setting rate and yield per plant.2.Os AOS1 is highly expressed by the infestation of BPH,but its expression cannot be induced by wounding,SSB infestation or phytohormones(jasmonic acid,salicylic acid,abscisic acid).The isolated full-length promoter of Os AOS1 is located at the translation initiation site ATG(+1)to upstream 1889 bp;The full-length promoter driven GUS reporter gene was cloned into expression vector(DX2181)(PAOS1)and transformed into Zhonghua 11,the GUS activity quantitative assay,histochemical GUS staining analysis and q RT-PCR were performed to detect the induced promoter expression activity,the results showed that after infestation by BPH,PAOS1 has increased expression activity in the sheath,but no induction of the PAOS1 expression was detected with SSB infestation or wounding.In order to identify the damage response elements of BPH in PAOS1,PAOS1 was gradually deleted from the 5' and transformed into Zhonghua 11,the results showed that the regions of-1573 to-1312,-979 to-647,and-426 to-181 contained positive regulatory elements for BPH damage induction,of which-1573 to-1312 region showed the strongest inductive and activated ability in the Os AOS1 promoter.Furthermore,the yeast one-hybrid and dual luciferase experiments proved that the-800 to-758 regions can bind to the LOC_Os01g1227 protein,and the-274 to-181 region can bind to the LOC_Os03g62790 protein.The results showed that LOC_Os01g1227 and LOC_Os03g62790 may be involved in the regulation of Os AOS1 expression induced by BPH damage.Summary:1.The Os HPL2 gene promoter and three positive regulatory regions inside were successfully isolated and cloned,which can be specificly induced by SSB infestation.The transgenic rice plants with Os HPL2 promoter and its positive regulatory regions driving cry1 C exhibited high insecticidal activity and fine seed setting rate and yield per plant.Therefore,the full-length Os HPL2 promoter and its positive regulatory regions have bright potential application prospects in developing new transgenic insect-resistant crops.2.The Os AOS1 gene promoter three positive regulatory regions inside were successfully isolated and cloned,which can be specificly induced by BPH infestation.Two binding proteins of the positive regulatory regions have also been identified.The application potential of the three positive regulatory regions needs to be further verified,and the interaction and regulatory mechanism between the positive regulatory regions and their binding proteins also need to be further studied.