| Plant indirect defense system had been built through co-evolution between plants and herbivores,which is mainly based on the emission of a blend of terpenoid volatiles,one of secondary metabolites of plants,with the aim to repel herbivores and recruit predators or parasitoids.At present,the strategy used in crops for insect resistant was still dominated by direct killing.In this study,the idea of indirect insect-resistance was applied for the first time in rice to control pests by regulating the behavior of insects.The volatile sesquiterpene(E)-β-Farnesene(EBF)can repel aphids and attract the natural enemies of aphids,such as ladybirds.Previous work with a peppermint EBF synthase gene had demonstrated that transgenic rice lines FS2-6 and FS3-14 showed a significant repelling effect on bird cherry-oat aphid(Rhopalosiphum padi)compared with the control.In this study,EβF production were first verified from volatiles of transgenic rice FS2-6 and FS3-14 by gas chromatography-mass spectrometry(GC-MS),but the amount was little.Then we found the synthesis efficiency of EBF was mainly limited by the supply of substrates FPP in cytoplasm.As there are two independent pathways of isoprenoids pathway: the mevalonate(MVA)pathway in the cytoplasm and the 2-C-methyl-Derythritol-4-phosphate(MEP)pathway in chloroplasts,we used the promoter of rice rbc S gene and its chloroplast transit peptide to drive EBF synthase gene(EBFS)for expressing EBF synthase in the chloroplast.In this way,EBF could synthesize from FPP in chloroplast,which overcame the deficiency of FPP in cytoplasm,probably due to a higher plasticity of the FPP pool in chloroplast.After we obtained the transgenic rice,we analyzed the expression of EBF synthase,the release of EBF and its attraction effect to the multicolored Asian lady beetle(Harmonia axyridis).The main results were as follows:1.The volatiles of the transgenic rice FS2-6 and FS3-14 were collected by headspace air collection method,and EBF in volatiles was confirmed by GC-MS analysis.2.EBFS-MBP fusion protein was obtained by prokaryotic expression in E.coli using p MAL-c2 x vector.The specific antibody could be used for detecting the expression of EBFS protein in transgenic plants.3.The transformation vector p DT-EBFStp was constructed by driving a peppermint EBFS gene with the promoter of rbc S gene and its chloroplast transit peptide.After transformation,we obtained the transgenic rice with EBF synthase located in chloroplast.PCR analysis and Southern blot were then used to select the homozygous line TP103-20 with single-copy insertion.The isolated flanking sequence revealed that T-DNA in TP103-20 was inserted into an intergenic region of rice chromosome 2.4.The expression of EBFS protein in homozygous line TP103-20 was 17.6 times that of FS2-6 by Western blot analysis.5.Volatiles of T3 transgenic plants at the tillering stage were detected by GC-MS.It shown that the highest release amount of TP103-20 was 2.6 μg/day,which was 43.7 times that of FS2-6.6.The attracting effect of TP103-20 on Harmonia axyridis was confirmed in a Y-tube olfactometer.At present,we are verifying the repellance of TP103-20 against aphids.The morphology and function of plastid are able to change following developmental and environmental cues during the growth of higher plants,and those changes are regulated by coordinated expression of plastid and nuclear genes.A large number of expression profile date about nuclear genes obtained in rice,have deepened our understanding of nuclear gene expression,and made great contribution to rice functional genomics.In contrast,very little is known about plastid gene expression in rice.In this study,we first constructed the plastid gene expression profile covering the whole life cycle of rice by using a custom microarray,in order to analyze the dynamic expression of plastid gene in different organs at different developmental stages and investigate the similarities and differences of the plastid transcriptomics between rice japonica Nipponbare and indica Minghui 63.Moreover,several candidate regions that may produce the non-coding RNA had been found by screening the non-coding regions of the entire plastid genome.The main results were as follows:1.A custom Affymetrix Exon/Tiling microarray,Mono Orgs520782 F,was constructed for studying the organellar gene expression profile of 4 important monocot crops in Gramineous,including rice,maize,sorghum and wheat.190096 probes on the microarray covered all the coding region as well as the non-coding region of the organellar genomes.Thus,the microarray could be not only used to analyze the expression profile of the plastid gene or the mitochondrial gene in these four crops,but also could be used to discover non-coding RNA in the organelles.2.The plastid gene expression profile during the whole growth period of rice was obtained for the first time,by microarray hybridization assay with 23 rice tissues collected from different organs at different developmental stages.By analyzing different functional plastid genes,it was found that the expression pattern of photosynthetic genes and genetic system genes was significantly different.The applicability of our custom microarray for use in maize was confirmed by hybridization assay with 3 distinct tissues of maize.3.Comparison between rice japonica Nipponbare and indica Minghui 63 showed that the expression patterns of the main functional plastid genes were similar in these two rice cultivars.However,the expression profile in stamens was species-specific.Moreover,the expression patterns of atp A,clp P,ndh I,psb N,rpl2,rpl16 and rpl32 were significantly different among the two cultivars.4.Seven of plastid genes with high levels of gene expression in callus were identified in our study.They were rps3,rps8,rps19,rpl14,rpl16,rpl22 and clp P.Expression of clp P and rpl20 at the same operon in callus was not lower than in the leaves according to the Northern blot results.Therefore,cis-regulatory elements in promoters of these genes may be used for efficient plastid transgene expression in callus,which will help to apply the transplastomic technology in rice.5.Based on hybridization data of tiling probes in all tissues,36 novel non-coding RNA candidate regions were obtained.The intergenic regions between trnf M and trn G,ndh C and trn V,psa I and ycf4,and the antisense regions of ndh H were constitutive expression in Nipponbare and Minghui 63. |
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