| Terpenoids(also known as isoprenoids)are natural products emit from a wide range of animal and plant species,which are widely distributed with important physiological and ecological functions.Terpenoids have various structures.A few of them are primary metabolites that regulate the growth and physiological functions of plants,and most of them play an important role as secondary metabolites in the regulation of inter species relationships.Terpenoids are one of the important components of Herbivore-induced plant volatiles(HIPVs),and mainly classified as monoterpenes,sesquiterpenes and homoterpenes.Plant can release a lot of terpenoid volatiles after being damaged by insects.Terpenoid volatiles play an important role in regulating plant-pest-natural enemy interaction and improving plant resistance.Aphids are the most economically important pests in wheat plant,which can affect the wheat photosynthesis,nutrient absorption and transportation.At the same time,aphids are also as vectors to spread a variety of plant virus diseases.After wheat heading,aphids are crowded in the ear,which result in unfulfilled Grain.The English green aphid Sitobion avenae(Fabricius),with high adaptability and productivity,seriously damage wheat at the ear stage.Under the condition of global warming,the damage of the aphid will be more serious.Breeding aphid resistant varieties is an effective way to control aphids.Recent studies showed that the terpenoids,such as S-linalool and DMNT in gramineous corn and rice can effectively improve the plant resistance to insects.In addition,methyl heptanone,the precursor of terpenoids with aphid resistance,has been identified in wheat plants.Therefore,in this study,the terpenoid volatiles were collected,isolated and identified from wheat germplasm Octoploid Tirtitrigia with and without S.avenae damages.And then the olfactory responses of S.avenae to terpenoids emitted from Octoploid Tirtitrigia were tested.In order to screen the synthase genes of the volatile terpenoids which showed resistance to the S.avenae,we conducted a de novo transcriptome analysis to compare RNA-seq profiles between the intact and damaged wheat plant ears of Octoploid Trititrigia.Finally,based on Chinese spring wheat genome,the terpene synthase gene family was analyzed by bioinformatics,including the identification of gene family members,evolutionary relationship,gene structure,protein structure and promoter element analysis.Expression analysis of Ta TPS genes in wheat damaged by aphids,could provide candidate genes for breeding of wheat resistance to aphid.Results are as follows:1.Terpenoid volatiles in the ears of Octoploid Trititrigia with and without S.avenae damage were identified.The results showed that S.avenae-damaged Octoploid Tirtitrigia emitted a higher amount of S-linalool,Ent-kaurene,(+)-delta-cadinene,(E)-nerolidol as compared to the intact plant.In addition,(E)-?-caryophyllene,??myrcene,(E)-4,8-dimethyl-1,3,7-nonatriene(DMNT)were new volatile terpenoids emitted by the damaged plant.2.The olfactory behavior of S.avenae to volatile terpenoids S-linalool was tested.The results showed that when S-linalool concentration was 1 ?g·ml-1 or 10 ?g·ml-1,it had a significant repellent effect on S.avenae.3.After de novo assembly and quantitative assessment,a total of 182348(74.8 %)unigenes were annotated by alignment with the public protein databases.Among these unigenes,2389 differentially expressed genes were identified between the intact and damaged ears of Octoploid Trititrigia.There are 2306 up-regulated genes and 83 down-regulated genes.The levels of transcripts encoding the TPSs biosynthesis,including Terpenoid backbone biosynthesis,Monoterpenoid biosynthesis,Diterpenoid biosynthesis,Sesquiterpenoid biosynthesis and cytochrome P450 of S.avenae damaged Octoploid Trititrigia ears were significantly higher than those of intact Octoploid Trititrigia ears.Ten randomly selected ten(terpene synthetase genes and modified enzyme genes)genes which expression levels significantly changed after damage by S.avenae were verified by RT-q PCR.The expression levels of these ten genes were consistent in the two methods.4.Candidate genes involved in terpenes biosynthesis were identified by showing the significant transcript changes between the intact and damaged plant ears of Octoploid Trititrigia.The transcript abundances of terpenes biosynthetic pathway-related genes were also positively correlated with the production of volatile terpenoids in the ears.A 1680 bp S-linalool synthase gene full-length open reading frame(ORF)of Octoploid Trititrigia was cloned.The predicted protein complete ORF sequence when compared with the S-linalool synthase gene of other species,contained an aspartate-rich region DDxx D motif.Its function was characterized as coordinating the divalent metal ions involved in substrate binding.Furthermore,the phylogenetic tree results indicated that the Octoploid Trititrigia S-linalool synthase and Tu NES1 are highly homologous.Prokaryotic expression analysis of the obtained S-linalool synthase gene sequence,Construction of prokaryotic expression using expression vector of p ET32a(+),S-linalool synthetase gene was digested with restriction enzyme and ligated to pet32a(+).The recombinant plasmid p ET32a-Ot LIS was successfully constructed.Then it was transferred into E.coli cell-free protein system for expression,and the soluble target protein was obtained.5.Terpene synthase(TPS)gene family is ubiquitous in eukaryotes and plays an important role in regulating various biological processes in plants,especially in pathogen and herbivore defense mechanisms.Also,little is known about this gene family in wheat.In this research,153 Ta TPS genes were identified and named according to their chromosome location.Ta TPS genes are randomly and unevenly distributed on chromosomes.Phylogenetic analysis of Ta TPS proteins and other homologous proteins corresponded to a six subgroups classification.The analysis of gene structure and motif showed that the Ta TPS gene was highly conserved in each subgroup,which suggested that whole genome duplication and segmental duplication events were the main expansion way of the Ta TPS gene family.A total of 111 homologous genes were identified,19 tandem duplication gene pairs with 40 Ta TPS genes and 91 homologous genes on different chromosomes were produced by segmental duplication.Expression profiles derived from transcriptome data results indicated that 30 Ta TPS gene expressions were significantly up-regulated,which distributed in TPS-a,TPS-b,TPS-c,TPS-e/f and TPS-g subfamilies.The number of TPS-e/f and TPS-g subfamily is the most.It is suggested that the Ta TPS genes may contribute to defense responses of wheat to pests such as aphids.The cis-regulatory elements(CREs)of Ta TPS gene were found to be diversified indicating that Ta TPS gene may participate in many physiological processes of wheat growth and development.Each Ta TPS gene contains at least two or more defense or hormone related CREs,suggesting an important role in wheat pest defense through hormone pathways.During the growth and development of plants,terpenoids play an important role in plant-pest interactions.Here,the terpenoids with defense effect on S.avenae were identified,terpene synthetase gene mining with direct or indirect defense against aphids in wheat germplasm resources was conducted.At the same time,through the study of Ta TPS gene family in Chinese spring wheat,it is useful to understand the evolution of TPS gene family while enriching TPS gene resources.In a word,the study of terpenoid synthase in wheat germplasm resources and wheat terpene synthase gene family in wheat could provide a basis for both breeding and genetic modification of the wheat varieties with resistant to wheat aphid. |
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