| Aphids (Aphididae) are major agricultural pests which cause significant yield losses of crop plantseach year by inflicting damage both through the direct effects of feeding and by vectoring harmful plantviruses. Each year, around62.5%of the26million hectares of the Chinese wheat growing area sufferedsevere aphid infestations, causing15-30%, and sometimes even as higher as50%substantial yieldlosses and great economic losses to the farmers. Along with the application of nitrogen fertilizer andelevation of atmospheric CO2concentration, aphid infestation becomes more serious. For many crops,insecticides provide a simple and effective strategy for aphid control. However, the application of suchchemicals is not desirable in the long term, because of the development of insecticide resistance and thepotential negative effects on non-target organisms, and the need for more sustainable agriculturalpractices with fewer external chemical inputs. Conventional breeding programs have been undertakenand considerable efforts have been expended in the search for aphid resistance in small grain germplasmworldwide. However, due to the complexity of plant-aphid interactions and the rapid development ofresistant pest biotypes, outbreak of aphids causing substantial losses are reported regularly. Breedersand growers are still struggling to find an efficient strategy for aphid control in wheat. Development ofaphid-resistant wheat through genetic engineering would be a good alternative strategy.Sesquiterpenoid produced from Mevalonate (MVA) pathway, is crucial in plant-herbivoreinteraction, involved in plant defensive responses. Farnesene pyrophosphate synthase (FPS) is the thekey enzyme in terpene biosynthetic pathway, whose catalytic product farnesene pyrophosphate (FPP) isthe substrate of (E)-β-farnesene synthase in MVA pathway.(E)-β-farnesene, a colorless, odorlessvolatile and one of the most important sesquiterpenoid, is the only component of alarm pheromone ofSitobion avenae Fabricius, Rhopalosi phum padi Linnaeus, Metopolophium dirhodum Walker andSchizaphis graminum Rondani, which could cause repulsion of aphids and also the attraction of naturalenemies and thus minimiz aphid infestation. Many plants contained the EβF synthase could release EβFand had the natural capacity of repellence to aphids. There is only FPS, but no EβF synthase, availiblein wheat, cotton, soybeans and other agronomic important crops, therefore, it was very valuable tounderstand the characteristics, distribution and supply situation of the FPS in the wheat and to create anew generation of genetically modified wheat which was aphid resistant and releasing aphid alarmpheromone, by regulation and improvement of the terpenoids metabolic pathways in wheat. In thisstudy, we isolated Tafps genes from common wheat for the first time, their sequence structure andexpression characteristics, enzyme catalytic activity, aphids’ resistance to the transgenic plants and soon were investigted thoroughly. The results are as following.1. Isolation, chromosome location and analyses on sequence structure of Tafps. Six FPP synthasegenes (Tafps-A1, Tafps-B1, Tafps-D1, Tafps-A2, Tafps-B2, Tafps-D2), which belong to two isoformswere isolated from common wheat variety Kenong199by homologous cloning for the first time. Themembers belong to the first isoform are assigned to the long arm of group3chromosomes, whereas the others belong to the second isoform were resided in the long arm of group1chromosomes. All thecDNA and genomic DNA of each gene were obtained, and three copies of each gene were predicted byrandomly sequencing. The similarity of each member of Tafps1and Tafps2was99.59%and98.39%,respectively. Genomic DNA of Tafps1contained11exons and10introns, and Tafps2contained12exons and11introns.The similarities of Tafps-B1and Atfps1, Tafps-B2and Atfps2was61.13%and67.62%, respectively. The similarity of Tafps-B1and Osfps1, Tafps-B2and Osfps2was87.98%and82.93%, respectively. Bioinformatics analysis and prediction results showed that wheat Tafps gene wasdivided into two isoforms, and had the typical two isopentenyl transferase functional domains.2. Analyses on the expression profiles of Tafps. Analyses of the expression profiles of the twoisoforms by real-time quantitative PCR indicated that the two isoforms expressed spatial and temporal,the expression levels of Tafps1were relatively higher in the green organs (including leaf, stem andglume) and the expression levels of Tafps2were relatively higher in the non-green organs (includingroot, influence and mature embryo) at the seeding, jointing and mature stage; whereas they remained thesame at earing stage. The expression levels of the two isoforms could be induced by aphids’ infestation.The expression levels of both genes was decreased in the root and increased in the leaf and stem atseeding stage with the level of Tafps1was significantly enhanced in the leaf（P<0.05） whereas that ofTafps2decreased in the roo（tP<0.01）. At earing stage, the level of Tafps1was significantly increased inthe lea（fP<0.01）and the stem（P<0.05）, a slightly increase in immature embryo, a significant decreasein glume（P<0.05）and a slightly decrease in the root after aphid infestation; the level of Tafps2wassignificantly enhanced in glume （P<0.001）and leaf（P<0.05） and a slight increase in the rest organsafter inoculation. Thus, it was speculated these genes was involved in the defensive response of wheatto aphid infestation.3. Analyses on enzyme catalytic activity and subcellular location of Tafps.After expression ofTafps-B1and Tafps-B2in protokaryon cell, TaFPS-B1and TaFPS-B2were purified by His-Tag andtheir catalytic activities were investigated by adding their substrates geranyl pyrophosphate (GPP) andisopentenyl pyrophosphate (IPP). After alkaline phosphatased, farnesol was detected by GC-MS, provedthe activity of farnesyl pyrophosphate synthase, with the activity of TaFPS-B1was about twice higherthan that of TaFPS-B2. After fusion Tafps-B1and Tafps-B2with GFP and transformed into Arabidopsismesophyll protoplasts induced by PEG, the fusion proteins hGFP with TaFPS-B1and TaFPS-B2weredetected in cytoplasm by confocal laser scanning microscope, corresponding with the fact that FPS isinvolved in the mevalonate (MVA) pathway in the cytoplasm.4. Functional analyses of Tafps in Arabidopsis. Three Tafps-B1and one Tafps-B2overexpressionplants, six Tafps-B1transgenic Atfps1mutants and three Tafps-B2transgenic Atfps2mutants wereobtained by floral dip method mediated with Agrobacterium. By contrast with wild-type Arabidopsis,β-Caryophyllene increased significantly in Tafps-B1and Tafps-B2transgenic Arabidopsis at seedingsatge. The emmison of β-Caryophyllene in Tafps-B1transgenic Arabidopsis was130.30ng per plant perday and5.5times higher than wild-type; the emmison of β-Caryophyllene in Tafps-B2transgenicArabidopsis was87.68ng per plant per day and3.7times higher than wild-type by GC-MS analysis. However, compared with wild-type, Atfps1mutants and Atfps2mutants decreased46.5%and53.3%seperately. These results indicated that fps was a key gene in the synthesis of sesquiterperen. Using the4-way olfactometer bioassay to detect the effects of the volatiles from transgenic Arabidopsis atflowering time on alate aphids (Myzus pesicae) behavior, the result of single factor experiment showedthat, in contrast with wild-type, transgenic Tafps-B1and Tafps-B2plants had significantly repellenteffect on aphids（Tafps-B1, P<0.01; Tafps-B2, P<0.05）; two choice experiments of4-way olfactometershowed that compared with wild type Arabidopsis, Tafps-B1transgenic Arabidopsis had significantrepellent effect on aphids（P<0.05）, Tafps-B2transgenic Arabidopsis also repelled aphids but notsignificant at statistical level. In addition, Atfps1and Atfps2mutants Arabidopsis were attracted to alateaphids and complementary plants showed faintly repellence to aphids. All the results demonstratedTafps has similar function as Atfps and over-expression of Tafps in Arabidopsis enhanced the repellenceto Myzus pesicae with Tafps1performed better than Tafps2.For further studies, crossings between transgenic Arabidopsis with Tafps-B1, Tafps-B2andtransgenic Arabidopsis with EβF synthase gene isolated from Mentha x piperita have been done todefine if the emission of EβF will be increased by over-expressing its substrate FPP. |
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