普通小麦倍半萜合成途径关键基因Tafps的鉴定和功能分析
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摘要
蚜虫是危害小麦生产的主要害虫之一，据统计，我国每年小麦蚜虫危害面积可达1667万公顷，造成减产15-30%，严重时可高达50%。近年来，由于全球气候变暖、耕作制度变化等因素，使麦蚜的繁殖能力和适应性显著增强，其危害日趋严重。目前，麦蚜防治以喷洒农药为主，但大量使用农药，不仅对人畜有害，而且造成严重的环境污染。培育抗虫小麦品种是防止蚜虫危害的最有效途径，但由于现有小麦种质资源中缺乏有效的抗蚜基因，抗性机制尚不明确，常规育种难以奏效。通过基因工程的方法创制小麦抗蚜新种质已成为目前研究的重要课题。
倍半萜类化合物合成于MVA途径，在植物与植食性昆虫互作中发挥重要作用，参与植物的防御反应。法尼烯焦磷酸合成酶（farnesyl pyrophosphate synthase，FPS）是萜类化合物合成途径中的关键酶之一，其催化合成的产物FPP可在细胞质中通过MVA(Mevalonate pathway)途径作为[反]-β-法尼烯【(E)-β-farnesene，EβF】合成酶的底物。EβF是重要的倍半萜类化合物，是一种无色无味挥发物，是小麦麦长管蚜（Sitobion avenae Fabricius）、禾谷缢管蚜（Rhopalosi phum padiLinnaeus）、麦无网长管蚜（Metopolophium dirhodum Walker）和麦二叉蚜（Schizaphis graminumRondani）等蚜虫报警信息素的唯一成份，可以使蚜虫产生骚动、从植株上脱落，并吸引蚜虫天敌，有效控制蚜虫危害。研究发现，许多植物中含有EβF合成酶，能释放EβF，具有天然的蚜虫驱避特性。但小麦、棉花、大豆等重要农作物中，只含有FPS，但不含有EβF合成酶。因此，了解小麦中FPS的特性、分布与供应状况，通过调控和改良小麦中倍半萜类化合物代谢途径，创制释放蚜虫报警信息素的新一代抗蚜转基因小麦，具有重要意义和应用价值。本项目分离克隆了小麦Tafps基因，并对其序列结构特征、表达模式、酶活性及其对蚜虫的抗性等进行了研究。取得以下研究结果。
1、小麦Tafps基因克隆、染色体定位及其结构特征分析：以普通小麦品种科农199为实验材料，采用同源克隆的方法，克隆得到6个法尼烯焦磷酸合成酶基因(Tafps-A1，Tafps-B1，Tafps-D1，Tafps-A2，Tafps-B2，Tafps-D2)，分别定位于第三同源群（Tafps1）和第一同源群（Tafps2）染色体的长臂上。随机测序获得了Tafps1和Tafps2各成员的基因组DNA和编码序列全长，初步表明各基因分别具有3个拷贝。Tafps1各成员的gDNA均含11个外显子，10个内含子；而Tafps2各成员的gDNA均含12个外显子，11个内含子。Tafps1和Tafps2各成员内部相似性分别为99.59%和98.39%。Tafps-B1与拟南芥Atfps1和Tafps-B2与拟南芥Atfps2的相似性分别为61.13%和67.62%。Tafps-B1与水稻Osfps1和Tafps-B2与水稻Osfps2的相似性分别为87.98%和82.93%。对各成员进行生物信息学分析和预测，结果表明小麦Tafps基因分为两种同工型，具有2个异戊烯基转移酶功能域。
2、小麦Tafps基因的表达模式分析：通过实时荧光定量PCR，对小麦科农199中Tafps1和Tafps2两个同工型表达特性进行分析表明两同工型在小麦中的表达具有时空特异性，在苗期、拔节期及成熟期，Tafps1在绿色器官（包括叶片、茎和颖壳）中表达量相对较高，Tafps2在非绿色器官（包括根、幼穗和种子）中表达量相对较高；抽穗期两基因表达趋势趋于一致。蚜虫咬食可诱导两基因表达量的变化。蚜虫咬食后，两基因在苗期的变化趋势相同，在根中表达量降低，在茎及叶片中表达量升高，其中，Tafps1的表达在叶片中显著升高（P<0.05）。抽穗期，蚜虫咬食后，Tafps1的表达在叶片（P<0.01）和茎（P<0.05）中的表达量显著升高，在幼胚中轻微升高，在颖壳中显著降低（P<0.05），根中轻微降低；Tafps2在颖壳（P<0.001）和叶片（P<0.05）中的表达量显著升高，在其余器官中均有不同程度的上升。推测Tafps基因与小麦对蚜虫侵害的应激抗性反应相关，具有防御反应诱导的表达特性。
3、小麦Tafps基因的酶活性及亚细胞定位分析：在大肠杆菌中表达Tafps-B1和Tafps-B2基因，通过组氨酸标签分离纯化获得的目的蛋白，与前体化合物异戊基焦磷酸（isopentenylpyrophosphate， IPP)和香叶草基焦磷酸（geranyl pyrophosphate， GPP)一起进行体外催化反应，GC-MS分析去磷酸化后的产物，结果显示， TaFPS-B1和TaFPS-B2均可IPP和GPP形成法尼醇，证明Tafps-B1和Tafps-B2基因在原核细胞中具有FPP合成活性，但TaFPS-B1的活性较高，约为TaFPS-B2的两倍。采用PEG介导方法，分别将Tafps-B1和Tafps-B2与hGFP的融合基因，转化拟南芥原生质体。激光共聚焦显微镜观察表明，TaFPS-B1和TaFPP-B2与hGFP的融合蛋白出现在胞质中，进一步为其参与胞质中的MVA途径提供了佐证。
4、小麦Tafps基因的功能分析：农杆菌介导的浸花法获得转Tafps-B1过表达拟南芥植株3棵，转Tafps-B2过表达植株1棵，转Tafps-B1Atfps1突变体拟南芥6棵，转Tafps-B2Atfps2突变体拟南芥植株3棵。GC-MS分析野生型、过表达、突变体以及互补表达拟南芥植株的挥发物。结果表明，与野生型拟南芥相比，苗期Tafps-B1和Tafps-B2过表达的拟南芥中倍半萜烯β-石竹烯（β-Caryophyllene）明显升高，转Tafps-B1拟南芥每株每天β-石竹烯的释放量为130.30ng，是野生型的5.5倍；转Tafps-B2拟南芥每株每天β-石竹烯的释放量为87.68ng，是野生型的3.7倍；与野生型相比，Atfps1拟南芥突变体、Atfps2拟南芥突变体中β-石竹烯的释放量分别降低46.5%和53.3%，说明fps基因控制倍半萜化合物的合成量。4通道嗅觉仪（4-way olfactometer bioassay）检测花期转基因拟南芥植株的挥发物对有翅桃蚜（Myzus pesicae）行为的影响，单因素检测实验结果表明，与野生型相比，Tafps-B1和Tafps-B2过表达拟南芥植株对蚜虫有明显的驱避作用（Tafps-B1，P<0.01; Tafps-B2，P<0.05）；4通道嗅觉仪双选择实验结果表明，与野生型拟南芥相比，Tafps-B1过表达拟南芥植株对蚜虫有显著的驱避作用（P<0.05），Tafps-B2过表达的拟南芥对蚜虫亦有驱避作用但统计分析不显著。fps1和fps2突变体拟南芥对有翅桃蚜均有明显的吸引作用，而互补植株对蚜虫有微弱的驱避作用。以上结果表明，小麦Tafps与拟南芥fps具有相似的功能，小麦Tafps在拟南芥中过表达，提高了拟南芥对桃蚜的驱避性（抗性），Tafps1基因的效果显著优于Tafps2。
为了近一步验证提高EβF合成酶的前体物质FPP是否能够提高EβF的释放量，将Tafps-B1和Tafps-B2过表达的拟南芥分别与转薄荷（Mentha x piperita）EβF合成酶基因拟南芥株系FS11-4.4进行杂交，后续的检测工作正在进行中。
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.
倍半萜类化合物合成于MVA途径，在植物与植食性昆虫互作中发挥重要作用，参与植物的防御反应。法尼烯焦磷酸合成酶（farnesyl pyrophosphate synthase，FPS）是萜类化合物合成途径中的关键酶之一，其催化合成的产物FPP可在细胞质中通过MVA(Mevalonate pathway)途径作为[反]-β-法尼烯【(E)-β-farnesene，EβF】合成酶的底物。EβF是重要的倍半萜类化合物，是一种无色无味挥发物，是小麦麦长管蚜（Sitobion avenae Fabricius）、禾谷缢管蚜（Rhopalosi phum padiLinnaeus）、麦无网长管蚜（Metopolophium dirhodum Walker）和麦二叉蚜（Schizaphis graminumRondani）等蚜虫报警信息素的唯一成份，可以使蚜虫产生骚动、从植株上脱落，并吸引蚜虫天敌，有效控制蚜虫危害。研究发现，许多植物中含有EβF合成酶，能释放EβF，具有天然的蚜虫驱避特性。但小麦、棉花、大豆等重要农作物中，只含有FPS，但不含有EβF合成酶。因此，了解小麦中FPS的特性、分布与供应状况，通过调控和改良小麦中倍半萜类化合物代谢途径，创制释放蚜虫报警信息素的新一代抗蚜转基因小麦，具有重要意义和应用价值。本项目分离克隆了小麦Tafps基因，并对其序列结构特征、表达模式、酶活性及其对蚜虫的抗性等进行了研究。取得以下研究结果。
1、小麦Tafps基因克隆、染色体定位及其结构特征分析：以普通小麦品种科农199为实验材料，采用同源克隆的方法，克隆得到6个法尼烯焦磷酸合成酶基因(Tafps-A1，Tafps-B1，Tafps-D1，Tafps-A2，Tafps-B2，Tafps-D2)，分别定位于第三同源群（Tafps1）和第一同源群（Tafps2）染色体的长臂上。随机测序获得了Tafps1和Tafps2各成员的基因组DNA和编码序列全长，初步表明各基因分别具有3个拷贝。Tafps1各成员的gDNA均含11个外显子，10个内含子；而Tafps2各成员的gDNA均含12个外显子，11个内含子。Tafps1和Tafps2各成员内部相似性分别为99.59%和98.39%。Tafps-B1与拟南芥Atfps1和Tafps-B2与拟南芥Atfps2的相似性分别为61.13%和67.62%。Tafps-B1与水稻Osfps1和Tafps-B2与水稻Osfps2的相似性分别为87.98%和82.93%。对各成员进行生物信息学分析和预测，结果表明小麦Tafps基因分为两种同工型，具有2个异戊烯基转移酶功能域。
2、小麦Tafps基因的表达模式分析：通过实时荧光定量PCR，对小麦科农199中Tafps1和Tafps2两个同工型表达特性进行分析表明两同工型在小麦中的表达具有时空特异性，在苗期、拔节期及成熟期，Tafps1在绿色器官（包括叶片、茎和颖壳）中表达量相对较高，Tafps2在非绿色器官（包括根、幼穗和种子）中表达量相对较高；抽穗期两基因表达趋势趋于一致。蚜虫咬食可诱导两基因表达量的变化。蚜虫咬食后，两基因在苗期的变化趋势相同，在根中表达量降低，在茎及叶片中表达量升高，其中，Tafps1的表达在叶片中显著升高（P<0.05）。抽穗期，蚜虫咬食后，Tafps1的表达在叶片（P<0.01）和茎（P<0.05）中的表达量显著升高，在幼胚中轻微升高，在颖壳中显著降低（P<0.05），根中轻微降低；Tafps2在颖壳（P<0.001）和叶片（P<0.05）中的表达量显著升高，在其余器官中均有不同程度的上升。推测Tafps基因与小麦对蚜虫侵害的应激抗性反应相关，具有防御反应诱导的表达特性。
3、小麦Tafps基因的酶活性及亚细胞定位分析：在大肠杆菌中表达Tafps-B1和Tafps-B2基因，通过组氨酸标签分离纯化获得的目的蛋白，与前体化合物异戊基焦磷酸（isopentenylpyrophosphate， IPP)和香叶草基焦磷酸（geranyl pyrophosphate， GPP)一起进行体外催化反应，GC-MS分析去磷酸化后的产物，结果显示， TaFPS-B1和TaFPS-B2均可IPP和GPP形成法尼醇，证明Tafps-B1和Tafps-B2基因在原核细胞中具有FPP合成活性，但TaFPS-B1的活性较高，约为TaFPS-B2的两倍。采用PEG介导方法，分别将Tafps-B1和Tafps-B2与hGFP的融合基因，转化拟南芥原生质体。激光共聚焦显微镜观察表明，TaFPS-B1和TaFPP-B2与hGFP的融合蛋白出现在胞质中，进一步为其参与胞质中的MVA途径提供了佐证。
4、小麦Tafps基因的功能分析：农杆菌介导的浸花法获得转Tafps-B1过表达拟南芥植株3棵，转Tafps-B2过表达植株1棵，转Tafps-B1Atfps1突变体拟南芥6棵，转Tafps-B2Atfps2突变体拟南芥植株3棵。GC-MS分析野生型、过表达、突变体以及互补表达拟南芥植株的挥发物。结果表明，与野生型拟南芥相比，苗期Tafps-B1和Tafps-B2过表达的拟南芥中倍半萜烯β-石竹烯（β-Caryophyllene）明显升高，转Tafps-B1拟南芥每株每天β-石竹烯的释放量为130.30ng，是野生型的5.5倍；转Tafps-B2拟南芥每株每天β-石竹烯的释放量为87.68ng，是野生型的3.7倍；与野生型相比，Atfps1拟南芥突变体、Atfps2拟南芥突变体中β-石竹烯的释放量分别降低46.5%和53.3%，说明fps基因控制倍半萜化合物的合成量。4通道嗅觉仪（4-way olfactometer bioassay）检测花期转基因拟南芥植株的挥发物对有翅桃蚜（Myzus pesicae）行为的影响，单因素检测实验结果表明，与野生型相比，Tafps-B1和Tafps-B2过表达拟南芥植株对蚜虫有明显的驱避作用（Tafps-B1，P<0.01; Tafps-B2，P<0.05）；4通道嗅觉仪双选择实验结果表明，与野生型拟南芥相比，Tafps-B1过表达拟南芥植株对蚜虫有显著的驱避作用（P<0.05），Tafps-B2过表达的拟南芥对蚜虫亦有驱避作用但统计分析不显著。fps1和fps2突变体拟南芥对有翅桃蚜均有明显的吸引作用，而互补植株对蚜虫有微弱的驱避作用。以上结果表明，小麦Tafps与拟南芥fps具有相似的功能，小麦Tafps在拟南芥中过表达，提高了拟南芥对桃蚜的驱避性（抗性），Tafps1基因的效果显著优于Tafps2。
为了近一步验证提高EβF合成酶的前体物质FPP是否能够提高EβF的释放量，将Tafps-B1和Tafps-B2过表达的拟南芥分别与转薄荷（Mentha x piperita）EβF合成酶基因拟南芥株系FS11-4.4进行杂交，后续的检测工作正在进行中。
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.
引文
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