摘要
植物萜类是植物挥发物的主要组成成分,主要有由半萜、单萜、倍半萜、二萜、四萜等组成。它们在植物香气组成、昆虫诱导、植物防御、生长调控等方面具有重要作用。α-法尼烯是存在于多种植物的叶片、花和果实中的一种倍半萜类挥发性有机化合物,已知与植物的抗冷性、昆虫的诱导及冷藏苹果和梨果实重大的生理病害-虎皮病的发生有关。α-法尼烯合成酶(AFS)是调控α-法尼烯合成的关键酶、最终酶,它催化FPP转变为α-法尼烯。近年来,通过外源萜类合酶在植物体的表达来研究萜类合成以及异戊二烯途径已经成为国内外研究的热点。
根据α-法尼烯合成酶基因全长序列设计特定引物,构建正义表达载体,转化拟南芥,研究α-法尼烯合酶的表达情况;利用多种蛋白质预测软件和原生质体定位研究α-法尼烯合酶在外源植物体中的定位,探讨功能发挥位点和α-法尼烯合酶参与的途径;检测转基因拟南芥、烟草次生代谢物的含量变化,研究α-法尼烯合酶的表达对拟南芥、烟草异戊二烯途径的影响。主要结果如下:
(1)构建了pBI-AFS正义表达载体,并转化拟南芥,得到转AFS基因的拟南芥植株。
(2)利用RT-PCR的方法和western杂交分析,检测α-法尼烯合成酶在转录水平和蛋白水平成功表达。
(3)利用多种蛋白质蛋白结构分析和定位预测软件(SignalP、TMHMM、TargetP、CLC ProteinWorkbench),对AFS蛋白进行蛋白结构分析和信号肽定位预测,发现α-法尼烯合成酶不具有跨膜区域和信号肽,该酶很可能定位在细胞质中。
(4)通过克隆青香蕉苹果的α-法尼烯合成酶基因(AFS)全长,构建pBI-AFS-GFP真核表达载体,制备拟南芥原生质体,利用GFP融合蛋白的亚细胞定位确定α-法尼烯合酶在细胞质中表达。
(5)纯化转基因烟草品系,提取转基因烟草T2代基因组,PCR筛选得到纯合的T2代转基因烟草。
(6)GC/MS检测挥发性萜类、烟草甾体物质的含量组成,发现与野生型相比,转基因烟草中单萜、二萜、三萜含量升高,倍半萜含量下降,甾体物质含量没有发生变化,烟草重要生理指标——尼古丁含量显著降低;从表型上看,转基因烟草植株明显高于野生型烟草;测定了野生烟草与转基因烟草中GA、IAA含量,发现转基因烟草具有较高的GA和IAA水平;转基因拟南芥和烟草中的色素含量与野生型没有明显差异。综合上述结果,AFS基因的表达对MEP和MVA途径有一定的促进作用。
Plant terpenoids are major component of plant volatiles, mainly consist of hemiterpenes, monoterpenes, sesquiterpenes and triterpenes, which play an important role in plant aroma composition, insect induction, plant defense and growth regulation. As a kind of sesquiterpene,α-farnesene is volatile organic compound, present in various plant leaves, flowers and fruit, and related to plant cold resistance, insects’attraction and physiological disorder -superficial scald of apple or pear fruit during cold storage.α-farnesene synthase is the key enzyme of converting FPP intoα-farnesene eventually. In recent years, researching terpenoids synthesis and isoprene pathways through the expression of exogenous terpenoids synthase in plant has become the hotspot research in the world.
In order to study the expression ofα-farnesene synthase, we designed specific primers according to the full sequence ofα-farnesene synthase gene, constructed pBI-AFS expression vector and transformed Arabidopsis thaliana. The subcellular localization ofα-farnesene synthase by protein prediction software,the protoplast localization, and its functional sites and pathways involved were studied. The changes of the secondary metabolites in transgenic Arabidopsis and tobaccos were detected,and the influence of the expression ofα-farnesene synthase on the isoprenoid pathways was discussed. Main results are as follows:
(1)A recombinant plant expression vector pBI-AFS was constructed and transformed into Arabidopsis.
(2) RT-PCR and western blot analysis showed the transcription and protein expression level in the transgenic Arabidopsis.
(3) Using protein structure analysis and signal peptide prediction software (TMHMM, TargetP, CLC, ProteinWorkbench) to analysis AFS protein structure and signal peptide, it was found thatα-farnesene synthase has no transmembrane area and signal peptides, which indicate the enzyme is probably targeted in the cytoplasm.
(4) To study the subcellular localization ofα-farnesene synthase. The cDNA of AFS was cloned and pBI-AFS-GFP eukaryotic expression vector was constructed and transformed into Aprotoplast. It was confirmed that GFP fusion protein is located in the cytoplasm.
(5) The T_2 homozygous transgenic tobaccos were got and confirmed by extraction of transgenic tobacco T_2 genome and PCR test.
(6) GC/MS analysis showed that monoterpenes, diterpene, triterpenes of transgenic tobaccos increased, while sesquiterpenes decreased in transgenic tobaccos compared with wild type. The content of steroid has not changed, the content of nicotine of the important physiological index of tobacco was significantly reduced in transgenic tobaccos. Looking from the phenotype, the transgenic tobacco plants are significantly higher than wild ones. Compared with wild ones, higher levels of GA, IAA were found in the transgenic tobaccos. There was no pigment difference found between transgenic plants and wild type. All the results showed that the expression of AFS gene in plants has a stimulus on the MEP and MVA pathways.
引文
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