Malla Ashwini, Shanmugaraj Balamurugan, Sharma Ashutosh, Ramalingam Sathishkumar
Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore 641 046, India.
Tecnologico de Monterrey, School of Engineering and Sciences, Centre of Bioengineering, Campus Queretaro, Av. Epigmenio González No. 500, Fracc. San Pablo, Queretaro 76130, Mexico.
Plants (Basel). 2021 Oct 27;10(11):2311. doi: 10.3390/plants10112311.
Isoflavonoids, the diverse group of secondary metabolites derived from the phenylpropanoid pathway, are distributed predominantly in leguminous plants. It has received considerable attention in recent days due to its health promoting benefits and is known to prevent certain diseases in humans. These isoflavonoids are synthesized from flavonoid intermediates of phenylpropanoid pathway by the enzyme isoflavone synthase. Metabolic engineering of isoflavonoid biosynthesis in non-legume crop plants could offer the health benefits of these compounds in diverse plant species further contributing for crop improvement. The transient expression of heterologous genes in the host is considered as an alternative to stable expression, that can provide a rapid way of studying the pathway engineering for metabolite production and could also act as a production platform for nutraceuticals and biopharmaceuticals. In this study, isoflavone genistein was produced in var. and by transiently expressing (). The gene was cloned in plant expression vector pEarleyGate 102 HA and pEAQ-HT-DEST 3 and transformed into plants by agroinfiltration. The presence of transgene in the agroinfiltrated leaves was confirmed by semiquantitative reverse-transcription polymerase chain reaction. The flavonoid substrate naringenin and isoflavonoid genistein were quantified using high performance liquid chromatography in both wild-type and infiltrated leaf samples of both the plants. The naringenin content varied in the range of 65.5-338.5 nM/g fresh weight, while the accumulation of genistein was observed with varying concentrations from 113 to 182.6 nM/g fresh weight in the agroinfiltrated leaf samples of both var. and . These results indicate that the transient expression of gene has led to the synthesis of isoflavonoid genistein in var. and providing an insight that stable expression of this gene could enrich the nutraceutical content in the crop plants. To the best of our knowledge, this is the first report on transient expression of gene for the production of genistein in var. and .
异黄酮是一类源自苯丙烷途径的多样化次生代谢产物，主要分布于豆科植物中。由于其对健康有益，近年来受到了广泛关注，并且已知其能预防人类的某些疾病。这些异黄酮是由苯丙烷途径的类黄酮中间体通过异黄酮合酶合成的。对非豆科作物植物中的异黄酮生物合成进行代谢工程改造，有望使这些化合物在多种植物物种中带来健康益处，进一步促进作物改良。在宿主中瞬时表达异源基因被认为是稳定表达的一种替代方法，它可以提供一种快速研究代谢产物生产途径工程的方式，还可以作为营养保健品和生物制药的生产平台。在本研究中，通过瞬时表达（）在变种和中产生了异黄酮染料木黄酮。该基因被克隆到植物表达载体pEarleyGate 102 HA和pEAQ-HT-DEST 3中，并通过农杆菌浸润法转化到植物中。通过半定量逆转录聚合酶链反应确认了农杆菌浸润叶片中转基因的存在。使用高效液相色谱法对野生型和两种植物浸润叶片样品中的类黄酮底物柚皮素和异黄酮染料木黄酮进行了定量。柚皮素含量在65.5 - 338.5 nM/克鲜重范围内变化，而在变种和的农杆菌浸润叶片样品中观察到染料木黄酮的积累浓度在113至182.6 nM/克鲜重之间变化。这些结果表明，基因的瞬时表达导致了变种和中异黄酮染料木黄酮的合成，这为该基因的稳定表达能够丰富作物植物中的营养成分含量提供了见解。据我们所知，这是关于在变种和中瞬时表达基因以生产染料木黄酮的首次报道。
