RIG/CAA: A road to more nutritious cassava: Cloning, functional characterization and over-expression of B-cyanoalanine synthase gene from cassava

标题：通往更有营养的木薯之路：木薯β -氰丙氨酸合成酶基因的克隆、功能表征和过表达最重要的产氰作物是热带块根作物木薯（Manihot esculenta, Crantz），热带地区每年收获超过1.53亿吨木薯根，主要供人类直接食用。木薯食品在食用前必须经过加工以去除氰化物，因为接触较低水平的氰化物会导致呕吐、恶心、心悸、头痛和视力受损。在非洲的一些地区，长期摄入大量加工不良的木薯也被证明与慢性氰化物相关的健康疾病有关。最近的研究结果还表明，氰化物参与了氨基酸含量和收获后的变质，补充了其对食草动物的威慑能力。β -氰丙氨酸合成酶（β - cas）的基因已经被确定，这是一种线粒体酶，可以将氰化物解毒为天冬酰胺。本研究计划通过将β - cas基因导入模式植物拟南芥，进一步表征木薯β - cas DNA序列，并阐明该酶的功能。在细胞质或线粒体中产生过表达β - cas基因的转基因木薯。细胞质中β - cas的表达将把根中储存的氰转移到氨基酸的合成中，使木薯品种氨基酸库增加，从而获得更高的蛋白质营养。此外，由于β - cas对氰化物的亲和力会导致可用活性氧的减少，因此将对过度表达β - cas的根进行收获后变质分析，从而延长保质期。该项目的更广泛影响是为全世界6亿人开发更有营养的食物来源，同时教育波多黎各马亚圭斯大学的本科生和研究生，这些学生都属于生物学中代表性不足的群体（西班牙裔）。

Name: Dimuth Siritunga, PhDProposal number: IOS 0641084Title: A road to more nutritious cassava: Cloning, functional characterization and over-expression of beta-cyanoalanine synthase gene from cassavaThe most agronomically important of the cyanogenic crops is the tropical root crop cassava (Manihot esculenta, Crantz) with over 153 million tons of cassava roots being harvested annually in the tropics, primarily for direct human consumption. Cassava foods must be processed to remove cyanogens prior to consumption as exposure to lower levels of cyanide causes vomiting, nausea, palpitations, headaches and impaired vision. The long-term ingestion of large quantities of poorly processed cassava has also been shown to be associated with chronic cyanide-associated health disorders in several areas of Africa. Recent findings have also demonstrated an involvement of cyanogens in both amino acid content and post-harvest deterioration complementing its herbivore deterrent capabilities. The gene for beta-cyanoalanine synthase (beta-CAS), a mitochondrial enzyme that detoxifies cyanide to asparagine has been identified. The research proposed here plans to further characterize the cassava beta-CAS DNA sequence as well as clarify the function of this enzyme by introducing the gene into model plant Arabidopsis. Furthermore transgenic cassava over-expressing beta-CAS gene in the cytoplasm or the mitochondria will be produced. The expression of beta-CAS in the cytoplasm would divert cyanogens from root storage to the synthesis of amino acids, producing a cassava variety with enhanced amino acid pool and thus higher protein nutrition. In addition, roots over-expressing beta-CAS will be analyzed for post-harvest deterioration since the affinity of beta-CAS for cyanide should lead to a reduction in the available reactive-oxygen species, and thus to a longer shelf-life. The broader impact of this project is to develop a more nutritious food source for 600 million people world-wide while educating undergraduate and graduate students attending the University of Puerto Rico Mayaguez, all of whom belong to an underrepresented group (Hispanic) in biology.