Table_5_Investigation of Baseline Iron Levels in Australian Chickpea and Evaluation of a Transgenic Biofortification Approach.DOCX

Iron deficiency currently affects over two billion people worldwide despite significant advances in technology and society aimed at mitigating this global health problem. Biofortification of food staples with iron (Fe) represents a sustainable approach for alleviating human Fe deficiency in developing countries, however, biofortification efforts have focused extensively on cereal staples while pulses have been largely overlooked. In this study we describe a genetic engineering (GE) approach to biofortify the pulse crop, chickpea (Cicer arietinum L.), with Fe using a combination of the chickpea nicotianamine synthase 2 (CaNAS2) and soybean (Glycine max) ferritin (GmFER) genes which function in Fe transport and storage, respectively. This study consists of three main components: (1) the establishment for baseline Fe concentration of existing germplam, (2) the isolation and study of expression pattern of the novel CaNAS2 gene, and (3) the generation of GE chickpea overexpressing the CaNAS2 and GmFER genes. Seed of six commercial chickpea cultivars was collected from four different field locations in Australia and assessed for seed Fe concentration. The results revealed little difference between the cultivars assessed, and that chickpea seed Fe was negatively affected where soil Fe bioavailability is low. The desi cultivar HatTrick was then selected for further study. From it, the CaNAS2 gene was cloned and its expression in different tissues examined. The gene was found to be expressed in multiple vegetative tissues under Fe-sufficient conditions, suggesting that it may play a housekeeping role in systemic translocation of Fe. Two GE chickpea events were then generated and the overexpression of the CaNAS2 and GmFER transgenes confirmed. Analysis of nicotianamine (NA) and Fe levels in the GE seeds revealed that NA was nearly doubled compared to the null control while Fe concentration was not changed. Increased NA content in chickpea seed is likely to translate into increased Fe bioavailability and may thus overcome the effect of the bioavailability inhibitors found in pulses; however, further study is required to confirm this. This is the first known example of GE Fe biofortified chickpea; information gleaned from this study can feed into future pulse biofortification work to help alleviate global Fe deficiency.

目前全球缺铁人群已超20亿，尽管旨在缓解这一全球性健康问题的技术与社会发展已取得显著进展。以铁（Fe）强化主食作物的生物强化（biofortification）策略，是缓解发展中国家人类缺铁问题的可持续路径，但此前的生物强化工作多聚焦于谷物类主食作物，豆类作物长期被忽视。本研究报道了一种基因工程（genetic engineering, GE）策略：通过联合使用鹰嘴豆（chickpea, Cicer arietinum L.）烟酰胺合酶2（nicotianamine synthase 2, CaNAS2）与大豆（soybean, Glycine max）铁蛋白（ferritin, GmFER）基因——二者分别参与铁的转运与储存——来实现豆类作物鹰嘴豆的铁生物强化。

本研究包含三大核心内容：（1）建立现有种质资源的基础铁浓度基线；（2）克隆新型CaNAS2基因并分析其表达模式；（3）创制过表达CaNAS2与GmFER基因的转基因鹰嘴豆。

研究人员从澳大利亚4个不同田间地点采集了6个商业鹰嘴豆品种的种子，对其种子铁浓度进行检测。结果显示，供试品种间的铁浓度差异极小，且当土壤铁生物利用度较低时，鹰嘴豆种子铁含量会受到负面影响。研究最终选取德西（desi）型品种HatTrick开展后续实验。从该品种中克隆得到CaNAS2基因，并检测其在不同组织中的表达情况，发现在铁充足条件下，该基因在多个营养组织中均有表达，提示其可能在铁的系统性转运中发挥持家功能。

随后研究人员成功创制了2个转基因鹰嘴豆事件，并验证了CaNAS2与GmFER外源基因的过表达。对转基因种子的烟酰胺（nicotianamine, NA）与铁含量进行分析后发现，与阴性对照（null control）相比，种子中的NA含量提升了近一倍，但铁浓度并未发生显著变化。鹰嘴豆种子中NA含量的提升有望转化为铁生物利用度的提升，从而克服豆类作物中存在的铁吸收抑制剂的负面影响，但仍需进一步研究加以验证。本研究是目前已知的首例转基因铁生物强化鹰嘴豆案例，本研究所得信息可为未来豆类生物强化工作提供参考，助力缓解全球性缺铁问题。