GENETIC TRANSFORMATION OF RICE FOR INCREASED IRON CONTENT

Abstract

Micronutrient deficiency or Hidden hunger is spread worldwide and affects large population in particularly in developing and poor countries. Among micronutrient deficiency, Fe and or Zn deficiency is most prominent and affects over a billion population worldwide. Fe and Zn deficiency in long term causes many adverse health problems to all age groups such as impairment in cognitive and physical development, reduced immunity and ultimately reduced work efficiency. Fe is a vital micronutrient and play multiple functions in human body including electron transport, DNA synthesis, synthesis of oxygen transport molecules hemoglobin and myoglobin and other enzymatic activities. On global scale, 29.9% women and 36.5% pregnant women suffered from anaemia in the year 2021. Similarly, 39.4% children were found anemic worldwide. In India, prevalence of anemia is alarmingly high with 66.4% women and 68.4% of children suffered from anemia in year 2019-20 as estimated by phase I of National Family Health Survey, Government of India. Interventions strategies like food supplementation, diet diversification and industrial food fortification are not adequately available to developing and poor countries, specially people living in rural areas. Alternatively, to increase the micronutrient content in edible part of crops, biofortification is an effective, sustainable and not expensive strategy to overcome micronutrient deficiency. Rice (2n=24) is a one of most important staple crop which feeds nearly half of the world population with providing about 20% calories to world population and up to 76% calories to south east Asian population. Generally, most of people consume the polished rice which lacks in many micronutrient and phytonutrients, therefore, rice is most suitable crop for biofortification to enhance the endospermic content of Fe and Zn. Ferritin is a natural iron storage protein and can theoretically accommodate up to 4500 iron atoms in its inner nanocavity core in the form of iron-protein complex. Therefore, Phytoferritin acts as a natural and alternative source of dietary iron, because it is less sensitive to chelators and readily bioavailable. In rice, iron homeostasis genes play very crucial role in acquisition of iron from soil, its internal translocation within plant body and loading into rice grain with the help of various specific metal transporter. Nicotianamine is natural metal chelator biosynthesized from trimerization of S-adenosyl methionine by action of nicotianamine synthase enzyme encoded by OsNAS gene. Nicotianamine is involved in transport of Fe as well as Zn in higher plants including rice. In present study, combined strategy has been employed with overexpression of OsNAS2 gene and targeted expression of Ferritin gene to enhance Fe content in rice endosperm. Ferritin gene from feed and food Pearl millet (Pennisetum glaucum) were cloned from developing seeds through sequencing of pooled RNA using illumina plateform and contigs of ferritin gene obtained from the I RNA seq data, which was used for iron biofortification of rice along with PvFer gene from Phaseolus vulgaris. Thus, three Ferritin genes from different sources namely, PvFer, feed PgFer and food PgFer was expressed under control of endosperm specific rice Globulin (OsGlb1) promoter in combination with rice homeostasis OsNAS2 gene which was overexpressed under control of maize Ubiquitin (ZmUbi) promoter. Transgenic development in rice was carried out with Agrobacterium-mediated transformation. The transgenic rice lines were produced in both japonica (cv. TP309) and indica (cv. IET10364) type of rice. The transgenic rice TP309 produced with PvFerOsNAS2 construct was denoted as TP309-PvFerOsNAS2 lines. Similarly, IET10364(indica) variety transformed with three construct and transgenic rice lines were denoted as IET10364 -PvFerOsNAS2, IET10364-feed PgFerOsNAS2 and IET10364-food PgFerOsNAS2 respectively. The confirmation of transgenic rice lines was performed by GUS histochemical assay of leaf tissue and transgene-specific PCR. Expression analysis of Ferritin gene and OsNAS2 gene in developing seeds of various transgenic (T1) rice lines was performed through semi-quantitative RT-PCR and quantitative real time-PCR. Further estimation of Fe and Zn concentration was performed in transgenic (T1) rice grains using ICP-MS technique and result showed significant increase in Fe and Zn concentration in unpolished as well as in polished grains as compared to respective NT control. The Fe concentration in transgenic TP309-PvFerOsNAS2 lines showed up to 2.6 fold increase and 2.8 fold increase in unpolished and polished rice grains respectively as compared to TP309 NT control. Similarly, IET10364-PvFerOsNAS2 transgenic (T1) showed up to 4.9 fold and 4.2 fold increase in Fe concentration in unpolished and polished grains respectively over NT control. The IET10364 feedPgFerOsNAS2 lines also showed up to 3.1 fold and 3.9 fold increase in Fe concentration in unpolished and polished grains respectively as compared to IET10364 control. Fe concentration in IET10364-food PgFerOsNAS2 lines showed up to 3.3 fold and 2.6 fold increase in unpolished and polished grains respectively over IET10364 control. Zn concentration of transgenic rice grains was also significantly increased with three gene construct which showed OsNAS2 gene are involved into transport of Zn also along with Fe. This study give insight into biofortification of rice with application of Ferritin gene in combination of rice metal homeostasis OsNAS2 gene to increase the Fe and Zn concentration in rice endosperm.