Cultivation Of Transgenic Rice With Enhanced Beta-carotene And Alpha-tocopherol Content

Vitamin A and vitamin E are essential micronutrients for the human body. Vitamin A deficiency causes a variety of symptoms, suce as night blindness. In the human diet, carotenoids are the sole precursor of vitamin A, and β-carotene is the main pro-vitamin A. While vitamin E plays a broad role in the prevention of cardiovascular and cerebrovascular diseases, cancer, central nervous system diseases, and maintaining the balance of the body’s immune system. Vitamin E comprises four different forms of tocopherols and tocotrienols(α-,β-,γ-,δ-tocopherols and α-,β-,γ-,δ-tocotrienols). Four forms of tocopherols and tocotrienols differ by the degree of methylation of the polar moiety. Among them, alfa-tocopherol has the highest vitamin E activity.Rice is one of the most important food crops, as the staple food of more than half of the population in the world. However rice is lack of vitamin A, and its contents of a-tocopherol are also low. Only comsuming the rice without supplementing other nutrients, will results in malnutrition. In this study, phytoene synthase gene (psy) from maize and phytoene dehydrogenase gene (crtI) from Erwinia uredovora are transformed into rice, under the control of rice endosperm-specific glutelin promoter, so that β-carotene can be accumulated in rice seeds. Gamma-tocopherol methyltransferase gene (y-TMT) from maize is transformed into rice in order to improve the contents of a-tocopherol in rice seeds. Then psylcrtI transformed plants crossed with y-TMT transformed plants to pyramid these transgennes.The main results acquired in this study are as follows:1) By Agrobacterium-mediated transformation, psy/crtI were transformed into japonica rice Zhonghuall and Kongyu131, and the maize y-TMT was transformed into Kongyu131. We tested the transformed plants by the analysis of PCR and Southern blotting. Thirty five putative psylcrtI Zhonghuall transformants,17putative psy/crtI Kongyu131transformants, and35putative y-TMT transformants were gotten by PCR screening. Through Southern blotting analysis,13single-copy inserted psy/crtl Zhonghuall transformants,6single-copy inserted psylcrtI Kongyu131transformants, and10single-copy inserted y-TMT transformants.2) Combining with PCR screening and Southern blotting analysis, single-copy inserted and marker-free psylcrtI transgenic homozygous line kyl-4was selected. The flanking genomic sequence on the right side of the T-DNA insertion site was determined by Tail-PCR method. In assistance of Southern blotting analysis, the flanking sequence was located on rice chromosome11. 3) The contents of β-carotene in rice seeds of psy/crtl transformed plants were meansured by ultra-high performance liquid chromatography (UPLC). The contents of β-carotene in different transformed lines ranged from1.99±0.11to4.41±0.30μg/g. The contents of β-carotene of the markerfree line ky1-4were3.11±0.08μg/g.4) The T-DNA insertion rice homozygous mutants of y-TMT,5A-3-1,5A-3-15,5A-5-2,5A-5-7,5A-5-16,5A-6-14,5A-6-17, and5A-7-12were selected by PCR analysis. Southern blotting analysis comfirmed that all homozygous mutants had two copies of the T-DNA. The contents of a-tocopherol in y-TMT mutants were measured. The contents of y-tocopherol and a-tocopherol in leaves of the Dongjin control were7.62±0.40μg/g,312.88±10.86μg/g. While the contents of a-tocopherol in leaves of5A-5-7,5A-5-16and5A-6-14were zero, and the contents of y-tocopherol in leaves of5A-5-7,5A-5-16and5A-6-14were200.63±4.25μg/g,229.49±10.52μg/g,204.85±24.00μg/g. The contents of y-tocopherol and a-tocopherol seeds of the Dongjin control were0.95±0.05μg/g,14.27.88±0.17μg/g. While the contents of y-tocopherol and a-tocopherol in seeds of5A-6-17were10.54±0.80μg/g and4.41±0.70μg/g; and those of5A-7-12were5.77±0.16μg/g and10.52±0.08μg/g.5) The contents of tocopherols of y-TMT transformed plants were measured by UPLC. The contents of a-tocopherol in leaves of the Zhonghual1control were247.88±5.46μg/g, and the maximum contents of a-tocopherol in y-TMT transformed plants were359.74±7.89μg/g; the contents of a-tocopherol of seeds in the Zhonghual1control were17.40±0.64μg/g, and the maximum contents of a-tocopherol in y-TMT transformed plants were20.46±0.79μg/g.