Rice Genome Sequencing and Genetically-Modified Rice

Rice Genome Sequencing and Genetically-Modified Rice

Rice is eaten by 3 billion people daily, and estimated to be consumed by nearly half (4 billion) of the human population on the earth in 20 years. (See also Fact Sheet.) It would be increasingly important to have high yield of rice production with resistance to disease and pests, with low cost by minimizing the use of fertilizers and insecticides, and to have high nutrients in the rice grain. Scientists have been eager to find genes that enhance such traits. The study of rice would also contribute to improve the value of other crops with similar genetic background such as wheat and maize.
Rice contains ca 30,000 genes in the genome of 430 million base pairs (12 chromosomes). In September, 1999, 10 countries agreed to form a consortium (the International Rice Genome Sequencing Project=IRGSP) for sequencing the genome of a Japanese rice cultivar called Nipponbare (GA3) by 2004. The funds for sequencing are estimated to be $200 mn, and initially $28 mn from Japan's Ministry of Agriculture, Forestry, and Fisheries and $12.3 mn from USA. For efficiency and speed, participants will share materials and databases of physical mapping information and annotated DNA. They also divide the sequencing efforts among participating groups, for example chromosome 9 (Thailand's National Center for Genetic Engineering and Biotechnology), chromosome 10 (Rutgers University, USA) and chromosome 11 (U of Wisconsin, USA).

In the meantime, Monsanto (now part of Pharmacia Corp) and the University of Washington (Leroy Hood et al, Seattle) announced on 4 April, 2000 a sequence draft covering 85% of the entire rice genome and finding many of the estimated genes. They used BAC (bacterial artificial chromosome) for copying genome fragments and worked out the nucleotide sequence of each BAC. The sequence data will be made accessible at Monsanto Web site for researchers who register with the company, but researchers may be obliged to give the first option to Monsanto for the negotiation of non-exclusive rights of the patents arising from the sequence data. At any rate, IRGSP consortium might end up with saving $100 mn and a few years of time thanks to the Monsanto data release. In other fronts, DuPont and Novartis also have their own rice databases established, while Celera Genomics of Rockville announced (spring, 1999) an ambitious plan to sequence the rice genome in 6 weeks.

There is of course much to be done before the genome sequence data be utilized to enhance crop productivity and nutrient enrichment. Ideally, useful genes from the same plant are multiplied for the genetically modified crops, and the modification should not entail hazardous components and/or metabolic trade-offs. Current criticism and concerns against genetically modified crops are unwarranted in many cases, and the setback due to misunderstanding in introducing the results of modern science into plant breeding is lamentable. However, because of its importance and with careful approach for better public acceptance, rice modified for better and beneficial traits would find the market and make significant contribution for feeding world population.

Rice is rather poor in many essential micronutrients and vitamins, particularly as it is usually milled and the remaining rice grains (endosperm) lack several essential nutrients, such as provitamin A (b-carotene) or any of its immediate precursors. Thus, predominant rice consumption promotes vitamin A deficiency, a serious public health problem in many countries, including highly populated areas of Asia, Africa, and Latin America. In Southeast Asia, 70% of children under the age of five suffer from vitamin A deficiency, leading to vision impairment and increased susceptibility to disease. It is estimated that a quarter of a million children go blind each year because of this nutritional deficiency, and UNICEF Vitamin A Global Initiative predicts that improved vitamin A nutrition could prevent 1 to 2 million deaths each year among children aged 1 to 4 years. See also Rockefeller Foundation News. It is quite reasonable that one of the first attempts to genetically modify rice is to enrich the grain with provitamin A.

Mammals make vitamin A from b-carotene that is one of the most abundant carotenoids found in plants. Carotenoids are yellow, orange, and red pigments that are essential components of the photosynthetic membranes of all plants. They serve as accessory light-harvesting pigments and as antioxidants that quench tissue-damaging free radicals. Carotenoids are synthesized in the central isoprenoid pathway within plastids. All isoprenoids are built from the common precursor isopentenyl diphosphate (IPP). IPP is thought to be synthesized in plastids from pyruvate, and immature rice endosperm synthesizes the carotenoid precursor geranyl geranyl diphosphate (GGPP) from IPP. Phytoene synthase (psy gene) condenses 2 GGPP to phytoene, which is desaturated to lycopene by phytoene desaturase (crt1 gene), and is cyclized by lycopene b-cyclase (lcy gene) to b-carotene. (GGPP is also the precursor of gibberellins, sterols, chlorophylls, and tocopherols in the general isoprenoid biosynthetic pathway).

Ye X et al (2000: uid=10634784) have carried out a single transformation of precultured immature rice embryos (n=800) by the use of Agrobacterium-mediated introduction of the entire b-carotene biosynthetic genes. The vector pB19hpc contained phytoene synthase (psy) from daffodil (Narcissus pseudonarcissus) combined with phytoene desaturase (crtI) from bacteria (Erwinia uredovora). These genes were placed under control of the endosperm-specific glutelin (Gt1) and the constitutive CaMV (cauliflower mosaic virus) 35S promoter, respectively. The selectable marker gene was aphIV for hygromycin resistance under CaMV 35S promoter. Hygromycin-resistant plants (n=50) were analyzed for the presence of the genes, and all found to have single gene insertions. Seeds (1 g) from individual lines were analyzed for carotenoids by photometric and by high-performance liquid chromatography (HPLC) analyses. The carotenoids found in the pB19hpc single transformants accounted for the yellow color, and none of these lines accumulated detectable amounts of lycopene. Instead, b-carotene, and to some extent lutein (from a-carotene) and zeaxanthin (from b-carotene), were formed. Thus, lycopene a- and b-cyclases and the hydroxylase appear to be expressed in normal rice endosperm to convert lycopene to b-carotene. In another experiment, immature rice embryos (n=500) were co-transformed with 2 vectors, one of which had psy and crt1 genes, whereas the other had lyc gene and the selectable marker. Of 60 selections, 12 had all the genes inserted, and the seeds contained b-carotene to a variable extent.

Line z11b contained b-carotene 1.6�μg/g in the endosperm as almost the only carotenoid thus the goal of providing at least 2�μg/g provitamin A (corresponding to 100�μg retinol equivalents at a daily intake of 300�g of rice per day) seems to be realistic. This successful example is only the beginning and much has to be done, such as field-testing to entail any metabolic trade-offs, to replace genes from other origins with those from rice, and to take all the precautions overcoming public concerns over genetically engineered crops. In these courses and with further genome sequence information, additional vitamins and other nutrients could be added to the rice grain for further nutrient enrichment and additional health benefits.

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