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Analysis on the molecular mechanism of the accumulation of seed storage materials and molecular breeding of phosphate storage

种子贮藏材料积累的分子机制分析及磷贮藏分子育种

基本信息

批准号：
13660004
负责人：
YOSHIDA Kaoru
金额：
$ 2.3万
依托单位：
THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Phosphates in seeds are stored as phytic acid (myo-inositol -1,2,3,4,5,6-hexakisphosphate). Phytic acid is not readily digested by monogastric animals, so that grains and legumes in feed result in a large amount of phosphorus in animal wastes. Reducing the phytic acid content of seeds is a major objective of breeding, both to increase the available nutrients and to reduce the environmental load. Myo-inositol 3-phosphate synthase (EC 5.5.1.4) catalyzes the first step of phytic acid biosynthesis. We isolated that gene from rice (RINO1), and demonstrated that it plays a key role in the synthetic pathway during seed development. In this study, we attempted to reduce the phytic acid content of grain by reducing RINO1 expression in developing rice seeds.To repress RINO1 expression, we introduced RINO1 cDNA in sense and antisense orientations under control of either the CaMV 35S, RINO1, or rice major storage protein glutelin (GluB-1) promoters, or the promoter of a transcriptional factor (RISBZ1) responsible for GluB-1 expression, into rice plants, using Agrobacterium tumefaciens-mediated transformation. To determine the effect of the transgene on phytic acid biosynthesis in seeds, the inorganic (Pi) and total phosphorus contents of the grain were examined. In non-transgenic rice seeds, the concentration of Pi is around 5% of the total phosphorus and the majority of the remaining phosphorus is phytic acid phosphorus. Several transgenic plants contained elevated Pi levels in T_1 seeds, varying from 12 to 63% Pi. The total phosphorus content of the seeds of transgenic plants remained constant, and equaled that of non-transgenic plants. The results suggest that the phytic acid content was reduced in these transgenic plants.

种子中的磷酸盐以植酸（肌醇-1,2,3,4,5,6-己基磷酸）的形式储存。植酸不容易被单胃动物消化，因此饲料中的谷物和豆类导致动物粪便中含有大量磷。降低种子植酸含量是育种的一个主要目标，既可以增加有效养分，又可以减少环境负荷。肌醇3-磷酸合成酶（EC 5.5.1.4）催化植酸生物合成的第一步。我们从水稻中分离出该基因（RINO1），并证实其在种子发育过程中在合成途径中起关键作用。在本研究中，我们试图通过降低发育中的水稻种子中RINO1的表达来降低籽粒中的植酸含量。为了抑制RINO1的表达，我们利用农杆菌介导的转化，在CaMV 35S、RINO1或水稻主要储存蛋白谷氨酸（GluB-1）启动子或负责GluB-1表达的转录因子（RISBZ1）启动子的控制下，以正义和反义方向将RINO1 cDNA导入水稻植株。为了确定转基因对种子中植酸生物合成的影响，测定了籽粒中无机磷（Pi）和总磷含量。在非转基因水稻种子中，π的浓度约占总磷的5%，剩余的磷大部分为植酸磷。一些转基因植物在T_1种子中含有较高的Pi水平，从12%到63%不等。转基因植物种子的总磷含量保持不变，与非转基因植物种子的总磷含量持平。结果表明，这些转基因植株的植酸含量有所降低。