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

种子中的磷酸盐以植酸(肌醇-1，2，3，4，5，6-六烷基磷酸)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有大量的磷。降低种子植酸含量是育种的主要目标，既是为了增加有效养分，又是为了减少环境负荷。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(RINO1)中分离到该基因，并证明它在种子发育的合成途径中发挥关键作用。在本研究中，我们试图通过降低RINO1在发育中的水稻种子中的表达来降低籽粒中的植酸含量。为了抑制RINO1的表达，我们利用根癌农杆菌介导法，在CaMV 35S、RINO1或水稻主要储存蛋白谷蛋白(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，将RINO1正、反义方向的cDNA导入水稻植株。为了确定转基因对种子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，磷的浓度约占总磷的5%，剩余磷的大部分是植酸磷。有几个转基因植株的T_1种子中含有较高的PI水平，从12%到63%不等。转基因植株种子的总磷含量保持不变，与非转基因植株持平。结果表明，转基因植株中的植酸含量降低。

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.