Molecular breeding for rice with low phytic acid grains.

低植酸水稻分子育种.

• 批准号: 15380004
• 负责人: YOSHIDA Kaoru T
• 金额: $ 10.11万
• 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 (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15380004/
• 关键词: phytoremediation; inositol phosphates; phosphorus; rice; phytic acid; molecular breeding; 1D-myo-inositol 3-phosphate synthase; seed storage compound; 環境負荷低減; フィチン

In most plant seeds, phosphorus is stored primarily as myo-inositol 1,2,3,4,5,6-hexakisphosphate (InsP_6), or phytic acid. Reducing the phytic acid content of seeds is a major breeding target both to increase the availability of mineral nutrients and to decrease the environmental load of phosphorus. The first step in phytic acid biosynthesis and inositol metabolism is catalyzed by 1D-myo-inositol 3-phosphate (Ins(3)P_1) synthase. In this study, we aimed to reduce the phytic acid levels in rice seeds by manipulating the expression of the rice Ins(3)p_1 synthase gene RINO1 using transgenic methods. RINO1 was transformed into rice plants in the antisense orientation under the control of the promoter of the glutelin gene GIuB-1, which encodes a seed storage protein and shows high and seed-specific expression during rice seed maturation. The T_4 generation of a stable transgenic line that contained four copies of the transgene showed no differences in morphology compared to non-transgenic p … More lants. The T_5 seeds of this line contained less RINO1 protein than did non-transgenic seeds during the late stages of ripening. Most of the T_5 seeds contained greater amounts of inorganic phosphates (Pi) than did non-transgenic seeds. Pi concentrations in non-transgenic and transgenic seeds were 2.77% and 17.29%, respectively. The expression patterns of RINO1 and GluB-1 were examined by quantitative real-time reverse transcriptase-polymerise chain-reaction (RT-PCR). These results suggested that higher effects could be obtained by using a promoter with the same temporal and spatial activity in the seeds as RINO1, thereby reducing the levels of RINO1 protein earlier in the process of seed maturation.Despite many physiological investigations of phytic acid accumulation and storage, little is known at the molecular level about the biosynthetic pathway in plants. Recent work has suggested two pathways for InsP_6 synthesis. One is an inositol lipid-independent pathway that occurs through the sequential phosphorylation of Ins(3)P_1. The other is a phosphoipase C-mediated inositol lipid-dependent pathway in which inositol 1,4,5-trisphosphate (Ins(1,4,5)P_3) is sequentially phosphorylated to InsP_6. We identified 12 genes from rice that code for the enzymes that may be involved in the metabolism of inositol phosphates. These enzymes include Ins(3)P_1 synthase, inositol monophosphatase, Ins(1,4,5)P_3 kinase, inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1), and inositol 1,3,4-teiskisphosphate 5/6-knase (ITP5/6K). The quantification of absolute amounts of mRNAs by real-time RT-PCR revealed the unique expression patterns of these genes. We showed that transcripts of RINO1, Ipk1, two genes out of six ITP5/6K are highly expressed in developing embryos, where vigorous synthesis of phytic acid occurs. The expression level was much lower in other organs This overexpression is consistent with the idea that these enzymes are needed to maintain the high level of physic acid synthesis in developing rice seeds. The results here also suggested that the inositol lipid-independent pathway is a major route of physic acid biosynthesis in rice seeds. Less

在大多数植物种子中，磷主要以肌醇1，2，3，4，5，6-六磷酸（InsP_6）或植酸的形式贮存。降低种子植酸含量是提高矿质营养有效性和降低磷环境负荷的主要育种目标。肌醇3-磷酸合成酶（1D-myo-inositol 3-phosphate synthase，Ins（3）P_1）是植酸生物合成和肌醇代谢的第一步。本研究旨在通过转基因技术调控水稻Ins（3）p_1合酶基因RINO 1的表达，降低水稻种子中植酸的含量。RINO 1在谷蛋白基因GIuB-1的启动子控制下以反义方向转化到水稻植株中，该基因编码种子贮藏蛋白，并在水稻种子成熟期间显示出高的和种子特异性的表达。含有4个拷贝的转基因的稳定转基因株系的T_4代与非转基因株系的T_4代相比在形态上没有差异。 ...更多信息 lants。转基因株系的T_5代种子在成熟后期的RINO 1蛋白含量低于非转基因株系。多数T_5代种子的无机磷含量高于非转基因种子。非转基因和转基因种子中的Pi浓度分别为2.77%和17.29%。采用实时定量逆转录聚合酶链反应（RT-PCR）检测RINO 1和GluB-1的表达模式。这些结果表明，可以获得更高的效果，通过使用启动子具有相同的时间和空间活性的种子中，RINO 1，从而降低RINO 1蛋白的水平在种子成熟的过程中的早期。尽管许多生理研究的植酸积累和存储，很少有人知道在分子水平上的生物合成途径在植物中。最近的研究表明InsP_6有两条合成途径。一种是通过Ins（3）P_1的顺序磷酸化发生的非肌醇脂质依赖性途径。另一条途径是磷脂酶C介导的肌醇脂质依赖性途径，其中肌醇1，4，5-三磷酸（Ins（1，4，5）P_3）被依次磷酸化为InsP_6。我们从水稻中鉴定了12个基因，这些基因编码可能参与肌醇磷酸代谢的酶。这些酶包括Ins（3）P_1合成酶、肌醇单磷酸酶、Ins（1，4，5）P_3激酶、肌醇1，3，4，5，6-五磷酸2-激酶（Ipk 1）和肌醇1，3，4-三磷酸5/6-激酶（ITP 5/6 K）。通过实时RT-PCR定量mRNA的绝对量揭示了这些基因的独特表达模式。我们发现，转录RINO 1，Ipk 1，两个基因的6个ITP 5/6 K的高度表达在发育中的胚胎，在那里发生了激烈的植酸合成。在其他器官中的表达水平要低得多。这种过表达与这些酶在发育中的水稻种子中维持高水平的油酸合成所需的想法一致。本研究结果还表明，非肌醇脂类依赖途径是水稻种子中植酸生物合成的一条主要途径。少

项目成果

Feng, X, Yoshida, KT: "Molecular approaches for producing low-phytic-acid grains in rice."Plant Biotechnology. (in press). (2004)

Feng, X, Yoshida, KT：“在水稻中生产低植酸谷物的分子方法。”植物生物技术。

cDNA microarray analysis of gene expression changes during pollination, pollen-tube elongation, fertilization, and early embryogenesis in rice pistils.

水稻雌蕊授粉、花粉管伸长、受精和早期胚胎发生过程中基因表达变化的 cDNA 微阵列分析。

• 发表时间: 2005
• 期刊: Sex. Plant Rep. 17
• 作者: Yoshida;K. T.;et al.
• 通讯作者: et al.

Evidence for an evolutionary force that prevents epigenetic silencing between tail-to-tail rice genes with a short spacer

证据表明存在一种进化力量，可以防止具有短间隔的尾对尾水稻基因之间的表观遗传沉默

• 发表时间: 2005
• 期刊: Gene 346
• 作者: H.Nakai;T.Ito;M.Hayashi;K.Kamiya;T.Yamamoto;K.Matsubara;Young-Min Kim;W.Jintanart;M.Okuyama;H.Mori;S.Chiba;Y.Sano;A.Kimura;Hironori Nagano;So Kobayashi
• 通讯作者: So Kobayashi