Molecular breeding for rice with low phytic acid grains.

在大多数植物种子中，磷主要以肌醇1，2，3，4，5，6-六烷基磷酸(InSP_6)或植酸的形式储存。降低种子植酸含量是提高矿质养分利用率和降低磷环境负荷的主要育种目标。植酸生物合成和肌醇代谢的第一步是由1D-肌醇3-磷酸(INS(3)P_1)合成酶催化的。本研究旨在通过转基因方法调控水稻INS(3)p_1合成酶基因RINO1的表达，从而降低水稻种子中的植酸水平。在谷蛋白基因GIuB-1启动子的控制下，将RINO1反义导入水稻植株，GIuB-1编码种子贮藏蛋白，在水稻种子成熟过程中表现出高表达和种子特异性表达。含有4个拷贝的稳定转基因株系的T_4代形态与非转基因p-…无明显差异更多的兰特。该品系的T_5种子成熟后期RINO1蛋白含量低于非转基因种子。大多数T_5种子的无机磷(PI)含量均高于非转基因种子。非转基因种子和转基因种子的PI含量分别为2.77%和17.29%。用实时定量逆转录聚合酶链式反应(RT-PCR)检测RINO1和Glub-1的表达。这些结果表明，在种子中使用与RINO1具有相同时间和空间活性的启动子可以获得更高的效果，从而在种子成熟的早期降低RINO1蛋白的水平。尽管有许多关于植酸积累和储存的生理研究，但在分子水平上对植物的生物合成途径知之甚少。最近的工作提出了两条合成InSP_6的途径。一种是通过INS(3)P_1的顺序磷酸化而发生的肌醇脂不依赖途径，另一种是由磷脂酶C介导的肌醇脂依赖途径，在该途径中，肌醇1，4，5-三磷酸(INS(1，4，5)P_3)被顺序磷酸化为InSP_6。我们从水稻中鉴定了12个基因，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1，3，4，5，6-五磷酸肌醇2-激酶(Ipk1)和肌醇1，3，4-teiskipate 5/6-Ken酶(ITP5/6K)。实时定量RT-PCR检测mRNAs的绝对量揭示了这些基因的独特表达模式。我们发现RINO1、Ipk1、六个ITP5/6K中的两个基因在发育中的胚胎中高表达，此时植酸的合成旺盛。在其他器官中的表达水平要低得多，这与这些酶是维持水稻种子发育中高水平的植酸合成所必需的观点是一致的。这一结果还表明，肌醇脂非依赖性途径是水稻种子植酸生物合成的主要途径。较少

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