Transgenic Crop Research and Development Center, National Institute of Agrobiological Sciences, Kannondai 3-1-3, Tsukuba, Ibaraki, Japan.
Plant Biotechnol J. 2011 Sep;9(7):729-35. doi: 10.1111/j.1467-7652.2010.00576.x. Epub 2010 Nov 16.
Novokinin (Arg-Pro-Leu-Lys-Pro-Trp, RPLKPW) is a new potent antihypertensive peptide based on the sequence of ovokinin (2-7) derived from ovalbumin. We previously generated transgenic rice seeds in which eight novokinin were fused to storage protein glutelins (GluA2 and GluC) for expression. Oral administration of these seeds to spontaneously hypertensive rats (SHRs) reduced systolic blood pressures at a dose of 1 g seed/kg of SHR. Here, 10- or 18-tandem repeats of novokinin with an endoplasmic reticulum (ER) retention signal (Lys-Asp-Glu-Leu, KDEL) at the C terminus were directly expressed in rice under the control of the glutelin promoter containing its signal peptide. Only small amounts of the 18-repeat novokinin accumulated, and it was unexpectedly deposited in the nucleolus. This abnormal intracellular localization was explained by an endogenous signal for nuclear localization. The GFP reporter protein fused to this sequence targeted to nuclei by a transient assay using onion epidermal cells. Transgenic seed expressing the 18-repeat novokinin exhibited significantly higher antihypertensive activity after a single oral dose to SHR even at one-quarter the amount (0.25 g/kg) of the transgenic rice seed expressing the fusion construct; though, its novokinin content was much lower (1/5). Furthermore, in a long-term administration for 5 weeks, even a smaller dose (0.0625 g/kg) of transgenic seeds could confer antihypertensive activity. This high antihypertensive activity may be attributed to differences in digestibility of expressed products by gastrointestinal enzymes and the unique intracellular localization. These results indicate that accumulation of novokinin as a tandemly repeated structure in transgenic rice is more effective than as a fusion-type structure.
诺伏考宁（Arg-Pro-Leu-Lys-Pro-Trp，RPLKPW）是一种基于卵白蛋白衍生的 Ovokinin（2-7）序列的新型强效抗高血压肽。我们之前生成了转基因水稻种子，其中 8 个诺伏考宁融合到储存蛋白谷蛋白（GluA2 和 GluC）中进行表达。将这些种子口服给予自发性高血压大鼠（SHR），以 SHR 每公斤 1 克种子的剂量降低收缩压。在这里，带有内质网（ER）保留信号（Lys-Asp-Glu-Leu，KDEL）的 10 或 18 个重复 Novokinin 在水稻中直接在含有信号肽的谷蛋白启动子控制下表达。只有少量的 18 个重复 Novokinin 积累，并且出乎意料地沉积在核仁中。这种异常的细胞内定位可以通过核定位的内源性信号来解释。GFP 报告蛋白与该序列融合，通过使用洋葱表皮细胞的瞬时测定靶向细胞核。表达 18 个重复 Novokinin 的转基因种子在单次口服 SHR 后表现出显著更高的抗高血压活性，即使是表达融合构建体的转基因水稻种子的四分之一量（0.25 g/kg）；尽管其 Novokinin 含量低得多（1/5）。此外，在 5 周的长期给药中，即使是较小的剂量（0.0625 g/kg）的转基因种子也可以赋予抗高血压活性。这种高抗高血压活性可能归因于胃肠道酶对表达产物的消化率的差异和独特的细胞内定位。这些结果表明，在转基因水稻中作为串联重复结构积累的 Novokinin 比作为融合型结构更有效。
