College of Food Science and Engineering, Hainan Tropical Ocean University, No. 1 Yucai Road, Sanya 572022, China.
Marine Food Engineering Technology Research Center of Hainan Province, No. 1 Yucai Road, Sanya 572022, China.
Genes (Basel). 2023 Sep 23;14(10):1850. doi: 10.3390/genes14101850.
The ( allele is an important target for the development of new high nitrogen-use efficiency (NUE) rice lines that would require less fertilizers. Detection of through PCR (polymerase chain reaction)-based assay is cumbersome and needs advanced laboratory skills and facilities. Hence, a method for conveniently and rapidly detecting on-field is a key requirement for further research and applications. In this study, we employed cleaved amplified polymorphic sequences (CAPs) and loop-mediated isothermal amplification (LAMP) techniques to develop a convenient visual detection method for high NUE gene ( from the rice line NM73). The TC→AA mutation at 1187-1188 bp loci was selected as the target sequence for the allele. We further employed this method of identification in 10 rice varieties that carried the gene and results revealed that one variety (NM73) carries the target allele, while other varieties did not possess the genotype. The optimal LAMP reaction using hydroxynaphthol blue (HNB), a chromogenic indicator, was carried out at 65 °C for 60 min, and the presence of allele was confirmed by color changes from violet to sky blue. The results of this study showed that the LAMP method can be conveniently and accurately used to detect the gene in rice.
该（等位基因）是开发新型高氮利用效率（NUE）水稻品系的重要目标，这些品系需要更少的肥料。通过聚合酶链反应（PCR）为基础的测定法检测（等位基因）很繁琐，需要先进的实验室技能和设备。因此，一种方便、快速地在田间检测（等位基因）的方法是进一步研究和应用的关键要求。在这项研究中，我们采用切割扩增多态性序列（CAPs）和环介导等温扩增（LAMP）技术，开发了一种方便的可视化检测方法，用于检测高氮利用效率基因（来自水稻品系 NM73）。在 1187-1188 bp 位点的 TC→AA 突变被选为（等位基因）的目标序列。我们进一步将这种鉴定方法应用于携带（基因）的 10 个水稻品种，结果表明，一个品种（NM73）携带目标（等位基因），而其他品种则没有该基因型。使用羟萘酚蓝（HNB）作为显色指示剂的最佳 LAMP 反应在 65°C 下进行 60 分钟，通过颜色从紫色变为天蓝色来确认（等位基因）的存在。本研究结果表明，LAMP 方法可方便、准确地用于检测水稻中的（基因）。
