LAMP法快速可视化检测水稻高直链淀粉基因Wxa  

Rapid and Visual Detection of High Amylose Gene Wxa in Rice(Oryza sativa)Using LAMP Method

作　　者：岱安其 梁祥帅 叶文伟 王和飞 吴先军 田永航 DAI An-Qi;LIANG Xiang-Shuai;YE Wen-Wei;WANG He-Fei;WU Xian-Jun;TIAN Yong-Hang(College of Food Science and Engineering,Hainan Tropic Ocean Univercity,Sanya 572022,China;Rice Research Institute,Sichuan Agricultural University,Wenjiang 611130,China)

机构地区：[1]海南热带海洋学院食品科学与工程学院,三亚572022 [2]四川农业大学水稻研究所,温江611130

出　　处：《农业生物技术学报》2023年第6期1325-1332,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31960399);海南省自然科学基金(321MS050)。

摘　　要：挖掘和利用高直链淀粉基因颗粒结合型淀粉合成酶(waxy,Wx)的等位变异Wx^(a)是当前水稻(Oryza sativa)育种的热点之一,而便捷地筛选基因Wx^(a)是关键。本研究设计水稻高直链淀粉基因Wx^(a)的可视化环介导恒温扩增(loop-mediated isothermal amplification,LAMP)功能标记,探索便捷的筛选方法。首先通过序列比对,找出Wx^(a)基因的Ex10-115(T/C)变异位点及上下游一致性序列作为目标序列。然后用Primer3 Input、NEBcutter V2.0和PrimerExplore V5等软件,在目标序列上设计用于基因型鉴定的酶切扩增多态性标记(cleaved amplified polymorphic sequences,CAPs)和LAMP标记,最后对标记的可视化检测进行测试。结果显示,在Ex10-115(T/C)变异位点附近有保守序列,据此设计出了CAPs标记(Wx^(a)-2F和Wx^(a)-2R)和LAMP标记(F3,B3,FIP和BIP)。用CAPs标记对10个水稻品种进行检测,结果显示,‘D62B’、‘G46B’、‘Ⅱ-32B’、‘ZS97B’和‘GLA4’等品种携带Wx^(a)基因,直链淀粉含量也较高,而其他5个品种不携带Wx^(a)基因,基因型与表型一致。用LAMP标记结合显色剂羟基萘酚蓝(hydroxynaphthol blue,HNB),63℃恒温反应60 min,裸眼可观察水稻品种是否携带Wx^(a)基因,与CAPs标记的检测结果一致。本研究设计了筛选水稻Wx^(a)基因的便捷方法,研究结果可为水稻其他重要基因的功能标记开发及应用提供参考。Mining and utilizing the allelic variation Wx^(a) of high amylose gene particle bound starch synthase(waxy,Wx)is one of the hot spots in rice(Oryza sativa)breeding,but the key is to screen the gene Wx^(a) conveniently.In this study,the visual loop-mediated isothermal amplification(LAMP)of rice high amylose gene Wx^(a) was designed to explore a convenient screening method.Firstly,the Ex10-115(T/C)variant site and upstream and downstream consistent sequences of Wx^(a) gene were identified as target sequences by sequence alignment.Then,using Primer3 Input,NEBcutter V2.0 and Primerexplore V5 software,cleaved amplified polymorphic sequences(CAPs)and LAMP markers were designed for genotyping.Finally,the visibility of the functional markers were tested.The results showed that there were conserved sequences near the Ex10-115(T/C)variant site,from which CAPs markers(Wx^(a)-2F and Wx^(a)-2R)and LAMP markers(F3,B3,FIP and BIP)were designed.Using the CAPs marker to detect the Ex10-115(T/C)variant site in 10 rice cultivars,the gene Wx^(a) was found in‘D62B',‘G46B',‘II-32B',‘ZS97B'and‘GLA4'those cultivars with higher amylose contents,while the other 5 cultivars did not carry Wx^(a) that there's concordance between genotype and phenotype.The Wx^(a) gene in rice was detected by LAMP marker combined with hydroxynaphthol blue(HNB)at 63℃ for 60 minutes that the Wx^(a) gene may be detected with the naked eye in rice varieties.The result was consistent with that of CAPs marker.In this study,a convenient method was designed to screen Wx^(a) genes in rice.The results can provide references for the development and application of functional markers for other important genes in rice.

关 键 词：高直链淀粉基因 酶切扩增多态性标记(CAPs) 环介导恒温扩增(LAMP) 可视化 功能标记 

分 类 号：S511.21[农业科学—作物学]