摘要
[目的]A类大豆皂苷是大豆及其制品产生苦涩味的主要原因,建立一种快速鉴定A类大豆皂苷缺失的大豆种质资源方法,对今后大豆品质育种具有重要的意义。[方法]通过比对大豆A类皂苷合成关键酶基因GmSg-5/Gmsg-5基因序列,找出单核苷酸多态性(SNP)位点,根据SNP位点分别设计了GmSg-5/Gmsg-5基因的特异探针,开发基于DNAzyme的缺口连接酶链式反应(Gap-LCR)鉴定GmSg-5/Gmsg-5基因特异性的可视化快速检测方法。[结果]根据GmSg-5/Gmsg-5基因1127 SNP位点的G/A变异,分别设计了GmSg-5和Gmsg-5基因的2套特异性互补探针L*、R、Ln、Rn*和L、R、Ln、Rn。通过优化Gap-LCR反应体系,在0.2μmol·L^-1模板浓度、缓冲液pH值8.4、探针浓度1.2μmol·L^-1条件下建立了可视化检测体系,显色反应10 min即可分别实现对GmSg-5/Gmsg-5基因的可视化特异检测。[结论]本研究从基因水平上建立了一种鉴定A类大豆皂苷缺失的可视化检测方法,该方法快速、简单、实用、经济。
[Objective]Group A saponins are the major contributor for undesirable bitter and astringent tastes in soybean products.Therefore,the establishment of a method to quickly identify soybeans that lack group A saponins is of important significance for soybean quality breeding.[Methods]The single nucleotide polymorphism(SNP)was detected through sequences alignment of GmSg-5/Gmsg-5 genes,the key enzyme genes of the group A saponins biosynthesis.Based on the SNP,specific probes for GmSg-5/Gmsg-5 genes were designed and a DNAzyme based Gap-LCR detection method for visually rapid distinction of GmSg-5/Gmsg-5 genes was developed.[Results]The probes(L*,R,Ln and Rn*)were designed to specifically amplify the GmSg-5 gene,and the other probes(L,R,Ln,and Rn)were designed to specifically amplify the Gmsg-5 gene.The visual detection method was developed under the concentration of 0.2μmol·L^-1 emplate,pH 8.4 LCR buffer,and 1.2μmol·L^-1 probes.The optimal time of detecting was 10 minutes after the chromogenic reaction.[Conclusion]The established visual DNAzyme based Gap-LCR method is capable of detecting the soybeans lacking group A saponins.This method is of efficient and economic benefits.
作者
谈美霞
李文帅
武国瑞
王敏
杜维俊
岳爱琴
赵晋忠
Tan Meixia;Li Wenshuai;Wu Guorui;Wang Min;Du Weijun;Yue Aiqin;Zhao Jinzhong(College of Arts and Science, Shanxi Agricultural University, Taigu 030801,China;College of Agriculture, Shanxi Agricultural University, Taigu 030801,China)
出处
《山西农业大学学报（自然科学版）》
CAS
北大核心
2020年第2期19-26,共8页
Journal of Shanxi Agricultural University(Natural Science Edition)
基金
山西省重点研发计划项目(201703D221004-5,201803D221020-2)
山西省重点研发计划重点项目(201703D211001)
山西省自然科学基金(2016011076,201901D111225)
山西农业大学科技创新基金项目(zdpy201706,zdpy201803,zdpy201905)。
