表面等离子体谐振(SPR)技术在水稻垩白基因Chalk5检测中的应用
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摘要
以垩白度低的明恢63与垩白度高的9311的基因组DNA为材料设计探针,构建基于表面等离子体谐振(Surface Plasmon Resonance,SPR)技术的水稻垩白基因检测方法,并用16个已知垩白度的水稻品种检验该方法的可靠性。结果表明:通过生物素修饰后单联DNA片段探针与羧基传感芯片上链霉亲和素(SA)的结合构建基因芯片,再以100μL 0.16μmol/L的PCR产物与探针进行分子杂交,可获得较好的SPR信号;以该方法获得的SPR信号差值的绝对值与水稻垩白成正相关关系,可定性判断水稻品种垩白度的高低;同时,SPR检测方法无需标记,分析过程完全自动化,可实现水稻垩白基因的快速检测。
Using genomic DNA of Minghui 63 with low chalkiness and 9311 with high chalkiness as probes, a method based on Surface Plasmon Resonance(SPR) was developed to detect Chalkiness in rice, and its reliability was tested by 16 rice varieties with known chalkiness. The results showed that single DNA fragment probes modified by biotin combining with streptavidin(SA) on carboxyl sensing chip to construct gene chip, PCR products of rice(100 μL 0.16 μmol/L) were subjected to molecular hybridization and get a more better SPR signal value; The difference of SPR signal obtained by this method is positively correlated with rice chalkiness, and the chalkiness of rice varieties can be qualitatively judged; at the same time, the SPR method did not need markers, and the analysis process was completely automated, which can realize the rapid detection of rice chalkiness gene.
Using genomic DNA of Minghui 63 with low chalkiness and 9311 with high chalkiness as probes, a method based on Surface Plasmon Resonance(SPR) was developed to detect Chalkiness in rice, and its reliability was tested by 16 rice varieties with known chalkiness. The results showed that single DNA fragment probes modified by biotin combining with streptavidin(SA) on carboxyl sensing chip to construct gene chip, PCR products of rice(100 μL 0.16 μmol/L) were subjected to molecular hybridization and get a more better SPR signal value; The difference of SPR signal obtained by this method is positively correlated with rice chalkiness, and the chalkiness of rice varieties can be qualitatively judged; at the same time, the SPR method did not need markers, and the analysis process was completely automated, which can realize the rapid detection of rice chalkiness gene.
引文
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[2]闵捷,朱智伟,章林平,等.中国超级杂交稻组合的稻米品质分析[J].中国水稻科学,2014,28(2):206-210.
[3]王颖姮,蔡秋华,廖长见,等.杂交水稻育种骨干亲本的米质分析[J].福建农业学报,2017,32(3):242-247.
[4]胡晋豪,徐庆国,黄琳.不同类型水稻品种米质的差异研究[J].作物研究,2014,28(5):461-466.
[5]李一博.水稻粒形、粒重基因GS5及垩白基因Chalk5的克隆与功能研究[D].武汉:华中农业大学,2011.
[6]王庆庆.水稻垩白基因定位及垩白遗传研究[D].天津:天津农学院,2014.
[7]《自然-遗传学》:首个调控稻米垩白的Chalk5基因成功克隆[J].现代生物医学进展,2014,14(15):3001.
[8]Li Y B,Fan C C,Xing Y Z,et al.Chalk5 encodes a vacuolar H+-translocating pyrophosphatase influencing grain chalkiness in rice[J].Nature Genetics,2014,46(4):398-404.
[9]吴世康.表面等离子共振传感器的原理与进展[J].影像科学与光化学,2017,35(1):15-25.
[10]蔡浩原.一种新型表面等离子体谐振(SPR)生化分析系统的研究[D].北京:中国科学院研究生院(电子学研究所),2003.
[11]何健.SPR生物传感器的优化和在生物材料生物相容性研究中的应用[D].南京:东南大学,2006.
[12]Murray M G,Thompson W F.Rapid isolation of high molecular weight plant DNA[J].Nucleic Acids Research,1980,8(19):4321.
[13]陈忠,刘宁,杨华卫,等.用表面等离子共振技术检测病毒的方法以及其中使用的芯片[P].中国专利:101464456B,2013-06-12.