WU Lin,WANG Yan,SUN Meng-yang,et al.Optimization of High Resolution Melting Analysis System for Fad3a Gene of Soybean Fatty Acid[J].Soybean Science,2012,31(06):882-886.[doi:10.3969/j.issn.1000-9841.2012.06.005]
大豆脂肪酸Fad3a基因HRM检测体系优化研究
- Title:
- Optimization of High Resolution Melting Analysis System for Fad3a Gene of Soybean Fatty Acid
- 文章编号:
- 1000-9841（2012）06-0882-05
- Keywords:
- Soybean; Fad3a; SNP; HRM systematic optimization
- 分类号:
- S565.1
- 文献标志码:
- A
- 摘要:
- 高分辨率熔解曲线分析法可准确检测单核苷酸多态性，具有稳定性和高效性的特点。建立基于HRM的SNP检测方法，确立最佳的反应体系和反应条件是后续基因分型研究的前提和基础。Fad3a基因为大豆脂肪酸合成的关键酶基因，以Fad3a基因为研究对象，通过测序获得大豆品种合丰25（亚麻酸含量5.94%）和L-5（亚麻酸含量2.75%）在Fad3a基因序列上的单核苷酸多态性位点，根据这2个品种的Fad3a基因测序结果，利用Lighter scanner primer design软件设计产物长度不同的SNP突变引物5对，探索PCR产物长度和不同的退火温度对HRM分型结果的影响。结果表明：HRM分型的扩增子适宜长度应小于150 bp，且在扩增成功的基础上使用相对较高的退火温度可使待测PCR产物熔解峰单一，熔解曲线平稳。
- Abstract:
- High-Resolution Melting(HRM)is an ideal approach for SNP(Single Nucleotide Polymorphism)genetic typing.An optimal detection system based on HRM should be established in order to obtain credible results on genetic typing studies.In this study,Fad3a gene,encoding a key enzyme for fatty acid synthesis of soybean,was cloned in two soybean cultivars(Hefeng25 and L-5)with different content of linolenic acid in soybean seed and sequenced to investigate the influence of PCR product length and annealing temperature on HRM genetic typing by designing five pairs of SNP mutation primers with different product lengths using LSPD software.The result indicated that melting peak and curve of PCR product were single and smooth under the condition that amplicon length was less than 150 bp and the annealing temperature was relatively high on the basis of successful amplification.
参考文献/References:
[1]Zhu Y,Song Q,Hyten D,et al.Single-nucleotide polymorphisms in soybean[J].Genetics,2003,163(3):1123-1134. [2]Gundry C,Vandersteen J,Reed G,et al.Amplicon melting analysis with labeled primers:a closed-tube method for differentiating homozygotes and heterozygotes[J].Journal of the Chinese Chemical Society,2003,49(3):396-406. [3]Montgomery J,Wittwer C T,Palais R,et al.Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis[J].Nature Protocols,2007,2:59-66. [4]Li J,Wang X,Dong R,et al.Evaluation of high-resolution melting for gene mapping in rice[J].Plant Molecular Biology Reporter,2011,29:979-985. [5]Hofinger B J,Jing H C,Hammond-Kosack K E,et al.High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley[J].Theoretical and Applied Genetics,2009,119:851-865. [6]Han Y,Khu D M,Monteros M J,et al.High-resolution melting analysis for SNP genotyping and mapping in tetraploid alfalfa(Medicago sativaL)[J].Molecular Breeding,2012,29:489-501. [7]朱振雷,束永俊,李勇,等.大豆种子DNA快速提取方法的改良及应用[J].东北农业大学学报，2009，40(10):60-63.(Zhu Z L,Shu Y J,Li Y,et al.Improvement and application of the rapid DNA extraction method from soybean seeds[J].Journal of Northeast Agricultural University,2009,40(10):60-63.） [8]尤崇革,李玉民,马克君,等.基于高分辨熔解技术进行单核苷酸多态性基因分型的方法学评价[J].中华临床医师杂志,2012,6(7):1786-1790.(You C G,Li Y M,Ma K J,et al.Evaluation on the methodology of SNP genotyping based on high resolution melting[J].Chinese Journal of Clinicians,2012,6(7):1786-1790.） [9]白牡丹,王彩虹,殷豪,等.苹果不同HRM反应体系分析效果评价[J].分子植物育种，2012,10(1):115-120.(Bai M D,Wang C X,Yin H,et al.Evaluation of different reaction systems for HRM analysis in apple[J].Molecular Plant Breeding,2012,10(1):115-120.） [10]焦丽,刘相国,王虎义,等.高分辨率溶解曲线分析技术在大豆Ecotilling中的应用[J].大豆科学,2011,30(5):843-846.(Jiao L,Liu X G,Wang H Y,et al.Application of high resolution melting analysis in soybean ecotilling[J].Soybean Science,2011,30(5):843-846.） [11]欧展辉.HRM分析检测单基因遗传病点突变及β-地中海贫血的临床检测[D].广州医学院第三附属医院,2011.(Ou Z H.Screening of gene mutations in hereditary disease by HRM and detecting β-thalassemia in clinic[D].Third Affiliated Hospital of Guangzhou Medical College,2011.） [12]Reed G H,Wittwer C T.Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis[J].Clinical Chemistry,2004,50(10):1748-1754. [13]Hofinger B J,Jing H C,Hammond-Kosack K E,et al.High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley[J].Theoretical and Applied Genetics,2009,119(5):851-865. [14]Ye M H,Chen J L,Zhao G P,et al.Sensitivity and specificity of high-resolution melting analysis in screening unknown SNPs and genotyping a known mutation[J].Animal Science Papers and Reports,2010,28(2):161-170. [15]Liew M,Seipp M,Durtschi J,et al.Closed-tube SNP genotyping without labeled probes/a comparison between unlabeled probe and amplicon melting[J].American Journal of Clinical Pathology,2007,127(3):341-348. [16]兰青阔,张桂华,王永,等.基于高分辨率熔解曲线技术快速筛选黄瓜SNP[J].分子植物育种,2011,9(5):642-647.(Lan Q K,Zhang G H,Wang Y,et al.Discovery of single nucleotide polymorphism in cucumber based on high resolution melting analysis[J].Molecular Plant Breeding,2011,9(5):642-647.） [17]刘阳,杨淑霞,李敏惠,等.引物浓度与退火温度不当导致巢式PCR非特异性扩增[J].成都医学院学报,2008,3(2):111-114.(Liu Y,Yang S X,Li H M,et al.Improper primer concentration and annealing temperature may cause nonspecific amplication in nested PCR[J].Journal of Chengdu Medical College,2008,3(2):111-114.）
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备注/Memo
基金项目：农业部大豆产业技术体系（CARS-04-PS04）；国家“十二五”科技支撑计划课题（2011BAD35B06）；国家重点基础研究发展计划“973计划”前期项目（2012CB126311）；国家自然科学基金项目（31201227）；中国博士后项目（20110491024）；黑龙江省博士后项目(LBH11220)；黑龙江省骨干教师项目（1252G014）。