Comparison of Five Endogenous Reference Genes for Specific PCR Detection and Quantification of Rice
|
摘要
Endogenous reference genes(ERGs) provide vital information regarding genetically modified organisms(GMOs). The successful detection of ERGs can identity GMOs and the source of genes, verify stability and reliability of the detection system, and calculate the level of genetically modified(GM)ingredients in mixtures. The reported ERGs in rice include sucrose-phosphate synthase(SPS),phospholipase D(PLD), RBE4 and rice root-specific GOS9 genes. Based on the characteristics of ERGs,a new ERG gene, phosphoenolpyruvate carboxylase(PEPC), was selected, and further compared with the four existing genes. A total of 18 rice varieties and 29 non-rice crops were used to verify the interspecies specificity, intraspecies consistency, sensitivity, stability and reliability of these five ERGs using qualitative and quantitative PCR. Qualitative detection indicated that SPS and PEPC displayed sufficient specificity, and the detection sensitivity was 0.05% and 0.005%, respectively. Although the specificity of both RBE4 and GOS9 were adequate, the amplicons were small and easily confused with primer dimers. Non-specific amplification of the PLD gene was present in maize and potato. Real-time quantitative PCR detection indicated that PLD, SPS and PEPC displayed good specificity, with R~2 of the standard curve greater than 0.98, while the amplification efficiency ranged between 90% and 110%. Both the detection sensitivities of PLD and PEPC were five copies and that of SPS was ten copies. RBE4 showed typical amplification in maize, beet and Arabidopsis, while GOS9 was found in maize, tobacco and oats. PEPC exhibited excellent detection sensitivity and species specificity, which made it a potentially useful application in GM-rice supervision and administration. Additionally, SPS and PLD are also suitable for GM-rice detection. This study effectively established a foundation for GMO detection,which not only provides vital technical support for GMO identification, but also is of great significance for enhancing the comparability of detection results, and the standardization of ERG testing in GM-rice.
Endogenous reference genes(ERGs) provide vital information regarding genetically modified organisms(GMOs). The successful detection of ERGs can identity GMOs and the source of genes, verify stability and reliability of the detection system, and calculate the level of genetically modified(GM)ingredients in mixtures. The reported ERGs in rice include sucrose-phosphate synthase(SPS),phospholipase D(PLD), RBE4 and rice root-specific GOS9 genes. Based on the characteristics of ERGs,a new ERG gene, phosphoenolpyruvate carboxylase(PEPC), was selected, and further compared with the four existing genes. A total of 18 rice varieties and 29 non-rice crops were used to verify the interspecies specificity, intraspecies consistency, sensitivity, stability and reliability of these five ERGs using qualitative and quantitative PCR. Qualitative detection indicated that SPS and PEPC displayed sufficient specificity, and the detection sensitivity was 0.05% and 0.005%, respectively. Although the specificity of both RBE4 and GOS9 were adequate, the amplicons were small and easily confused with primer dimers. Non-specific amplification of the PLD gene was present in maize and potato. Real-time quantitative PCR detection indicated that PLD, SPS and PEPC displayed good specificity, with R~2 of the standard curve greater than 0.98, while the amplification efficiency ranged between 90% and 110%. Both the detection sensitivities of PLD and PEPC were five copies and that of SPS was ten copies. RBE4 showed typical amplification in maize, beet and Arabidopsis, while GOS9 was found in maize, tobacco and oats. PEPC exhibited excellent detection sensitivity and species specificity, which made it a potentially useful application in GM-rice supervision and administration. Additionally, SPS and PLD are also suitable for GM-rice detection. This study effectively established a foundation for GMO detection,which not only provides vital technical support for GMO identification, but also is of great significance for enhancing the comparability of detection results, and the standardization of ERG testing in GM-rice.
Endogenous reference genes(ERGs) provide vital information regarding genetically modified organisms(GMOs). The successful detection of ERGs can identity GMOs and the source of genes, verify stability and reliability of the detection system, and calculate the level of genetically modified(GM)ingredients in mixtures. The reported ERGs in rice include sucrose-phosphate synthase(SPS),phospholipase D(PLD), RBE4 and rice root-specific GOS9 genes. Based on the characteristics of ERGs,a new ERG gene, phosphoenolpyruvate carboxylase(PEPC), was selected, and further compared with the four existing genes. A total of 18 rice varieties and 29 non-rice crops were used to verify the interspecies specificity, intraspecies consistency, sensitivity, stability and reliability of these five ERGs using qualitative and quantitative PCR. Qualitative detection indicated that SPS and PEPC displayed sufficient specificity, and the detection sensitivity was 0.05% and 0.005%, respectively. Although the specificity of both RBE4 and GOS9 were adequate, the amplicons were small and easily confused with primer dimers. Non-specific amplification of the PLD gene was present in maize and potato. Real-time quantitative PCR detection indicated that PLD, SPS and PEPC displayed good specificity, with R~2 of the standard curve greater than 0.98, while the amplification efficiency ranged between 90% and 110%. Both the detection sensitivities of PLD and PEPC were five copies and that of SPS was ten copies. RBE4 showed typical amplification in maize, beet and Arabidopsis, while GOS9 was found in maize, tobacco and oats. PEPC exhibited excellent detection sensitivity and species specificity, which made it a potentially useful application in GM-rice supervision and administration. Additionally, SPS and PLD are also suitable for GM-rice detection. This study effectively established a foundation for GMO detection,which not only provides vital technical support for GMO identification, but also is of great significance for enhancing the comparability of detection results, and the standardization of ERG testing in GM-rice.
引文
Bawa A S,Anilakumar K R.2013.Genetically modified foods:Safety,risks and public concerns:A review.J Food Sci Technol,50(6):1035-1046.
Chaouachi M,Alaya A,Ali I B H,Hafsa A B,Nabi N,Bérard A,Romaniuk M,Skhiri F,Sa?d K.2013.Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet“Beta vulgaris L.”:GMO application.Plant Cell Rep,32(1):117-128.
Chen H Y,Liang X M,Chen S X,Wang Z H,Li L,Huang W S,Hu X Z,Zhu S F,Chen H J.2010.Entry-exit inspection and quarantine industry standard of P.R.China for the detection of GM rice(SN/T 2584-2010):Protocol of real time polymerase chain reaction for detecting genetically modified components in rice and its derived products.Beijing:Chinese Standard Publishing.
Cheng N,Shang Y,Xu Y C,Zhang L,Luo Y B,Huang K L,Xu WT.2017.On-site detection of stacked genetically modified soybean based on event-specific TM-LAMP and a DNAzymelateral flow biosensor.Biosens Bioelectron,91:408-416.
Ding J Y,Jia J W,Yang L T,Wen H B,Zhang C M,Liu W X,Zhang D B.2004.Validation of a rice specific gene,sucrose phosphate synthase,used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes.J Agric Food Chem,52(11):3372-3377.
Gryson N.2010.Effect of food processing on plant DNAdegradation and PCR-based GMO analysis:A review.Anal Bioanal Chem,396(6):2003-2022.
Hernández M,Esteve T,Pla M.2005.Real-time polymerase chain reaction based assays for quantitative detection of barley,rice,sunflower,and wheat.J Agric Food Chem,53(18):7003-7009.
Jeong S C,Pack I S,Cho E Y,Youk E S,Park S,Yoon W K,Kim C G,Choi Y D,Kim J K,Kim H M.2007.Molecular analysis and quantitative detection of a transgenic rice line expressing a bifunctional fusion TPSP.Food Control,18(11):1434-1442.
Jiang L X,Yang L T,Zhang H B,Guo J C,Mazzara M,van den EG,Zhang D B.2009.International collaborative study of the endogenous reference gene,sucrose phosphate synthase(SPS),used for qualitative and quantitative analysis of genetically modified rice.J Agric Food Chem,57(9):3525-3532.
Jin S B,Jin W W,Ding Y,Song Y C.2000.Genomic homology comparison of maize and rice using in situ hybridization.Chin Sci Bull,45(22):2431-2434.(in Chinese with English abstract)
JRC(Joint Research Centre).2006.Event-specific method for the quantitation of rice line LLRICE62 using real-time PCR.Community Reference Laboratory for GM Food and Feed.Italy.
Kou J P,Tang Q L,Zhang X F.2015.Agricultural GMO safety administration in China.J Integr Agric,14(11):2157-2165.
Li Y H,Peng Y F,Hallerman E M,Wu K M.2014.Biosafety management and commercial use of genetically modified crops in China.Plant Cell Rep,33(4):565-573.
Pray C,Huang J,Hu R F,Deng H Y,Yang J,Morin X K.2018.Prospects for cultivation of genetically engineered food crops in China.Glob Food Secur AGR,16:133-137.
Shang Y,Zhu P Y,Xu W T,Guo T X,Tian W Y,Luo Y B,Huang K L.2013.Single universal primer multiplex ligation-dependent probe amplification with sequencing gel electrophoresis analysis.Anal Biochem,443(2):243-248.
Wang F,Dang C,Chang X F,Tian J C,Lu Z B,Chen Y,Ye G Y.2017.Variation among conventional cultivars could be used as a criterion for environmental safety assessment of Bt rice on nontarget arthropods.Sci Rep,7:41918.
Wang W X,Zhu T H,Lai F X,Fu Q.2011.Event-specific qualitative and quantitative detection of transgenic rice Kefeng-6by characterization of the transgene flanking sequence.Eur Food Res Technol,232(2):297-305.
Wu G,Zhang L,Wu Y H,Cao Y L,Lu C M.2010.Comparison of Five endogenous reference genes for specific PCR detection and quantification of Brassica napus.J Agric Food Chem,58(5):2812-2817.
Xu W T,Bai W B,Guo F,Luo Y B,Yuan Y F,Huang K L.2008.A papaya-specific gene,papain,used as an endogenous reference gene in qualitative and real-time quantitative PCRdetection of transgenic papayas.Eur Food Res Technol,228:301.
Xu W T,Shang Y,Zhu P Y,Zhai Z F,He J,Huang K L,Luo Y B.2013.Randomly broken fragment PCR with 5′end-directed adaptor for genome walking.Sci Rep,3:3465.
Yang L T,Pan A H,Jia J W,Ding J Y,Chen J X,Cheng H,Zhang C M,Zhang D B.2005.Validation of a tomato-specific gene,LAT52,used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic tomatoes.JAgric Food Chem,53(2):183-190.
Chaouachi M,Alaya A,Ali I B H,Hafsa A B,Nabi N,Bérard A,Romaniuk M,Skhiri F,Sa?d K.2013.Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet“Beta vulgaris L.”:GMO application.Plant Cell Rep,32(1):117-128.
Chen H Y,Liang X M,Chen S X,Wang Z H,Li L,Huang W S,Hu X Z,Zhu S F,Chen H J.2010.Entry-exit inspection and quarantine industry standard of P.R.China for the detection of GM rice(SN/T 2584-2010):Protocol of real time polymerase chain reaction for detecting genetically modified components in rice and its derived products.Beijing:Chinese Standard Publishing.
Cheng N,Shang Y,Xu Y C,Zhang L,Luo Y B,Huang K L,Xu WT.2017.On-site detection of stacked genetically modified soybean based on event-specific TM-LAMP and a DNAzymelateral flow biosensor.Biosens Bioelectron,91:408-416.
Ding J Y,Jia J W,Yang L T,Wen H B,Zhang C M,Liu W X,Zhang D B.2004.Validation of a rice specific gene,sucrose phosphate synthase,used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes.J Agric Food Chem,52(11):3372-3377.
Gryson N.2010.Effect of food processing on plant DNAdegradation and PCR-based GMO analysis:A review.Anal Bioanal Chem,396(6):2003-2022.
Hernández M,Esteve T,Pla M.2005.Real-time polymerase chain reaction based assays for quantitative detection of barley,rice,sunflower,and wheat.J Agric Food Chem,53(18):7003-7009.
Jeong S C,Pack I S,Cho E Y,Youk E S,Park S,Yoon W K,Kim C G,Choi Y D,Kim J K,Kim H M.2007.Molecular analysis and quantitative detection of a transgenic rice line expressing a bifunctional fusion TPSP.Food Control,18(11):1434-1442.
Jiang L X,Yang L T,Zhang H B,Guo J C,Mazzara M,van den EG,Zhang D B.2009.International collaborative study of the endogenous reference gene,sucrose phosphate synthase(SPS),used for qualitative and quantitative analysis of genetically modified rice.J Agric Food Chem,57(9):3525-3532.
Jin S B,Jin W W,Ding Y,Song Y C.2000.Genomic homology comparison of maize and rice using in situ hybridization.Chin Sci Bull,45(22):2431-2434.(in Chinese with English abstract)
JRC(Joint Research Centre).2006.Event-specific method for the quantitation of rice line LLRICE62 using real-time PCR.Community Reference Laboratory for GM Food and Feed.Italy.
Kou J P,Tang Q L,Zhang X F.2015.Agricultural GMO safety administration in China.J Integr Agric,14(11):2157-2165.
Li Y H,Peng Y F,Hallerman E M,Wu K M.2014.Biosafety management and commercial use of genetically modified crops in China.Plant Cell Rep,33(4):565-573.
Pray C,Huang J,Hu R F,Deng H Y,Yang J,Morin X K.2018.Prospects for cultivation of genetically engineered food crops in China.Glob Food Secur AGR,16:133-137.
Shang Y,Zhu P Y,Xu W T,Guo T X,Tian W Y,Luo Y B,Huang K L.2013.Single universal primer multiplex ligation-dependent probe amplification with sequencing gel electrophoresis analysis.Anal Biochem,443(2):243-248.
Wang F,Dang C,Chang X F,Tian J C,Lu Z B,Chen Y,Ye G Y.2017.Variation among conventional cultivars could be used as a criterion for environmental safety assessment of Bt rice on nontarget arthropods.Sci Rep,7:41918.
Wang W X,Zhu T H,Lai F X,Fu Q.2011.Event-specific qualitative and quantitative detection of transgenic rice Kefeng-6by characterization of the transgene flanking sequence.Eur Food Res Technol,232(2):297-305.
Wu G,Zhang L,Wu Y H,Cao Y L,Lu C M.2010.Comparison of Five endogenous reference genes for specific PCR detection and quantification of Brassica napus.J Agric Food Chem,58(5):2812-2817.
Xu W T,Bai W B,Guo F,Luo Y B,Yuan Y F,Huang K L.2008.A papaya-specific gene,papain,used as an endogenous reference gene in qualitative and real-time quantitative PCRdetection of transgenic papayas.Eur Food Res Technol,228:301.
Xu W T,Shang Y,Zhu P Y,Zhai Z F,He J,Huang K L,Luo Y B.2013.Randomly broken fragment PCR with 5′end-directed adaptor for genome walking.Sci Rep,3:3465.
Yang L T,Pan A H,Jia J W,Ding J Y,Chen J X,Cheng H,Zhang C M,Zhang D B.2005.Validation of a tomato-specific gene,LAT52,used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic tomatoes.JAgric Food Chem,53(2):183-190.