欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2018, Vol. 44 ›› Issue (7): 949-955.doi: 10.3724/SP.J.1006.2018.00949

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

抗病转基因水稻M12及其产品成分的定性、定量PCR检测方法

李鹏1,2,张琳3,叶吉妮4,贺诗瑶4,贾军伟1,潘爱虎1,*(),唐雪明1,*()   

  1. 1 上海市农业科学院生物技术研究所 / 上海市农业遗传育种重点实验室, 上海 201106
    2 复旦大学生命科学学院, 上海 200433
    3 莱芜职业技术学院, 山东莱芜 271100
    4 台州学院生命科学学院, 浙江台州 318000
  • 收稿日期:2017-12-03 接受日期:2018-03-26 出版日期:2018-07-10 网络出版日期:2018-04-16
  • 通讯作者: 潘爱虎,唐雪明
  • 基金资助:
    本研究由国家自然科学基金项目(31500461), 上海市科技兴农重点攻关项目[沪农科攻字(2015)第4-3], 上海市农业科学院卓越团队项目[农科创2017(B-07)]和上海市农业转基因生物安全监管专项资助

A Qualitative and Quantitative PCR Detection Method for Disease-resistant Genetically Modified Rice M12 and Its Derivates

Peng LI1,2,Lin ZHANG3,Ji-Ni YE4,Shi-Yao HE4,Jun-Wei JIA1,Ai-Hu PAN1,*(),Xue-Ming TANG1,*()   

  1. 1 Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences / Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201106, China
    2 School of Life Science, Fudan University, Shanghai 200433, China
    3 Laiwu Vocational and Technical College, Laiwu 200433, Shandong, China
    4 School of Life Science, Taizhou University, Taizhou 318000, Zhejiang, China
  • Received:2017-12-03 Accepted:2018-03-26 Published:2018-07-10 Published online:2018-04-16
  • Contact: Ai-Hu PAN,Xue-Ming TANG
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (31500461), the Key Technologies Program of Shanghai Agricultural Commission [2015(4-3)], SAAS Program for Excellent Research Team [2017(B-07)], and the Agricultural GMO Safety Supervision Program of Shanghai.

RichHTML

78

PDF

912

PDF

42

摘要：

通过定性PCR扩增了转基因水稻M12转化体特异性片段和水稻内标基因(sps)片段, 将PCR产物酶切连接后构建到T载体, 得到适于转基因水稻M12品系特异性PCR检测的质粒分子pM12, 建立了事件特异性定性、定量PCR检测方法。定性PCR结果表明, 本方法可特异性检测出M12, 检测限可达100拷贝单倍体水稻基因组; 定量PCR结果表明, M12和sps定量PCR标准曲线R 2为0.998和0.997, 扩增效率为95.3%和108.4%, 重复性分析标准偏差范围为0.043~0.276, 定量极限和检测极限可达100和10拷贝。对转基因含量为1.0%的混合样品定量结果的相对偏差在8.0%以内。本研究建立的方法可用于转基因水稻M12及其加工产品成分的检测。

关键词: 品系特异性, 定性PCR, 定量PCR, M12

Abstract:

In this study, the specific sequence of genetically modified Rice M12 and the endogenous reference gene sps were amplified to construct a T vector as the plasmid pM12 for establishing the qualitative and quantitative PCR detection method of transgenic Rice M12 and its derivates. The qualitative PCR method could specifically quantify the samples of M12 with the detection sensitivity about 100 copies of the rice haploid genome. On the basis of SYBR Green qPCR assay, R 2 values of standard curves of M12 and sps were 0.998 and 0.997, the amplification efficiency was 95.3% and 108.4%, respectively. Moreover, the standard deviations (SD) of repeatability ranged from 0.043 to 0.276. The limit of quantification (LOQ) and limit of detection (LOD) were 100 and 10 copies, respectively. The mixed rice sample containing 1.0% gene transforming into rice was exactly quantified by the developed quantitative PCR method, and the quantified bias between the true value and tested value was below 8.0%. In conclusion, these methods can be used for identifying and quantifying M12 and its derivatives.

Key words: event-specific, qualitative PCR, quantitative PCR, M12

表1

定性、定量PCR反应所用引物"

引物
Primer		 引物序列
Primer sequence (5°-3°)
sps-F TTGCGCCTGAACGGATAT [13]
sps-R CGGTTGATCTTTTCGGGATG [13]
M12-SF GTTGGAGATTTTGGGCTTG [2]
M12-SR ATAGCCTCTCCACCCAAGCG [2]
M12-F GTTGGAGATTTTGGGCTTGC
M12-R CGGCAAGAAACCATCCAGTT

图1

抗病水稻M12特异性序列箭头代表引物的位置和方向。"

图2

pM12质粒的PCR鉴定M: DNA marker (DL1000); 1~2: 引物M12-F/R; 3~4: 引物sps-F/R; 5: 阴性对照。"

图3

M12事件特异性定性PCR的特异性检测M12品系特异性扩增(A)和水稻内标准基因sps扩增(B)。M: DNA marker (DL1000); 1: M12质粒pM12; 2: M12; 3: 花优14; 4: 华恢1号; 5: 旱优3号; 6: 旱恢3T; 7: 阴性对照。"

图4

M12事件特异性定性PCR的灵敏度分析M12品系特异性扩增(A)和水稻内标准基因sps扩增(B)。M: DNA marker (DL1000); 1~5: 10 000、1000、100、10、5个拷贝单倍体水稻基因组; 6: 阴性对照。"

表2

M12定量PCR检测方法的检测极限和定量极限分析"

拷贝数
Copy		 出现次数/检测次数
Signal rate (positive signals)		 循环数平均值
Ct mean		 标准差
SD
10000000 9/9 6.54 0.256
1000000 9/9 9.72 0.324
100000 9/9 13.15 0.236
10000 9/9 17.53 0.103
1000 9/9 20.82 0.265
100 9/9 24.12 0.231
10 2/9

图5

M12事件特异性扩增曲线及标准曲线质粒DNA拷贝数分别为10 000 000、1 000 000、100 000、10 000、1000、100拷贝。"

图6

内标基因sps扩增曲线及标准曲线质粒DNA拷贝数分别为10 000 000、1 000 000、100 000、10 000、1000、100拷贝。"

表3

M12和sps基因定量PCR检测体系的重复性分析"

质粒分子DNA拷贝数
Copies of plasmid DNA		 sps M12
循环数平均值
Ct mean		 循环数标准差
Ct SD		 循环数平均值
Ct mean		 循环数标准差
Ct SD
1000000 12.15 0.087 6.52 0.276
100000 15.30 0.076 9.72 0.154
10000 18.62 0.067 13.13 0.186
10000 21.91 0.043 17.52 0.133
1000 24.32 0.123 20.71 0.165
100 27.60 0.124 24.05 0.134

表4

转基因水稻M12定量PCR检测的混合样品分析"

混合样本
Mixed samples (%)		 平均值1
Mean 1		 平均值2
Mean 2		 平均值3
Mean 3		 平均值
Mean (%)		 标准差
SD
1.0 0.95 0.95 0.88 0.92 0.04
0 0 0 0 0 —
[1] 杨剑波, 倪大虎, 吴家道, 许传万, 贾士荣, 唐益雄, Fauquet C, 张世平. , 转Xa21基因杂交水稻选育和评价. 分子植物育种, 2006,4:174-180
doi: 10.3969/j.issn.1672-416X.2006.02.005
Yang J B, Ni D H, Wu J D, Xu C W, Jia S R, Tang Y X, Fauquet C, Zhang S P . Breeding and food safety evaluation of transgenic hybrid rice harboring Xa21 gene. Mol Plant Breed, 2006,4:174-180 (in Chinese with English abstract)
doi: 10.3969/j.issn.1672-416X.2006.02.005
[2] 中华人民共和国国家标准——农业部1485号公告-5-2010 转基因植物及其产品成分检测, 抗病水稻M12及其衍生品种定性PCR方法
National Standards of People’s Republic of China—Ministry of Agriculture Notice No.1485-5-2010, Detection of Genentically Modified Plants and Derived Products: Qualitative PCR Methods for Disease-resistant Rice M12 and Its Derivates
[3] Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T . Loop-mediated isothermal amplification of DNA. Nucl Acids Res, 2000,28:e63
doi: 10.1097/RLU.0b013e3181f49ac7 pmid: 102748
[4] Turkec A, Lucas S J, Karacanli B, Baykut A, Yuksel H . Assessment of a direct hybridization microarray strategy for comprehensive monitoring of genetically modified organisms (GMOs). Food Chem, 2016,194:399-409
doi: 10.1016/j.foodchem.2015.08.030 pmid: 26471572
[5] Vogelstein B, Kinzler K W . Digital PCR Proc Natl Acad Sci USA, 1999,96:9236-9241
doi: 10.1073/pnas.96.16.9236
[6] Feriotto G, Gardenghi S, Bianchi N, Gambari R . Quantitation of Bt-176 maize genomic sequences by surface plasmon resonance-based biospecific interaction analysis of multiplex polymerase chain reaction (PCR). J Agric Food Chem, 2003,51:4640-4646
doi: 10.1021/jf0341013 pmid: 14705890
[7] Li Y Q, Sun L, Qian J, Long L L, Li H N, Liu Q, Cai J R, Wang K . Fluorescent “on-off-on” switching sensor based on CdTe quantum dots coupled with multiwalled carbon nanotubes@graphene oxide nanoribbons for simultaneous monitoring of dual foreign DNAs in transgenic soybean. Biosens Bioelectron, 2017,92:26-32
doi: 10.1016/j.bios.2017.01.057 pmid: 28182975
[8] 胡宗悦, 须周恒, 卢亦愚 . 环介导等温扩增技术的常见问题分析与研究进展. 病毒学报, 2016,32:659-665
Hu Z R, Xu Z H, Lu Y Y . Analysis of common issues and research progress in loop mediated isothermal amplication. J Virol, 2016,32:659-665 (in Chinese with English abstract)
[9] 阮先乐, 张杰 . 转基因成分的检测方法综述. 江苏农业科学, 2017,45(5):12-15
doi: 10.15889/j.issn.1002-1302.2017.05.003
Ruan X L, Zhang J . Progress in techniques for the detection and analysis of genetically modified ingredients. Jiangsu Agric Sci, 2017,45(5):12-15 (in Chinese)
doi: 10.15889/j.issn.1002-1302.2017.05.003
[10] 蔡军, 李慧, 胡梦龙, 傅洋, 汪磊, 王璐 . 转基因成分分析检测技术研究进展. 食品安全质量检测学报, 2016,7:706-714
Cai J, Li H, Hu M L, Fu Y, Wang L, Wang L . Research progress in techniques for the detection and analysis of genetically modified ingredients. J Food Safety Qual, 2016,7:706-714 (in Chinese with English abstract)
[11] 王荣谈, 张建中, 刘冬儿, 张大兵, 杨立桃 . 转基因产品检测方法研究进展. 上海农业学报, 2010,26(1):116-119
doi: 10.3969/j.issn.1000-3924.2010.01.029
Wang R T, Zhang J Z, Liu D E, Zhang D B, Yang L T . Advance in detection methods of genetically modified organisms and their derived products. Acta Agric Shanghai, 2010,26(1):116-119 (in Chinese with English abstract)
doi: 10.3969/j.issn.1000-3924.2010.01.029
[12] Li P, Jia J W, Bai L, Pan A H, Tang X M . Identification and quantification of genetically modified Moonshade carnation lines using conventional and TaqMan real-time polymerase chain reaction methods. Appl Biochem Biotechnol, 2013,170:1151-1162
doi: 10.1007/s12010-013-0254-7 pmid: 23645416
[13] Jiang L X, Yang L T, Zhang H B, Guo J C, Mazzara M, Van den Eede G, Zhang D B, . 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, 2009,57:3525-3532
doi: 10.1038/423231a pmid: 19326953
[14] Zhang H, Yang L, Guo J, Li X, Jiang L, Zhang D . Development of one novel multiple-target plasmid for duplex quantitative PCR analysis of Roundup Ready soybean. J Agric Food Chem, 2008,56:5514-5520
doi: 10.1016/j.jbiotec.2008.07.533 pmid: 18570432
[15] Corbisier P, Broeders S, Charels D, Trapmann S, Vincent S, Emons H . Validation report. Certification of plasmidic DNA containing MON 810 maize DNA fragments. European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, EUR22948EN- 2007
[16] Zhang H B, Yang L T, Guo J C, Li X, Zhang D B . Development of one novel multiple-target plasmid for duplex quantitative PCR analysis of Roundup ready soybean. J Agric Food Chem, 2008,56:5514-5520
doi: 10.1016/j.jbiotec.2008.07.533 pmid: 18570432
[17] Yang L T, Guo J C, Pan A H, Zhang H B, Zhang K W, Wang Z M, Zhang D B . Event-specific quantitative Detection of nine genetically modified maizes using one novel standard reference molecule. J Agric Food Chem, 2007,55:15-24
doi: 10.1021/jf0615754 pmid: 17199308
[1] 李增强, 丁鑫超, 卢海, 胡亚丽, 岳娇, 黄震, 莫良玉, 陈立, 陈涛, 陈鹏. 铅胁迫下红麻生理特性及DNA甲基化分析[J]. 作物学报, 2021, 47(6): 1031-1042.
[2] 卢海, 李增强, 唐美琼, 罗登杰, 曹珊, 岳娇, 胡亚丽, 黄震, 陈涛, 陈鹏. 红麻DNA甲基化响应镉胁迫及甲基化差异基因的表达分析[J]. 作物学报, 2021, 47(12): 2324-2334.
[3] 郑清雷,余陈静,姚坤存,黄宁,阙友雄,凌辉,许莉萍. 甘蔗Rieske Fe/S蛋白前体基因ScPetC的克隆及表达分析[J]. 作物学报, 2020, 46(6): 844-857.
[4] 高世武,傅志伟,陈云,林兆里,许莉萍,郭晋隆. 甘蔗热带种金属硫蛋白家族基因的克隆及响应重金属胁迫的表达分析[J]. 作物学报, 2020, 46(02): 166-178.
[5] 孙婷婷,王文举,娄文月,刘峰,张旭,王玲,陈玉凤,阙友雄,许莉萍,李大妹,苏亚春. 甘蔗脂氧合酶基因ScLOX1的克隆与表达分析[J]. 作物学报, 2019, 45(7): 1002-1016.
[6] 王作敏,刘瑾,孙士超,张新宇,薛飞,李艳军,孙杰. 彩色棉多药和有毒化合物输出蛋白MATE家族基因的鉴定及表达分析[J]. 作物学报, 2018, 44(9): 1380-1392.
[7] 王玲,刘峰,戴明剑,孙婷婷,苏炜华,王春风,张旭,毛花英,苏亚春,阙友雄. 甘蔗ScWRKY4基因的克隆与表达特性分析[J]. 作物学报, 2018, 44(9): 1367-1379.
[8] 段方猛, 罗秋兰, 鲁雪莉, 齐娜伟, 刘宪舜, 宋雯雯. 玉米油菜素甾醇生物合成关键酶基因ZmCYP90B1的克隆及其对逆境胁迫的响应[J]. 作物学报, 2018, 44(03): 343-356.
[9] 苏亚春,黄珑,凌辉,王竹青,刘峰,苏炜华,黄宁,吴期滨,高世武,阙友雄. 甘蔗CDK基因的cDNA全长克隆与表达分析[J]. 作物学报, 2017, 43(01): 42-50.
[10] 翟玉山,邓宇晴,董萌,徐倩,程光远,彭磊,林彦铨*,徐景升*. 甘蔗捕光叶绿素a/b 结合蛋白基因ScLhca3 的克隆及表达[J]. 作物学报, 2016, 42(09): 1332-1341.
[11] 强治全,梁雅珺,于正阳,杜娅,张帅,朱维宁,张林生. 小麦wzy2-1基因的克隆及功能分析[J]. 作物学报, 2016, 42(08): 1253-1258.
[12] 苏炜华,刘峰,黄珑,苏亚春,黄宁,凌辉,吴期滨,张华,阙友雄. 甘蔗Ca2+/H+反向运转体基因的克隆与表达分析[J]. 作物学报, 2016, 42(07): 1074-1082.
[13] 刘峰,苏炜华,黄珑,肖新换,黄宁,凌辉,苏亚春,张华,阙友雄. 甘蔗Na+/H+逆转运蛋白基因的克隆与表达分析[J]. 作物学报, 2016, 42(04): 501-512.
[14] 丛亚辉，王婷婷，柳聚阁，王宁，高萌萌，李艳,盖钧镒. 大豆耐铝毒候选基因GmSTOP1的克隆与表达分析[J]. 作物学报, 2015, 41(12): 1802-1809.
[15] 成伟,郑艳茹,葛丹凤,程光远,翟玉山,邓宇晴,彭磊,谭向尧,徐景升*. 甘蔗转录激活因子ScCBF1基因的克隆与表达分析[J]. 作物学报, 2015, 41(05): 717-724.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2