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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.

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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 —
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