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作物学报 ›› 2022, Vol. 48 ›› Issue (5): 1059-1070.doi: 10.3724/SP.J.1006.2022.13025

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

转基因玉米NK603基体标准物质研制

单露英1(), 李俊2, 李亮3, 张丽2, 王颢潜1, 高佳琪3, 吴刚2, 武玉花2,*(), 张秀杰1,*()   

  1. 1农业农村部科技发展中心, 北京 100025
    2中国农业科学院油料作物研究所 / 农业农村部油料作物生物学与遗传育种重点实验室, 湖北武汉 430062
    3中国农业科学院生物技术研究所, 北京 100081
  • 收稿日期:2021-03-19 接受日期:2021-09-09 出版日期:2022-05-12 网络出版日期:2021-10-11
  • 通讯作者: 武玉花,张秀杰
  • 作者简介:E-mail: 2581063925@qq.com
  • 基金资助:
    国家转基因生物新品种培育重大专项资助(2016ZX08012003)

Development of genetically modified maize (Zea mays L.) NK603 matrix reference materials

SHAN Lu-Ying1(), LI Jun2, LI Liang3, ZHANG Li2, WANG Hao-Qian1, GAO Jia-Qi3, WU Gang2, WU Yu-Hua2,*(), ZHANG Xiu-Jie1,*()   

  1. 1Development Center of Science and Technology, Ministry of Agriculture and Rural Affairs, Beijing 100025, China
    2Oil Crops Research Institute, Chinese Academy of Agricultural Sciences / Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture and Rural Affairs, Wuhan 430062, Hubei, China
    3Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2021-03-19 Accepted:2021-09-09 Published:2022-05-12 Published online:2021-10-11
  • Contact: WU Yu-Hua,ZHANG Xiu-Jie
  • Supported by:
    National Major Project for Developing New GM Crops(2016ZX08012003)

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摘要：

转基因安全管理和标识制度的实施需要标准化的检测方法和转基因检测标准物质, 转基因标准物质是获得准确、可靠、可比检测结果的保证。目前, 转基因玉米NK603已在我国批准进口用作加工原料, 其安全监管亟须制备标准物质。本研究将筛选后的转基因玉米NK603杂合种子和对应的非转基因受体种子, 按转基因质量分数为60.0 mg g-1、100.0 mg g-1、1000 mg g-1 (理论值)的比例配置了3个梯度浓度水平的转基因基体标准物质。利用二重微滴数字聚合酶链式反应(droplet digital polymerase chain reaction, ddPCR)对研制的标准物质进行均匀性与稳定性检测。结果表明: 研制的标准物质均匀性良好, 可在60℃下运输14 d, 稳定性不低于6个月。同时, 由8家实验室采用二重ddPCR方法联合测定标准物质NK603转化体与内标基因的拷贝数比值, 其标准值及不确定度为: (2.75±0.26)%、(4.68±0.39)%、(51.8±3.7)%。本批标物使用时最小取样量100 mg。可用于转基因食品和饲料中转基因成分NK603的定性和定量检测以及转基因玉米特异性检测方法的评价和实验室质量控制等领域。

关键词: 转基因玉米NK603, 基体标准物质, 二重ddPCR方法, 实时荧光PCR方法, 联合定值

Abstract:

The implementation of genetically modified organisms (GMO) safety management and labeling system requires standardized testing methods and reference materials (RMs). Furthermore, the RMs are the guarantee of obtaining accurate, reliable and comparable testing results. At present, genetically modified (GM) maize (Zea mays L.) NK603 has been approved to be imported as processing raw material in China, and its safety supervision urgently needs to prepare RMs. In this study, the heterozygous seeds of GM maize NK603 and the non-GM maize counterpart after identification were well milled and then gravimetrically mixed to prepare three levels of powder RMs according to the mass fractions of 60.0, 100.0, and 1000 mg g-1. The homogeneity and stability test by duplex ddPCR method showed that the RMs of GM maize NK603 were homogenous and stable for not less than six months. The property values were collaboratively characterized by eight laboratories using duplex ddPCR method. The certified values together with their expanded uncertainties were (2.75±0.26)%, (4.68±0.39)%, and (51.8±3.7)%, respectively. The minimum sample intake was determined to be 100 mg. This batch of RMs can be used for the qualitative and quantitative detection of NK603 event in food and feed, as well as for the evaluation of GM maize NK603-specific assays and laboratory quality control.

Key words: genetically modified maize NK603, reference materials, ddPCR, real-time fluorescent quantitative PCR, collaborative characterization

表1

引物探针信息表"

靶标
Target		 引物/探针名称Primer/probe name 序列
Primer sequence (5°-3°)		 产物大小Amplicon size (bp) 参考文献
Reference
NK603插入位点
NK603 insert site		 603CH-F GGAGACAAGAAGGGGAGGAGGTAAACAGAT 267 本研究
This study
603CH-R GGCAGGGTGTTGTTGTCCATTTTATGG
NK603 NK603F ATGAATGACCTCGAGTAAGCTTGTTAA 108 [12]
NK603R AAGAGATAACAGGATCCACTCAAACACT
NK603 P TGGTACCACGCGACACACTTCCACTC
zSSIIb zSSIIbF CGGTGGATGCTAAGGCTGATG 88 [13]
zSSIIbR AAAGGGCCAGGTTCATTATCCTC
zSSIIbP TAAGGAGCACTCGCCGCCGCATCTG
TC1507 TC1507F TAGTCTTCGGCCAGAATGG 58 [14]
TC1507R CTTTGCCAAGATCAAGCG
TC1507P TAACTCAAGGCCCTCACTCCG
MON863 MON863F TGTTACGGCCTAAATGCTGAACT 84 [15]
MON863R GTAGGATCGGAAAGCTTGGTAC
MON863P TGAACACCCATCCGAACAAGTAGGGTCA
GA21 GA21F CTTATCGTTATGCTATTTGCAACTTTAGA 112 [16]
GA21R TGGCTCGCGATCCTCCT
GA21P CATATACTAACTCATATCTCTTTCTCAACAGCAGGTGGGT
MON88017 MON88017F GAGCAGGACCTGCAGAAGCT 95 [17]
MON88017R TCCGGAGTTGACCATCCA
MON88017P FAM-TCCCGCCTTCAGTTTAAACAGAGTCGGGTRA

图1

实时荧光PCR方法对转基因玉米NK603和非转基因玉米的真实性鉴定 A: 转基因玉米NK603真实性鉴定; B: 非转基因玉米真实性鉴定。"

图2

转基因玉米NK603纯度鉴定结果"

图3

转基因玉米NK603基因型鉴定电泳图 泳道1~4、9: 均用NK603受体插入位点引物603CH-F/R扩增; 泳道5~8、10: 均用NK603转化体特异性引物NK603F/R扩增。"

图4

基体基因组DNA提取率电泳图 A: 试剂盒法; B: CTAB法; 1~9: 非转基因玉米材料; 10~18: 转基因玉米材料; N: 空白对照; M: DNA marker DL1000。"

表2

基体基因组DNA提取率结果"

样品
Sample		 转基因玉米
GM maize NK603		 非转基因玉米受体
Non-transgenic receptor maize
1 12.12 11.48
2 12.30 12.24
3 12.00 11.94
4 12.84 13.01
5 13.14 13.20
6 12.42 12.84
7 11.76 12.36
8 12.18 12.12
9 12.60 12.68
t检验t-test 0.64
平均抽提效率比
Average extraction ratio (%)		 99.5

表3

转基因玉米NK603a种子粉末基体标准物质的重量配比结果"

重复
Repetitive		 m1 (g) m2 (g) Δm1 (g) Δm2 (g) m (g) 转基因含量
MF (mg g-1)
1 12.005 187.995 0.145 2.181 200.000 60.0
2 12.004 187.994 0.145 2.181 199.998 60.0
3 12.006 187.995 0.145 2.181 200.001 60.0
4 12.005 187.996 0.145 2.181 200.001 60.0
5 12.004 187.996 0.145 2.181 200.000 60.0
6 12.005 187.995 0.145 2.181 200.000 60.0
平均值Mean 12.005 187.995 0.145 2.181 200.000 60.0

表4

转基因玉米NK603b种子粉末基体标准物质的重量配比结果"

重复
Repetitive		 m1 (g) m2 (g) Δm1 (g) Δm2 (g) m (g) 转基因含量
MF (mg g-1)
1 25.011 224.999 0.303 2.610 250.010 100.0
2 25.010 225.001 0.303 2.610 250.011 100.0
3 25.010 224.998 0.303 2.610 250.008 100.0
4 25.012 224.999 0.303 2.610 250.011 100.0
5 25.011 225.001 0.303 2.610 250.012 100.0
6 25.012 225.002 0.303 2.610 250.014 100.0
平均值Mean 25.011 225.000 0.303 2.610 250.011 100.0

图5

转基因玉米NK603基体标准物质均匀性检验测试结果"

表5

转基因玉米NK603基体标准物质检验测试数据的方差分析结果"

样品
Sample		 差来源
Soruces of variation		 差方和
Q		 自由度
df		 方差
S		 统计量
F		 F0.05 (14,30) 比较结果
Test data		 结论
Result
NK603a 瓶间Among bottles 0.43 14 0.031 1.93 2.04 F < F0.05 (14,30) 均匀Homogeneous
瓶内In the bottle 0.48 30 0.016
NK603b 瓶间Among bottles 1.18 14 0.084 1.76 F < F0.05 (14,30) 均匀Homogeneous
瓶内In the bottle 1.43 30 0.048
NK603c 瓶间Among bottles 16.87 14 1.200 0.85 F < F0.05 (14,30) 均匀Homogeneous
瓶内In the bottle 42.57 30 1.420

表6

转基因玉米NK603基体标准物质短期稳定性考察结果"

项目
Item		 NK603a NK603b NK603c
25℃ 37℃ 60℃ 25℃ 37℃ 60℃ 25℃ 37℃ 60℃
0 d 2.84 2.84 2.84 4.62 4.74 4.80 51.68 51.68 51.68
3 d 2.84 2.89 2.92 4.75 4.65 4.62 51.24 51.33 51.78
7 d 2.88 2.74 2.79 4.63 4.79 4.59 53.01 52.11 50.41
14 d 2.72 2.66 2.82 4.84 4.77 4.62 51.32 52.12 51.79
平均值Mean 2.82 2.78 2.84 4.71 4.74 4.66 51.8108 51.81 51.41
斜率(β1) -0.0082 -0.0150 0.0039 0.0121 0.0052 -0.0100 -0.0022 0.0458 -0.0060
与斜率相关的不确定度s(β1) 0.006 0.005 0.006 0.008 0.006 0.009 0.096 0.030 0.078
t0.95,n-2 4.3027 4.3027 4.3027 4.3027 4.3027 4.3027 4.3027 4.3027 4.3027
t0.95,n-2×s(β1) 0.0237 0.0226 0.0270 0.0350 0.0265 0.0375 0.4135 0.1300 0.3364
稳定性判断
Stability judgment		 |β1|≤t0.95,n-2×s(β1) |β1|≤t0.95,n-2×s(β1) |β1|≤t0.95,n-2×s(β1)
判断结果
Conclusion		 稳定
Stable		 稳定
Stable		 稳定
Stable		 稳定
Stable		 稳定
Stable		 稳定
Stable		 稳定
Stable		 稳定
Stable		 稳定
Stable

表7

转基因玉米NK603基体标准物质长期稳定性考察结果"

项目
Item		 NK603a NK603b NK603c
4℃ -20℃ 4℃ -20℃ 4℃ -20℃
0个月0 month 2.76 2.80 4.84 4.71 50.85 51.20
1个月1 month 2.83 2.86 4.75 4.76 50.61 51.36
2个月 2 months 2.81 2.77 4.78 4.79 50.67 51.73
4个月 4 months 2.74 2.86 4.81 4.83 51.86 50.69
6个月 6 months 2.81 2.87 4.82 4.77 51.59 51.64
平均值Mean 2.79 2.83 4.80 4.77 51.12 51.32
斜率(β1) 0.0011 0.0109 0.0023 0.0112 0.1910 0.0085
与斜率相关的不确定度s(β1) 0.0092 0.0093 0.0082 0.0083 0.0812 0.0984
t0.95,n-2 3.1824 3.1824 3.1824 3.1824 3.1824 3.1824
t0.95,n-2×s(β1) 0.0292 0.0296 0.0261 0.0265 0.2584 0.3132
稳定性判断Stability judgment |β1|≤t0.95,n-2×s(β1) |β1|≤t0.95,n-2×s(β1) |β1|≤t0.95,n-2×s(β1)
判断结果Conclusion 稳定Stable 稳定Stable 稳定Stable 稳定Stable 稳定Stable 稳定Stable

表8

转基因玉米NK603基体标准物质定值结果统计"

参数
Parameter		 结果Result
NK603a NK603b NK603c
标准值Certified value (copy/copy) (%) 2.75 4.68 51.80
组间SD SD between groups 0.03 0.06 0.63
组间RSD RSD between groups (%) 1.19 1.31 1.21

表9

转基因玉米NK603基体标准物质量值和扩展不确定度"

标准物质
名称
RMs name		 标准值Certified value Y (%) 定值相对
不确定度
urel.c		 均匀性相对不确定度urel,bb 短期稳定性相对不确定度urel,ss 长期稳定性相对不确定度urel,ls 相对标准不确定度urel,CRM 相对扩展不确定度Urel,CRM UCRM (k=2) (%)
NK603a 2.75 0.0202 0.025 0.028 0.020 0.047 0.094 0.26
NK603b 4.68 0.0210 0.024 0.025 0.011 0.041 0.082 0.39
NK603c 51.80 0.0203 0.007 0.026 0.010 0.035 0.071 3.70
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