中国农业科学 ›› 2020, Vol. 53 ›› Issue (7): 1322-1337.doi: 10.3864/j.issn.0578-1752.2020.07.003
李俊1,李夏莹2,王颢潜2,翟杉杉1,陈子言2,高鸿飞1,李允静1,吴刚1,张秀杰2,武玉花1
收稿日期:
2019-08-07
接受日期:
2019-11-13
出版日期:
2020-04-01
发布日期:
2020-04-14
作者简介:
李俊,E-mail:lijuner126@126.com。
基金资助:
Jun LI1,Xia-ying LI2,Jing-qian WANG2,Shanshan Zhai1,Zi-yan CHEN2,Hong-fei GAO1,YunJing LI1,Gang WU1,Xiu-jie ZHANG2,Yu-hua WU1
Received:
2019-08-07
Accepted:
2019-11-13
Online:
2020-04-01
Published:
2020-04-14
摘要: 【目的】 转基因油菜是四大转基因作物之一,是中国转基因生物安全监管的重要对象,转基因检测为转基因安全监管提供技术支撑。转基因筛查是转基因检测的第一步,筛查靶标设置不合理会导致漏检部分转基因成分。建立转基因油菜筛查策略,并研制与筛查策略配套的阳性质粒分子,将为中国的转基因油菜安全监管提供强有力的技术支撑。【方法】 通过收集数据库中登记的转基因油菜品种的外源基因元件信息,分析转基因油菜品种中常用的调控元件和标记基因,基于最大筛查覆盖率原则,确定转基因油菜的筛查靶标。通过检索数据库或查询专利,收集筛查元件的核苷酸序列。一个筛查元件通常有多个标准方法,查阅各筛查元件的检测标准,分析各标准中普通PCR引物对和实时荧光PCR引物/探针组合在筛查元件核苷酸序列中的位置,根据引物探针的结合位点,确定拟构建到质粒上的各筛查元件的核苷酸序列。人工合成各筛查元件和油菜内标基因的融合序列,克隆到常用质粒pUC18,构建阳性质粒分子。采用各筛查元件的普通PCR和实时荧光PCR方法,评估阳性质粒分子的适用性。【结果】 建立了转基因油菜的筛查策略,通过检测CaMV 35S启动子、FMV 35S启动子、Bar、PAT、CP4-EPSPS、NPTⅡ、HPT、NOS终止子和PinⅡ终止子共9个基因元件,可实现已知信息转基因油菜品种的全覆盖。构建出聚合9个筛查元件和2个油菜内标基因HMG I/Y和CruA的转基因油菜筛查质粒分子pYCSC-1905。9个筛查元件和2个油菜内标基因的扩增效率均在90%—110%,证明质粒分子上的不同靶标序列没有相互干扰,影响PCR的扩增效率。质粒分子pYCSC-1905可用作9个筛查元件和2个油菜内标基因的通用阳性对照,适用于国家标准(GB/T和农业农村部公告)、出入境检验检疫行业标准(SN/T)和欧盟标准。【结论】 提出的转基因油菜筛查策略涵盖9个基因元件,可实现从商业化到安全评价各阶段转基因油菜的筛查检测,显著降低转基因油菜的筛查漏检率。研制的配套质粒分子pYCSC-1905为转基因油菜筛查和各基因元件标准方法的应用提供了通用标准样品,保证检测机构间检测数据的准确性和可比性。
李俊,李夏莹,王颢潜,翟杉杉,陈子言,高鸿飞,李允静,吴刚,张秀杰,武玉花. 转基因油菜筛查阳性质粒分子的研制及应用[J]. 中国农业科学, 2020, 53(7): 1322-1337.
Jun LI,Xia-ying LI,Jing-qian WANG,Shanshan Zhai,Zi-yan CHEN,Hong-fei GAO,YunJing LI,Gang WU,Xiu-jie ZHANG,Yu-hua WU. Development and Application of Plasmid Reference Molecule for Genetically Modified Rapeseed Screening[J]. Scientia Agricultura Sinica, 2020, 53(7): 1322-1337.
表1
ISAAA数据库中登记的转基因油菜品种"
类型 Type | 数目 Number | 名称 Name |
---|---|---|
独立转化体 Event | 26 | 23-18-17、23-198、61061、73496、DHA Canola、GT200、GT73、Topas 19/2(HCN10、HCN92)、T45、MON88302、MPS961、MPS962、MPS963、MPS964、MPS965、MS1、MS11、MS8、OXY235、PHY14、PHY23、PHY35、PHY36、RF1、RF2、RF3 |
复合性状品种 Gene-stacked variety | 14 | 73496×RF3、HCN28×MON88302、MON88302×MS8×RF3、MON88302×RF3、MS1×MON88302、MS1×RF1、MS1×RF2、MS1×RF3、MS8×MON88302、MS8×RF3、MS8×RF3×GT73、RF1×MON88302、HCN92×MON88302、RF2×MON88302 |
表2
9个筛查元件在油菜转化体中的分布"
编号 No. | 转化体 Event | P-CaMV35S | P-FMV35S | Bar | mCP4-EPSPS | HPT | NPTⅡ | PAT | T-NOS | T-PinⅡ |
---|---|---|---|---|---|---|---|---|---|---|
1 | T45 | √ | √ | |||||||
2 | GT73 / RT73 | √ | √ | |||||||
3 | GT200 | √ | √ | |||||||
4 | MS8 | √ | √ | |||||||
5 | MS1 | √ | √ | √ | ||||||
6 | RF1 | √ | √ | √ | ||||||
7 | RF2 | √ | √ | √ | ||||||
8 | RF3 | √ | √ | |||||||
9 | OXY235 | √ | √ | |||||||
10 | TOPAS19/2 | √ | √ | √ | ||||||
11 | Falcon GS40/ 90pHoe6/A | √ | √ | |||||||
12 | Liberator L62 | √ | √ | |||||||
13 | 23-18-17, 23-198 | √ | √ | √ | ||||||
14 | HCN10 | √ | √ | |||||||
15 | PHY14,PHY35 | √ | √ | |||||||
16 | PHY36 | √ | √ | |||||||
17 | 73496 | √ | ||||||||
18 | MON88302 | √ | ||||||||
19 | 61061 | √ | √ | |||||||
20 | MS11 | √ |
表3
各筛查元件和油菜内标基因的核苷酸序列、标准方法信息"
元件/基因 Element/Gene | 序列长度 Length (bp) | 登录号 Accession No. | 靶标长度 Target size (bp) | PCR类型 PCR type | 引物/探针 Primer/Probe | 序列 Sequence(5′-3′) | 产物大小 Amplicon size (bp) | 标准 Standards |
---|---|---|---|---|---|---|---|---|
P-CaMV35Sq | 835 | AF485783.1 | 250 | CT-PCR | F | GCTCCTACAAATGCCATCATTGC | 195 | 1782-3-2012 [ SN/T 1197-2016 [ ISO 21569:2005 [ |
R | GATAGTGGGATTGTGCGTCATCCC | |||||||
35S-cf3 | CCACGTCTTCAAAGCAAGTGG | 123 | QL-ELE-00-004[ | |||||
35S-cr4 | TCCTCTCCAAATGAAATGAACTTCC | |||||||
RT-PCR | 35S-QF | CGACAGTGGTCCCAAAGA | 74 | 1782-3-2012[ SN/T 1201-2014[ SN/T 2705-2010[ | ||||
35S-QR | AAGACGTGGTTGGAACGTCTTC | |||||||
35S-QP | TGGACCCCCACCCACGAGGAGCATC | |||||||
F | GCCTCTGCCGACAGTGGT | 82 | GB/T 19495.4-2018[ SN/T 1197-2016[ SN/T 1204-2016[ QL-ELE-00-012[ | |||||
R | AAGACGTGGTTGGAACGTCTTC | |||||||
P | CAAAGATGGACCCCCACCCACG | |||||||
p35s-F | ATTGATGTGATATCTCCACTGACGT | 101 | GB/T 33526-2017[ | |||||
p35s-R | CCTCTCCAAATGAAATGAACTTCCT | |||||||
p35s-P | CCCACTATCCTTCGCAAGACCCTTCCT | |||||||
NPTⅡ | 795 | AF485783.1 | 669 | CT-PCR | TN5-1 | GGATCTCCTGTCATCT | 173 | QL-ELE-00-003[ |
TN5-2 | GATCATCCTGATCGAC | |||||||
APH2 short | CTCACCTTGCTCCTGCCGAGA | 215 | SN/T 1197-2016[ | |||||
APH2 reverse | CGCCTTGAGCCTGGCGAACAG | |||||||
NPTIIF68 | ACTGGGCACAACAGACAATCG | 289 | 1782-2-2012[ | |||||
NPTIIR356 | GCATCAGCCATGATGGATACTTT | |||||||
RT-PCR | F | AGGATCTCGTCGTGACCCAT | 183 | GB/T 19495.4-2018[ SN/T 1197-2016[ SN/T 2705-2010[ SN/T 1201-2014[ SN/T 1204-2016[ | ||||
R | GCACGAGGAAGCGGTCA | |||||||
P | CACCCAGCCGGCCACAGTCGAT | |||||||
qNPTIIF63 | CTATGACTGGGCACAACAGACA | 101 | 1782-2-2012[ | |||||
qNPTIIR163 | CGGACAGGTCGGTCTTGACA | |||||||
qNPTIIFP90 | CTGCTCTGATGCCGCCGTGTTCCG | |||||||
P-FMV35S | 596 | JN400388.1 | 393 | CT-PCR | FMV-1 | AAGCCTCAACAAGGTCAG | 196 | QL-ELE-00-010[ |
FMV-2 | CTGCTCGATGTTGACAAG | |||||||
FMV35S-F1 | AAGACATCCACCGAAGACTTA | 210 | 1782-3-2012 [ SN/T 1197-2016 [ ISO 21569:2005 [ | |||||
FMV35S-R1 | AGGACAGCTCTTTTCCACGTT | |||||||
RT-PCR | pFMV-F | CAAAATAACGTGGAAAAGAGCT | 78 | QL-ELE-00-015[ SN/T 1197-2016[ | ||||
pFMV-R | TCTTTTGTGGTCGTCACTGC | |||||||
pFMV | CTGACAGCCCACTCACTAATGC | |||||||
FMV35S-QF | AAGACATCCACCGAAGACTTA | 210 | 1782-3-2012[ SN/T 1201-2014[ SN/T 2705-2010[ | |||||
FMV35S-QR | AGGACAGCTCTTTTCCACGTT | |||||||
FMV35S-Qp | TGGTCCCCACAAGCCAGCTGCTCGA | |||||||
F | CGAAGACTTAAAGTTAGTGGGCATCT | 79 | GB/T 19495.4-2018[ SN/T 1204-2016[ | |||||
R | TTTTGTCTGGTCCCCACAA | |||||||
P | TGAAAGTAATCTTGTCAACATCGAGCAGCTGG | |||||||
mCP4-EPSPS | 1368 | JN400388.1 | 793 | CT-PCR | CP4 Synthetic F | GCATGCTTCACGGTGCAA | 108 | QL-ELE-00-019[ |
CP4 Synthetic R | TGAAGGACCGGTGGGAGAT | |||||||
F | GACTTGCGTGTTCGTTCTTC | 204 | SN/T 1197-2016[ | |||||
R | AACACCGTTGAGCTTGAGAC | |||||||
mCP4ESF | ACGGTGAYCGTCTTCCMGTTAC | 333 | 1861-5-2012[ | |||||
mCP4ESR | GAACAAGCARGGCMGCAACCA | |||||||
RT-PCR | F | GCAAATCCTCTGGCCTTTCC | 146 | GB/T 19495.4-2018[ | ||||
R | CTTGCCCGTATTGATGACGTC | SN/T 1204-2016[ | ||||||
P | TCATGTTCGGCGGTCTCGCG- | SN/T 1197-2016[ | ||||||
Bar | 552 | MH973511.1 | 433 | CT-PCR | Pat-Bar Fwd | CGTCAACCACTACATCGAGACAA | 69 | QL-ELE-00-022[ |
Pat-Bar Rev | GTCCACTCCTGCGGTTCCT | |||||||
bar-F | GAAGGCACGCAACGCCTACGA | 262 | 1782-6-2012[ | |||||
bar-R | CCAGAAACCCACGTCATGCCA | |||||||
F | ACCATCGTCAACCACTACATCG | 430 | SN/T 1197-2016[ | |||||
R | GCTGCCAGAAACCCACGTCAT | |||||||
RT-PCR | RapB-F1 | ACAAGCACGGTCAACTTCC | 60 | QL-ELE-00-014 [28] SN/T 1204-2016 [ | ||||
RapB-R1 | GAGGTCGTCCGTCCACTC | |||||||
RapB-S1 | TACCGAGCCGCAGGAACC | |||||||
F | ACAAGCACGGTCAACTTCC | 175 | GB/T 19495.4-2018[ SN/T 1197-2016[ SN/T 2705-2010[ SN/T 1201-2014[ | |||||
R | ACTCGGCCGTCCAGTCGTA | |||||||
P | CCGAGCCGCAGGAACCGCAGGAG | |||||||
PAT | 579 | DQ156557.1 | 499 | CT-PCR | Pat-Pat Fwd | CCGCGGTTTGTGATATCGTT | 109 | QL-ELE-00-021[ |
Pat-Pat Rev | TCTTGCAACCTCTCTAGATCATCAA | |||||||
PAT-F | GAAGGCTAGGAACGCTTACGA | 262 | 1782-6-2012[ | |||||
PAT-R | CCAAAAACCAACATCATGCCA | |||||||
F | GTCGACATGTCTCCGGAGAG | 191 | SN/T 1197-2016[ | |||||
R | GCAACCAACCAAGGGTATC | |||||||
RT PCR | Pat-F | CGCGGTTTGTGATATCGTTAAC | 108 | QL-ELE-00-025[ | ||||
Pat-R | TCTTGCAACCTCTCTAGATCATCAA | |||||||
Pat-P | AGGACAGAGCCACAAACACCACAAGAGTG | |||||||
PAT-KVM-5 | TTGAGGGTGTTGTGGCTGGTA | 68 | QT-ELE-00-002[ | |||||
Pat1-p | TGTCCAATCGTAAGCGTTCCT | |||||||
PAT-KVM-6 | CTTCCAGGGCCCAGCGTAAGCA | |||||||
F | GTCGACATGTCTCCGGAGAG | 191 | GB/T 19495.4-2018 [ SN/T 1197-2016 [ SN/T 2705-2010 [ SN/T 1201-2014 [ SN/T 1204-2016 [ | |||||
R | GCAACCAACCAAGGGTATC | |||||||
P | TGGCCGCGGTTTGTGATATCGTTAA | |||||||
T-NOS | 256 | AF485783.1 | 256 | CT-PCR | tNOS_NN_Fwd | GATTAGAGTCCCGCAATTATACATTTAA | 69 | QL-ELE-00-018[ |
tNOS D REV | TTATCCTAGKTTGCGCGCTATATTT | |||||||
HA-nos118-f | GCATGACGTTATTTATGAGATGGG | 118 | SN/T 1197-2016[ | |||||
HA-nos118-r | GACACCGCGCGCGATAATTTATCC | |||||||
NOS-F1 | GAATCCTGTTGCCGGTCTTG | 180 | SN/T 1197-2016[ | |||||
NOS-R1 | TTATCCTAGTTTGCGCGCTA | 1782-3-2012[ | ||||||
RT-PCR | 180-F | CATGTAATGCATGACGTTATTTATG | 84 | QL-ELE-00-011[ SN/T 1204-2016[ | ||||
180-R | TTGTTTTCTATCGCGTATTAAATGT | |||||||
Tm-180 | ATGGGTTTTTATGATTAGAGTCCCGCAA | |||||||
NOS-F2 | ATCGTTCAAACATTTGGCA | 165 | 1782-3-2012 [ GB/T 19495.4-2018 [ GB/T 33526-2017 [ SN/T 1197-2016 [ SN/T 2705-2010 [ | |||||
NOS-R2 | ATTGCGGGACTCTAATCATA | |||||||
NOS-P | CATCGCAAGACCGGCAACAGG | |||||||
T-PINⅡ | 310 | KP784700.1 | 310 | RT-PCR | F | GACTTGTCCATCTTCTGGATTGG | 105 | 未发表 Unpublished |
R | CACACAACTTTGATGCCCACAT | |||||||
P | AGTGATTAGCATGTCACTATGTGTGCATCC | |||||||
HPT | 1026 | AF234296.1 | 474 | CT-PCR | HptF226 | GAAGTGCTTGACATTGGGGAGT | 472 | 1782-2-2012[ |
HptR697 | AGATGTTGGCGACCTCGTATT | |||||||
RT-PCR | qHptF286 | CAGGGTGTCACGTTGCAAGA | 110 | |||||
qHptR395 | CCGCTCGTCTGGCTAAGATC | |||||||
qHptFP308 | TGCCTGAAACCGAACTGCCCGCTG | |||||||
HMG I/Y | 950 | AF127919 | 206 | RT-PCR | HMG I/Y-F | GGTCGTCCTCCTAAGGCGAAAG | 99 | 2031-9-2013 [ |
HMG I/Y-R | CTTCTTCGGCGGTCGTCCAC | |||||||
HMG I/Y-P | CGGAGCCACTCGGTGCCGCAACTT | |||||||
CT-PCR | hmg-F | TCCTTCCGTTTCCTCGCC | 206 | |||||
hmg-R | TTCCACGCCCTCTCCGCT | |||||||
CruA | 3113 | X14555 | 150 | RT-PCR | QCRUAF | GGCCAGGGCTTCCGTGAT | 101 | |
QCRUAR | CCGTCGTTGTAGAACCATTGG | |||||||
QCRUAP | AGTCCTTATGTGCTCCACTTTCTGGTGCA | |||||||
CT-PCR | CruAF398 | GGCCAGGGCTTCCGTGAT | 150 | |||||
CruAR547 | CTGGTGGCTGGCTAAATCGA |
[1] | HOLST-JENSEN A, RØNNING S B, LOVSETH A, BERDAL K G. PCR technology for screening and quantification of genetically modified organisms (GMOs). Analytical and Bioanalytical Chemistry, 2003,375:985-993. |
[2] | 黄新, 高宏伟, 李想, 凌杏园, 朱水芳, 陈洪俊, 潘良文, 曹际娟, 章桂明 . GB/T 19495.4-2018 转基因产品检测实时荧光定性聚合酶链式反应(PCR)检测方法. 北京: 中国标准出版社, 2018. |
HUANG X, GAO H W, LI X, LING X Y, ZHU S F, CHEN H J, PAN L W, CAO J J, ZHANG G M. GB/T 19495.4-2018 Detection of genetically modified organisms and derived products - Qualitative real-time polymerase chain reaction (PCR) methods. Beijing: China Standards Press, 2018. (in Chinese) | |
[3] | 付伟, 杜智欣, 王勤, 许文涛, 吴刚, 朱水芳, 刘晓飞 . GB/T 33526-2017转基因植物产品数字PCR检测方法. 北京: 中国标准出版社, 2017. |
FU W, DU Z X, WANG Q, XU W T, WU G, ZHU S F, LIU X F. GB/T 33526-2017 Genetically modified organism detection method by digital PCR. Beijing: China Standards Press, 2017. (in Chinese) | |
[4] | 谢家建, 沈平, 彭于发, 李葱葱, 宋贵文, 孙爻 . 农业部1782号公告-3-2012转基因植物及其产品成分检测调控原件CAMV35S启动子、FMV35S启动子、NOS终止子和CaMV35S终止子定性PCR方法. 北京: 中国农业出版社, 2012. |
XIE J J, SHEN P, PENG Y F, LI C C, SONG G W, SUN Y. Announcement by the Ministry of Agriculture No. 1782-3-2012 Detection of genetically modified plants and derived products - Qualitative PCR methods for the regulatory elements CaMV 35Spromoter, FMV35S promoter, NOS promoter, NOS terminator and CaMV35 terminator. Beijing: China Agriculture Press, 2012. (in Chinese) | |
[5] | 杨立桃, 厉建萌, 刘勇, 张大兵, 宋贵文, 兰青阔, 郭金超, 朱君 . 农业部1861号公告-5-2012转基因植物及其产品成分检测CP4-epsps基因定性PCR方法. 北京: 中国农业出版社, 2012. |
YANG L T, LI J M, LIU Y, ZHANG D B, SONG G W, LAN Q K, GUO J C, ZHU J. Announcement by the Ministry of Agriculture No. 1861-5-2012 Detection of genetically modified plants and derived products - Qualitative PCR method for CP4-epsps gene. Beijing: China Agriculture Press, 2012. (in Chinese) | |
[6] | 路兴波, 宋贵文, 李凡, 沈平, 杨立桃, 孙红炜, 武海斌, 王敏, 王鹏 . 农业部1782号公告-6-2012, 转基因植物及其产品成分检测bar或pat基因定性PCR方法. 北京: 中国农业出版社, 2012. |
LU X B, SONG G W, LI F, SHEN P, YANG L T, SUN H W, WU H B, WANG M, WANG P. Announcement by the Ministry of Agriculture No.1782-6-2012 Detection of genetically modified plants and derived products - Qualitative PCR method of bar or pat gene. Beijing: China Agriculture Press, 2012. (in Chinese) | |
[7] | 卢长明, 宋贵文, 吴刚, 武玉花, 曹应龙, 厉建萌, 罗军玲 . 农业部1782号公告-2-2012 转基因植物及其产品成分检测标记基因NPTII、HPT和PMI. 北京: 中国农业出版社, 2012. |
LU C M, SONG G W, WU G, WU Y H, CAO Y L, LI J M, LUO J L. Announcement by the Ministry of Agriculture No.1782-2-2012. 2012-06-06 Detection of genetically modified plants and derived products - Qualitative PCR methods for the marker genes NPTII, HPT and PMI. Beijing: China Agriculture Press, 2012. (in Chinese) | |
[8] | 卢长明, 刘信, 武玉花, 吴刚, 沈平, 杨立桃, 张大兵 . 农业部2031号公告-9-2013 转基因植物及其产品成分检测油菜内标准基因定性PCR方法. 北京: 中国农业出版社, 2013. |
LU C M, LIU X, WU Y H, WU G, SHEN P, YANG L T, ZHANG D B. Announcement by the Ministry of Agriculture No.2031-9-2013 Detection of genetically modified plants and derived products - Target-taxon-specific qualitative PCR method for rapeseed. Beijing: China Agriculture Press, 2012. (in Chinese) | |
[9] | 章桂明, 凌杏园, 潘广, 向才玉, 程颖慧, 康林, 余道坚, 龙海, 郑耘, 陈枝楠, 杨伟东 . SN/T 1201-2014 饲料中转基因植物成份PCR检测方法. 北京: 中国标准出版社, 2014. |
ZHANG G M, LING X Y, PAN G, XIANG C Y, CHENG Y H, KANG L, YU D J, LONG H, ZHENG Y, CHEN Z N, YANG W D. SN/T 1201-2014. Protocol of PCR for detection of genetically modified feed. Beijing: China Standards Press, 2014. (in Chinese) | |
[10] | 李丹宁, 高东微, 钟玉清, 张隽, 万宇平 . SN/T 2705-2010 调味品中转基因植物成分实时荧光PCR定性检测方法. 北京: 中国标准出版社, 2010. |
LI D N, GAO D W, ZHONG Y Q, ZHANG X, WAN Y P. SN/T 2705-2010 Protocol of the Real-time fluorescence qualitative polymerase chain reaction for detecting genetically modified plant components in condiments. Beijing: China Standards Press, 2010. (in Chinese) | |
[11] | 潘良文, 李想, 吕蓉, 杨捷琳, 刘月明, 高琴 . SN/T 1197-2016 油菜中转基因成分检测普通PCR和实施荧光PCR方法. 北京: 中国标准出版社, 2016. |
PAN L W, LI X, LV R, YANG J L, LIU Y M, GAO Q. SN/T 1197-2016 Detection of genetically modified ingredients in rapeseed conventional and real-time PCR methods. Beijing: China Standards Press, 2016. (in Chinese) | |
[12] | 黄新, 高宏伟, 李想, 潘良文, 朱水芳, 陈洪俊, 段胜男 . SN/T 1204-2016 植物及其加工产品中转基因成分实时荧光PCR定性检验方法. 北京: 中国标准出版社, 2016. |
HUANG X, GAO H W, LI X, PAN L W, ZHU S F, CHEN H J, DUAN S N. SN/T 1204-2016 Protocol of the real-time PCR for detecting genetically modified plants and their derived products. Beijing: China Standards Press, 2016. (in Chinese) | |
[13] | 张丽, 武玉花, 吴刚, 曹应龙, 李均, 卢长明 . 转基因油菜筛查检测策略研究. 中国油料作物学报, 2012,34(1):74-81. |
ZHANG L, WU Y H, WU G, CAO Y L, LI J, LU C M . Strategy of transgenic rapeseed screening based on exogenous gene elements. Chinese Journal of Oil Crop Sciences, 2012,34(1):74-81. (in Chinese) | |
[14] | 刘冰 . 油菜中转基因成分的筛查检测策略. 西北农业学报, 2018,27(1):62-68. |
LIU B . Screening strategy of genetically modified rapeseed based on inserted genetic elements. Acta Agriculturae Boreali-occidentalis Sinica, 2018,27(1):62-68. (in Chinese) | |
[15] | WU Y, LI J, LI X, ZHAI S S, GAO H F, LI Y, ZHANG X, WU G . Development and strategy of reference materials for the DNA-based detection of genetically modified organisms. Analytical and Bioanalytical Chemistry, 2019,411(9):1729-1744. |
[16] | LI Z, LI X, WANG C, SONG G, PI L, ZHENG L, ZHANG D, YANG L . One novel multiple-target plasmid reference molecule targeting eight genetically modified canola events for genetically modified canola detection. Journal Agricultural and Food Chemistry, 2017,65(38):8489-8500. |
[17] | WU Y, LI J, WANG Y, LI X, LI Y, ZHU L, LI J, WU G . Development and application of a general plasmid reference material for GMO screening. Plasmid, 2016,87(88):28-36. |
[18] | 徐俊锋, 汪小福, 陈笑芸, 彭城, 徐晓丽, 朱青, 缪青梅 . 用于四种主要作物转基因筛查检测的标准质粒分子的构建及应用. 农业生物技术学报, 2015,23(9):1167-1177. |
XU J F, WANG X F, CHEN X Y, PENG C, XU X L, ZHU Q, MIAO Q M . Construction and application of standard plasmid molecules for screening detection of four major genetically modified crops. Journal of Agricultural Biotechnology, 2015,23(9):1167-1177. (in Chinese) | |
[19] | 牛丛丛, 文雯, 龙海飞, 张禹佳, 吴小文 . 基于文献计量学分析的我国转基因油菜研究进展. 科技情报开发与经济, 2015(25):156-157. |
NIU C C, WEN W, LONG H F, ZHANG Y J, WU X W . Research progress of genetically modified rapeseed in China based on bibliometric analysis. Science and Technology Information Development and Economy, 2015(25):156-157. (in Chinese) | |
[20] | ISO. Foodstuffs-Methods of analysis for the detection of genetically modified organisms and derived products-Qualitative nucleic acid based methods. ISO 21569: 2005, 1-69. |
[21] | EUROPEAN COMMISSION. JRC compendium of reference methods for GMO analysis - Qualitative PCR method for detection of Cauliflower Mosaic Virus 35S promoter. 2010, . Accessed 14 Aug 2018. |
[22] | EUROPEAN COMMISSION. JRC compendium of reference methods for GMO analysis - Qualitative duplex PCR method for detection of Cauliflower Mosaic Virus 35S promoter and nopaline synthase terminator (partim CaMV P-35S). 2010, . Accessed 14 Aug 2018. |
[23] | EUROPEAN COMMISSION. GMOMETHODS: EU database of reference methods - Qualitative PCR method for detection of neomycin phosphotransferase II gene. 2018, . Accessed 14 Aug 2018. |
[24] | EUROPEAN COMMISSION. GMOMETHODS: EU database of reference methods - Qualitative PCR method for detection of Figwort Mosaic Virus 35S promoter. . Accessed 14 Aug 2018. |
[25] | EUROPEAN COMMISSION. JRC compendium of reference methods for GMO analysis - Qualitative PCR method for detection of Figwort Mosaic Virus 35S promoter. . Accessed 14 Aug 2018. |
[26] | EUROPEAN COMMISSION. JRC compendium of reference methods for GMO analysis - Qualitative PCR method for detection of CP4 epsps gene. 2014, . Accessed 14 Aug 2018. |
[27] | EUROPEAN COMMISSION. JRC compendium of reference methods for GMO analysis - Qualitative PCR method for detection of phosphinothricin N-acetyltransferase (bar) gene. . Accessed 14 Aug 2018. |
[28] | EUROPEAN COMMISSION. JRC compendium of reference methods for GMO analysis - Qualitative PCR method for detection of phosphinothricin N-acetyltransferase gene. . Accessed 14 Aug 2018. |
[29] | EUROPEAN COMMISSION. JRC compendium of reference methods for GMO analysis - Qualitative PCR method for detection of phosphinothricin N-acetyltransferase (pat) gene. 2014, . Accessed 14 Aug 2018. |
[30] | EUROPEAN COMMISSION. JRC compendium of reference methods for GMO analysis - Qualitative duplex PCR method for detection of pat gene; bar gene (partimpat). . Accessed 14 Aug 2018. |
[31] | EUROPEAN COMMISSION. JRC compendium of reference methods for GMO analysis - Quantitative PCR method for detection of phosphinothricin N-acetyltransferase gene. 2010, . Accessed 14 Aug 2018. |
[32] | EUROPEAN COMMISSION. JRC compendium of reference methods for GMO analysis - Qualitative PCR method for detection of nopaline synthase terminator. 2014. . Accessed 14 Aug 2018. |
[33] | EUROPEAN COMMISSION. GMOMETHODS: EU database of reference methods - Qualitative PCR method for detection of nopaline synthase terminator. 2014, . Accessed 14 Aug 2018. |
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