转基因油菜筛查阳性质粒分子的研制及应用

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

Development and Application of Plasmid Reference Molecule for Genetically Modified Rapeseed Screening

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

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

Table 3 Information of nucleotide sequence and corresponding standardized method of screening elements and rapeseed reference genes

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 [4] SN/T 1197-2016 [11] ISO 21569:2005 [20] R GATAGTGGGATTGTGCGTCATCCC 35S-cf3 CCACGTCTTCAAAGCAAGTGG 123 QL-ELE-00-004[21] 35S-cr4 TCCTCTCCAAATGAAATGAACTTCC RT-PCR 35S-QF CGACAGTGGTCCCAAAGA 74 1782-3-2012[4] SN/T 1201-2014[9] SN/T 2705-2010[10] 35S-QR AAGACGTGGTTGGAACGTCTTC 35S-QP TGGACCCCCACCCACGAGGAGCATC F GCCTCTGCCGACAGTGGT 82 GB/T 19495.4-2018[2] SN/T 1197-2016[11] SN/T 1204-2016[12] QL-ELE-00-012[22] R AAGACGTGGTTGGAACGTCTTC P CAAAGATGGACCCCCACCCACG p35s-F ATTGATGTGATATCTCCACTGACGT 101 GB/T 33526-2017[3] p35s-R CCTCTCCAAATGAAATGAACTTCCT p35s-P CCCACTATCCTTCGCAAGACCCTTCCT NPTⅡ 795 AF485783.1 669 CT-PCR TN5-1 GGATCTCCTGTCATCT 173 QL-ELE-00-003[23] TN5-2 GATCATCCTGATCGAC APH2 short CTCACCTTGCTCCTGCCGAGA 215 SN/T 1197-2016[11] APH2 reverse CGCCTTGAGCCTGGCGAACAG NPTIIF68 ACTGGGCACAACAGACAATCG 289 1782-2-2012[7] NPTIIR356 GCATCAGCCATGATGGATACTTT RT-PCR F AGGATCTCGTCGTGACCCAT 183 GB/T 19495.4-2018[2] SN/T 1197-2016[11] SN/T 2705-2010[10] SN/T 1201-2014[9] SN/T 1204-2016[12] R GCACGAGGAAGCGGTCA P CACCCAGCCGGCCACAGTCGAT qNPTIIF63 CTATGACTGGGCACAACAGACA 101 1782-2-2012[7] qNPTIIR163 CGGACAGGTCGGTCTTGACA qNPTIIFP90 CTGCTCTGATGCCGCCGTGTTCCG P-FMV35S 596 JN400388.1 393 CT-PCR FMV-1 AAGCCTCAACAAGGTCAG 196 QL-ELE-00-010[25] FMV-2 CTGCTCGATGTTGACAAG FMV35S-F1 AAGACATCCACCGAAGACTTA 210 1782-3-2012 [4] SN/T 1197-2016 [11] ISO 21569:2005 [20] FMV35S-R1 AGGACAGCTCTTTTCCACGTT RT-PCR pFMV-F CAAAATAACGTGGAAAAGAGCT 78 QL-ELE-00-015[24] SN/T 1197-2016[11] pFMV-R TCTTTTGTGGTCGTCACTGC pFMV CTGACAGCCCACTCACTAATGC FMV35S-QF AAGACATCCACCGAAGACTTA 210 1782-3-2012[4] SN/T 1201-2014[9] SN/T 2705-2010[10] FMV35S-QR AGGACAGCTCTTTTCCACGTT FMV35S-Qp TGGTCCCCACAAGCCAGCTGCTCGA F CGAAGACTTAAAGTTAGTGGGCATCT 79 GB/T 19495.4-2018[2] SN/T 1204-2016[12] R TTTTGTCTGGTCCCCACAA P TGAAAGTAATCTTGTCAACATCGAGCAGCTGG mCP4-EPSPS 1368 JN400388.1 793 CT-PCR CP4 Synthetic F GCATGCTTCACGGTGCAA 108 QL-ELE-00-019[26] CP4 Synthetic R TGAAGGACCGGTGGGAGAT F GACTTGCGTGTTCGTTCTTC 204 SN/T 1197-2016[11] R AACACCGTTGAGCTTGAGAC mCP4ESF ACGGTGAYCGTCTTCCMGTTAC 333 1861-5-2012[5] mCP4ESR GAACAAGCARGGCMGCAACCA RT-PCR F GCAAATCCTCTGGCCTTTCC 146 GB/T 19495.4-2018[2] R CTTGCCCGTATTGATGACGTC SN/T 1204-2016[12] P TCATGTTCGGCGGTCTCGCG- SN/T 1197-2016[11] Bar 552 MH973511.1 433 CT-PCR Pat-Bar Fwd CGTCAACCACTACATCGAGACAA 69 QL-ELE-00-022[27] Pat-Bar Rev GTCCACTCCTGCGGTTCCT bar-F GAAGGCACGCAACGCCTACGA 262 1782-6-2012[6] bar-R CCAGAAACCCACGTCATGCCA F ACCATCGTCAACCACTACATCG 430 SN/T 1197-2016[11] R GCTGCCAGAAACCCACGTCAT RT-PCR RapB-F1 ACAAGCACGGTCAACTTCC 60 QL-ELE-00-014 [28] SN/T 1204-2016 [12] RapB-R1 GAGGTCGTCCGTCCACTC RapB-S1 TACCGAGCCGCAGGAACC F ACAAGCACGGTCAACTTCC 175 GB/T 19495.4-2018[2] SN/T 1197-2016[11] SN/T 2705-2010[10] SN/T 1201-2014[9] R ACTCGGCCGTCCAGTCGTA P CCGAGCCGCAGGAACCGCAGGAG PAT 579 DQ156557.1 499 CT-PCR Pat-Pat Fwd CCGCGGTTTGTGATATCGTT 109 QL-ELE-00-021[29] Pat-Pat Rev TCTTGCAACCTCTCTAGATCATCAA PAT-F GAAGGCTAGGAACGCTTACGA 262 1782-6-2012[6] PAT-R CCAAAAACCAACATCATGCCA F GTCGACATGTCTCCGGAGAG 191 SN/T 1197-2016[11] R GCAACCAACCAAGGGTATC RT PCR Pat-F CGCGGTTTGTGATATCGTTAAC 108 QL-ELE-00-025[30] Pat-R TCTTGCAACCTCTCTAGATCATCAA Pat-P AGGACAGAGCCACAAACACCACAAGAGTG PAT-KVM-5 TTGAGGGTGTTGTGGCTGGTA 68 QT-ELE-00-002[31] Pat1-p TGTCCAATCGTAAGCGTTCCT PAT-KVM-6 CTTCCAGGGCCCAGCGTAAGCA F GTCGACATGTCTCCGGAGAG 191 GB/T 19495.4-2018 [2] SN/T 1197-2016 [11] SN/T 2705-2010 [10] SN/T 1201-2014 [9] SN/T 1204-2016 [12] R GCAACCAACCAAGGGTATC P TGGCCGCGGTTTGTGATATCGTTAA T-NOS 256 AF485783.1 256 CT-PCR tNOS_NN_Fwd GATTAGAGTCCCGCAATTATACATTTAA 69 QL-ELE-00-018[32] tNOS D REV TTATCCTAGKTTGCGCGCTATATTT HA-nos118-f GCATGACGTTATTTATGAGATGGG 118 SN/T 1197-2016[11] HA-nos118-r GACACCGCGCGCGATAATTTATCC NOS-F1 GAATCCTGTTGCCGGTCTTG 180 SN/T 1197-2016[11] NOS-R1 TTATCCTAGTTTGCGCGCTA 1782-3-2012[4] RT-PCR 180-F CATGTAATGCATGACGTTATTTATG 84 QL-ELE-00-011[33] SN/T 1204-2016[12] 180-R TTGTTTTCTATCGCGTATTAAATGT Tm-180 ATGGGTTTTTATGATTAGAGTCCCGCAA NOS-F2 ATCGTTCAAACATTTGGCA 165 1782-3-2012 [4] GB/T 19495.4-2018 [2] GB/T 33526-2017 [3] SN/T 1197-2016 [11] SN/T 2705-2010 [10] 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[7] 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 [8] 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