Scientia Silvae Sinicae ›› 2020, Vol. 56 ›› Issue (10): 45-52.doi: 10.11707/j.1001-7488.20201005
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Lei Zhang,Jianjun Hu*
Received:
2019-10-30
Online:
2020-10-25
Published:
2020-11-26
Contact:
Jianjun Hu
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Lei Zhang,Jianjun Hu. An Analysis of T-DNA Insertion Loci and Detection of the Locus-Specific of Transgenic Populus nigra Lines with BtCry1Ac[J]. Scientia Silvae Sinicae, 2020, 56(10): 45-52.
Table 1
Primer sequences for insertion sites"
引物名称 Primer name | 引物序列 Primer sequence (5′—3′) |
Bt-F | GAATTCGCTAGGAACCAAGCCATT |
Bt-R | AAGTATATCCATCAAATGTGGACT |
LAD1 | ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA |
LAD2 | ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT |
LAD3 | ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA |
LAD4 | ACGATGGACTCCAGAGCGGCCGCBDNBNNNCGGT |
AC1 | ACGATGGACTCCAGAG |
LB-0A | CCCAACTTAATCGCCTTGCAGCACATC |
LB-1A | ACGATGGACTCCAGTCCGGCCGATTTGGGTGATGG TTCACGTAGTGGG |
LB-2A | TGGACCGCTTGCTGCAACTCTCTC |
RB-0A | GGATACCGAGGGGAATTTATGGAACG |
RB-1A | ACGATGGACTCCAGTCCGGCCGATAGTGACCTTAG GCGACTTTTGAACG |
RB-2A | GTCATCGGCGGGGGTCATAACG |
RB-F | TGTGGAGCATTGGTGTGAAG |
RB-R | TTGAGTATGCCCTGCTGACA |
LB-F | CCAACAATCCCCCAGATGAA |
LB-R | AGGACACGGAAACCTTCGGA |
Table 2
Primer sequence for qRT-PCR"
引物名称 Primer name | 引物序列(正向) Primer sequence (Forward)(5′—3′) | 引物序列(反向) Primer sequence (Reverse)(5′—3′) |
Actin | AAACTGTAATGGTCCTCCCTCCG | GCATCATCACAATCACTCTCCGA |
qBt | TGTATGGGGACCGGATTCTA | CGAGCCTCGAAAACTACCAT |
qSPK | TTTGGAACGATCACAGCTGC | GCTCTCCAGTTCCAGCACTA |
qATM | AGGGGAAGGAATGCGAAAGA | GTCCCCAATGCCAACCTTG |
qIBG | ACTCTACCAAGTCAGCCACC | AGGAGTGCATGGGAAAGTGA |
qPIF1 | TGCCGGCTCTTAGAATTTGC | GACCCAAAGACTTGCAAGCA |
Fig.1
PCR detection of transgenic plants and expression level of BtCry1Ac A: T-DNA of vector pBIN437; B: PCR detection of transgenic poplar; C: Semi-quantitative PCR analysis of the BtCry1Ac. M: DL2000 DNA marker; 1-8: CK3, Populus deltoides 'Danhong', n12, n222, B3-44, B3-102, B3-132, B3-153 (B3:P. deltoides 'Danhong'×n222). "
Fig.2
Right boundary sequence of transgenic P. nigra n12 with BtCry1Ac A: The hiTAIL-PCR of right boundary in n12(M: DL2000 DNA marker; LAD1, LAD2, LAD3, LAD4 are degenerate primers; 2: The PCR products of AC1 and RB-1A; 3: The PCR products of AC1 and RB-2A); B: Right-boundary sequence and splicing position characteristics of n12. "
Fig.3
Left boundary sequence of transgenic P. nigra n222 A: The hiTAIL-PCR of left boundary in n222(M: DL2000 DNA marker; LAD1, LAD2, LAD3, LAD4 are degenerate primers; 2: The PCR products of AC1 and LB-1A; 3: The PCR products of AC1 and LB-2A); B: Left-boundary sequence and splicing position characteristics of n222. "
Fig.5
Specific PCR assay for insertion site of transgenic P. nigra n12 and n222 A: Specific PCR detection on right boundary in n12(M: DL2000 DNA marker; 1-4: CK3, n12, n12, n222); B: Specific PCR detection on left boundary in n222(M: DL2000 DNA marker; 1-8: CK3, P. deltoides 'Danhong', n12, n222, B3-44, B3-102, B3-132, B3-153). "
Fig.6
The expression levels of genes located at the T-DNA insertion sites A: Tissue specific expression of SPK, ATM and IBG; B: Relative expression level of SPK and ATM in transgenic P. nigra n12; C: Relative expression level of IBG and PIF in transgenic P. nigra n222 and hybrids. DH:P. deltoides 'Danhong'. "
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