Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (9): 114-124.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1482
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SUN Rui-fen1(), ZHANG Yan-fang2, NIU Su-qing1, GUO Shu-chun1, LI Su-ping1, YU Hai-feng1, NIE Hui1, MOU Ying-nan1
Received:
2020-12-07
Online:
2021-09-26
Published:
2021-10-25
SUN Rui-fen, ZHANG Yan-fang, NIU Su-qing, GUO Shu-chun, LI Su-ping, YU Hai-feng, NIE Hui, MOU Ying-nan. Expression Analysis and Functional Verification of the HaACO1 Gene in Sunflower[J]. Biotechnology Bulletin, 2021, 37(9): 114-124.
名称 Name | 编号 No. | 序列 Sequence(5'-3') | 用途 Application |
---|---|---|---|
qHaACO1 | F1 | GCTTCAAAGAAATGGTGGCT | HaACO1的qRT-PCR qRT-PCR of HaACO1 |
R1 | GGGAGATGGCGGAGATAGA | ||
18S rRNA | F2 | AGAAACGGCTACCACATCCA | 向日葵内参基因18S rRNA Reference gene 18S rRNA in sunflower |
R2 | TTGTTATTTATTGTCACTACCTCCC | ||
sHaACO1 | F3 | CGGGGTACCATGGAGGAGGAGACATTTCCAG Kpn I | HaACO1编码区扩增(引入KpnⅠ/BamⅠ位点) Amplification of HaACO1 coding region(carrying KpnⅠ/BamH I sites) |
R3 | CGGGATCCAGCAGTTGCAATGGGGTC Bam Ⅰ | ||
zHaACO1 | F4 | CGGGATCCATGGAGGAGGAGACATTTCCAG Bam HI | HaACO1编码区扩增(引入Bam HⅠ/KpnⅠ位点) Amplification of HaACO1 coding region(carrying BamHⅠ/KpnⅠ sites) |
R4 | CGGGGTACCTTAAGCAGTTGCAATGGGGTC Kpn I | ||
CaMV35S | p-F | AGACGTTCCAACCACGTCTTCA | 转基因烟草中HaACO1基因检测(CaMV 35S启动子近3'端序列) HaACO1 detection in transgenic tobacco(near 3' terminal sequence of CaMV35S promoter) |
NtEF-1α | F5 | TGAGATGCACCACGAAGCTC | 烟草内参基因EF-1α Reference gene EF-1α in tobacco |
R5 | CCAACATTGTCACCAGGAAGTG | ||
P5CS | F6 | TGGTCGTCAGCGGCTTAGAT | 烟草P5CS的qRT-PCR qRT-PCR of P5CS in tobacco |
R6 | TGCCAAACTGTCATTGTCCC | ||
POD | F7 | CCCTGGTGTTGTTTCTTGTG | 烟草POD的qRT-PCR qRT-PCR of POD in tobacco |
R7 | CCTGAGCCTGAACTTCTTGG | ||
MnSOD | F8 | GCAGACGGACCTTAGCAACA | 烟草MnSOD的qRT-PCR qRT-PCR of MnSOD in tobacco |
R8 | GGGAGCCAAAGTTAGTGTCG | ||
GuZnSOD | F9 | CGGGACCACATTACAATCCT | 烟草GuZnSOD的qRT-PCR qRT-PCR of GuZnSOD in tobacco |
R9 | ATCAGCGTGAACAACCACAG |
Table 1 Primer information
名称 Name | 编号 No. | 序列 Sequence(5'-3') | 用途 Application |
---|---|---|---|
qHaACO1 | F1 | GCTTCAAAGAAATGGTGGCT | HaACO1的qRT-PCR qRT-PCR of HaACO1 |
R1 | GGGAGATGGCGGAGATAGA | ||
18S rRNA | F2 | AGAAACGGCTACCACATCCA | 向日葵内参基因18S rRNA Reference gene 18S rRNA in sunflower |
R2 | TTGTTATTTATTGTCACTACCTCCC | ||
sHaACO1 | F3 | CGGGGTACCATGGAGGAGGAGACATTTCCAG Kpn I | HaACO1编码区扩增(引入KpnⅠ/BamⅠ位点) Amplification of HaACO1 coding region(carrying KpnⅠ/BamH I sites) |
R3 | CGGGATCCAGCAGTTGCAATGGGGTC Bam Ⅰ | ||
zHaACO1 | F4 | CGGGATCCATGGAGGAGGAGACATTTCCAG Bam HI | HaACO1编码区扩增(引入Bam HⅠ/KpnⅠ位点) Amplification of HaACO1 coding region(carrying BamHⅠ/KpnⅠ sites) |
R4 | CGGGGTACCTTAAGCAGTTGCAATGGGGTC Kpn I | ||
CaMV35S | p-F | AGACGTTCCAACCACGTCTTCA | 转基因烟草中HaACO1基因检测(CaMV 35S启动子近3'端序列) HaACO1 detection in transgenic tobacco(near 3' terminal sequence of CaMV35S promoter) |
NtEF-1α | F5 | TGAGATGCACCACGAAGCTC | 烟草内参基因EF-1α Reference gene EF-1α in tobacco |
R5 | CCAACATTGTCACCAGGAAGTG | ||
P5CS | F6 | TGGTCGTCAGCGGCTTAGAT | 烟草P5CS的qRT-PCR qRT-PCR of P5CS in tobacco |
R6 | TGCCAAACTGTCATTGTCCC | ||
POD | F7 | CCCTGGTGTTGTTTCTTGTG | 烟草POD的qRT-PCR qRT-PCR of POD in tobacco |
R7 | CCTGAGCCTGAACTTCTTGG | ||
MnSOD | F8 | GCAGACGGACCTTAGCAACA | 烟草MnSOD的qRT-PCR qRT-PCR of MnSOD in tobacco |
R8 | GGGAGCCAAAGTTAGTGTCG | ||
GuZnSOD | F9 | CGGGACCACATTACAATCCT | 烟草GuZnSOD的qRT-PCR qRT-PCR of GuZnSOD in tobacco |
R9 | ATCAGCGTGAACAACCACAG |
Fig.3 HaACO1 expression at different time under different abiotic stresses N:NaCl stress. J:Mechanical damage stress. T:Low temperature stress. S:Salicylic acid stress. *,**:Significant difference(P<0.05)and extremely significant difference(P<0.01),respectively,compared with 0 d
Fig. 7 HaACO1 expression analysis in transgenic plants under abiotic stresses CK: Untreatment; DW: lower temperature treatment under 4℃; PEG: the treatment with 20% PEG6000; NaCl: the treatment with 120 mol/L NaCl; **: extremely significant difference of transgenetic line T-10 compared with WT under stress (P<0.01)
Fig.8 Analyses of physiological parameters related to stress tolerance in transgenic tobacco **:Extremely significant difference of transgenic line T-11 compared with WT under stress(P<0.01). The same below
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