Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (9): 1895-1906.doi: 10.16420/j.issn.0513-353x.2021-0700
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HU Ruolin1, WANG Jiali1, YANG Huiqin1, YUAN Chao1, NIU Yi1,*(), TANG Qinglin1, WEI Dayong1, TIAN Shibing2, YANG Yang2, WANG Zhimin1,*(
)
Received:
2021-10-16
Revised:
2022-05-17
Online:
2022-09-25
Published:
2022-10-08
Contact:
NIU Yi,WANG Zhimin
E-mail:niuy2001134@163.com;minzniwang_555@163.com
CLC Number:
HU Ruolin, WANG Jiali, YANG Huiqin, YUAN Chao, NIU Yi, TANG Qinglin, WEI Dayong, TIAN Shibing, YANG Yang, WANG Zhimin. Cloning and Functional Analysis of Auxin Response Factor Gene SmARF5 in Solanum melongena[J]. Acta Horticulturae Sinica, 2022, 49(9): 1895-1906.
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URL: https://www.ahs.ac.cn/EN/10.16420/j.issn.0513-353x.2021-0700
用途 Purpose | 引物 Primer | 序列(5′-3′) Sequence(5′-3′) | 限制酶 Restriction enzyme |
---|---|---|---|
全长克隆 Full-cloning | SmARF5-F SmARF5-R | ATGGGGTCTGTTGAAGTAAAGA GTGGAAGGCAAAGTTCAATAAAAGC | |
亚细胞定位 Subcellular localization | pCAMBIA1300-GFP-SmARF5-F pCAMBIA1300-GFP-SmARF5-R | CAAATCGACTCTAGAAAGCTTATGGGGTCTGTTGAAGTAAAGAACA GCCCTTGCTCACCATGGTACCGTTCGGAAATTCTGATGTTGAGC | HindⅢ KpnⅠ |
遗传转化 Genetic transformation | SmARF5-2301G-F SmARF5-2301G-R | GCTCTAGAGCATGGGGTCTGTTGAAGTAAA CGAGCTCGTCAGTTCGGAAATTCTGATG | XbaⅠ SacⅠ |
酵母双杂交 Yeast two- hybrid assay | pGADT7-SmARF5-F pGADT7-SmARF5-Full-R pGADT7-SmARF5-674aa-R pGBKT7-SmARF5-F pGBKT7-SmARF5-R pGBKT7-SmIAA16-F pGBKT7-SmIAA16-R pGBKT7-SmIAA26-F pGBKT7-SmIAA26-R | CGCCATATGATGGGGTCTGTTGAAGTAAAGAACA TCCCCCGGGTCAGTTCGGAAATTCTGATGTTGA CCGCTCGAGGCTATCTGCTACAGCTTGATAA CGCCATATGATGGGGTCTGTTGAAGTAAAGAACA TCCCCCGGGTCAGTTCGGAAATTCTGATGTTGA TCCCCCGGGAATGGTAGGGGAAGAAGAAA GCGTCGACTCAGTTAATTCTGTTCTTGC TCCCCCGGGAATGGAAGGTTATTCAAGAA GCGTCGACCTATGGCTGCTGCTTAGTTC | NdeⅠ SmaⅠ XhoⅠ NdeⅠ SmaⅠ SmaⅠ SalⅠ SmaⅠ SalⅠ |
qRT-PCR | qRT-SmARF5-F qRT-SmARF5-R α-tubuin-F α-tubuin-R | ACTGTCCGTTGGTATGAGGT CAGGCCATCTCAGTGGATCC ATGAGAGAGTGCATATCGAT TTCACTGAAGAAGGTGTTGAA |
Table 1 The list of primer sequence
用途 Purpose | 引物 Primer | 序列(5′-3′) Sequence(5′-3′) | 限制酶 Restriction enzyme |
---|---|---|---|
全长克隆 Full-cloning | SmARF5-F SmARF5-R | ATGGGGTCTGTTGAAGTAAAGA GTGGAAGGCAAAGTTCAATAAAAGC | |
亚细胞定位 Subcellular localization | pCAMBIA1300-GFP-SmARF5-F pCAMBIA1300-GFP-SmARF5-R | CAAATCGACTCTAGAAAGCTTATGGGGTCTGTTGAAGTAAAGAACA GCCCTTGCTCACCATGGTACCGTTCGGAAATTCTGATGTTGAGC | HindⅢ KpnⅠ |
遗传转化 Genetic transformation | SmARF5-2301G-F SmARF5-2301G-R | GCTCTAGAGCATGGGGTCTGTTGAAGTAAA CGAGCTCGTCAGTTCGGAAATTCTGATG | XbaⅠ SacⅠ |
酵母双杂交 Yeast two- hybrid assay | pGADT7-SmARF5-F pGADT7-SmARF5-Full-R pGADT7-SmARF5-674aa-R pGBKT7-SmARF5-F pGBKT7-SmARF5-R pGBKT7-SmIAA16-F pGBKT7-SmIAA16-R pGBKT7-SmIAA26-F pGBKT7-SmIAA26-R | CGCCATATGATGGGGTCTGTTGAAGTAAAGAACA TCCCCCGGGTCAGTTCGGAAATTCTGATGTTGA CCGCTCGAGGCTATCTGCTACAGCTTGATAA CGCCATATGATGGGGTCTGTTGAAGTAAAGAACA TCCCCCGGGTCAGTTCGGAAATTCTGATGTTGA TCCCCCGGGAATGGTAGGGGAAGAAGAAA GCGTCGACTCAGTTAATTCTGTTCTTGC TCCCCCGGGAATGGAAGGTTATTCAAGAA GCGTCGACCTATGGCTGCTGCTTAGTTC | NdeⅠ SmaⅠ XhoⅠ NdeⅠ SmaⅠ SmaⅠ SalⅠ SmaⅠ SalⅠ |
qRT-PCR | qRT-SmARF5-F qRT-SmARF5-R α-tubuin-F α-tubuin-R | ACTGTCCGTTGGTATGAGGT CAGGCCATCTCAGTGGATCC ATGAGAGAGTGCATATCGAT TTCACTGAAGAAGGTGTTGAA |
Fig. 6 PCR identification of T0 generation of SmARF5 transgenic tobacco WT:Wild type;D:ddH2O;P:Recombinant plasmid;1-16:Transgenic plants;M:Trans 2K plus DNA marker.
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