Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (1): 77-85.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0181
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LV Di(), CHEN Ru-mei, ZHOU Xiao-jin()
Received:
2021-02-13
Online:
2022-01-26
Published:
2022-02-22
Contact:
ZHOU Xiao-jin
E-mail:lvdi1@foxmail.com;zhouxiaojin@caas.cn
LV Di, CHEN Ru-mei, ZHOU Xiao-jin. Interactions Between ZmJAZ and ZmMYC2 Using Bimolecular Fluorescence Complementation Assay[J]. Biotechnology Bulletin, 2022, 38(1): 77-85.
基因名称 Gene name | 引物名称 Primer name | 引物序列 Primer sequence(5'-3') |
---|---|---|
ZmActin | ZmActin-qF | ATGTTTCCTGGGATTGCCGAT |
ZmActin-qR | CCAGTTTCGTCATACTCTCCCTTG | |
ZmJAZ4 | ZmJAZ4-qF | CGCTCAGGCTCTGAAACAGTGAAACC |
ZmJAZ4-qR | CGACGACAGACACAGTGCCTAAGAAT | |
ZmJAZ8 | ZmJAZ8-qF | TTGAGAAACGCCGTGACAGGGT |
ZmJAZ8-qR | GTCAGCCTTCACCGATGGGACT | |
ZmJAZ12 | ZmJAZ12-qF | CTGATGCTAAGAAGCCTACTCGC |
ZmJAZ12-qR | GCGTCTGAAGGAGAAGTTTGGTA |
Table 1 Primers used in qRT-PCR
基因名称 Gene name | 引物名称 Primer name | 引物序列 Primer sequence(5'-3') |
---|---|---|
ZmActin | ZmActin-qF | ATGTTTCCTGGGATTGCCGAT |
ZmActin-qR | CCAGTTTCGTCATACTCTCCCTTG | |
ZmJAZ4 | ZmJAZ4-qF | CGCTCAGGCTCTGAAACAGTGAAACC |
ZmJAZ4-qR | CGACGACAGACACAGTGCCTAAGAAT | |
ZmJAZ8 | ZmJAZ8-qF | TTGAGAAACGCCGTGACAGGGT |
ZmJAZ8-qR | GTCAGCCTTCACCGATGGGACT | |
ZmJAZ12 | ZmJAZ12-qF | CTGATGCTAAGAAGCCTACTCGC |
ZmJAZ12-qR | GCGTCTGAAGGAGAAGTTTGGTA |
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 扩增产物 Product |
---|---|---|
ZmMYC2-F | ATGAACCTGTGGACGGAC | ZmMYC2-ORF |
ZmMYC2-R | TTACCTGCCCATGGCAGACCCG | |
ZmJAZ4-F | ATGGCCGCCTCCGGGAACAACA | ZmJAZ4-ORF |
ZmJAZ4-R | TCAGAGCCTGAGCGCGAGCCACGA | |
ZmJAZ8-F | ATGGATCTGTTGGAGCGGAATATT | ZmJAZ8-ORF |
ZmJAZ8-R | TTACTGGGCCTTGCCCTCCGCTGT | |
ZmJAZ12-F | ATGGCGGCGTCCGCGAGGCCCG | ZmJAZ12-ORF |
ZmJAZ12-R | CTAGCTCAGGTTCAGGTTGGACTT | |
ZmMYC2-GF | TCATTTGGAGAGGACCTCGAGATGAACCTGTGGACGGACGACAA | ZmMYC2-GFP |
ZmMYC2-GR | CCTCGCCCTTGCTCACCATTCTAGACCTGCCCATGGCAGACCCGGGCT | |
ZmJAZ4-GF | ATTTCATTTGGAGAGGACCTCGAGATGGCCGCCTCCGGGAACAACA | ZmJAZ4-GFP |
ZmJAZ4-GR | CCTCGCCCTTGCTCACCATTCTAGAGAGCCTGAGCGCGAGCCAC | |
ZmJAZ8-GF | ATTTCATTTGGAGAGGACCTCGAGATGGATCTGTTGGAGCGGAATA | ZmJAZ8- GFP |
ZmJAZ8-GR | CCTCGCCCTTGCTCACCATTCTAGACTGGGCCTTGCCCTCCGCT | |
ZmJAZ12-GF | ATTTCATTTGGAGAGGACCTCGAGATGGCGGCGTCCGCGAGGCC | ZmJAZ12-GFP |
ZmJAZ12-GR | CCTCGCCCTTGCTCACCATCTAGAGCTCAGGTTCAGGTTGGACT | |
ZmMYC2-NF | ATTTGGAGAGGACCTCGAGATGAACCTGTGGACGGAC | ZmMYC2-YFPN |
ZmMYC2-NR | CTTTTGCTCCATTCTAGACCTGCCCATGGCAGACCC | |
ZmMYC2-NF | ATTTGGAGAGGACCTCGAGATGAACCTGTGGACGGAC | ZmMYC2-YFPC |
ZmMYC2-CR | GTATGGGTACATTCTAGACCTGCCCATGGCAGACCC | |
ZmJAZ4-NF | CATTTCATTTGGAGAGGACCTCGAGATGGCCGCCTCCGGGAACAA | ZmJAZ4-YFPN |
ZmJAZ4-NR | AACTTTTGCTCCATTCTAGAGAGCCTGAGCGCGAGCCACGAGG | |
ZmJAZ4-NF | CATTTCATTTGGAGAGGACCTCGAGATGGCCGCCTCCGGGAACAA | ZmJAZ4-YFPC |
ZmJAZ4-CR | CATCGTATGGGTACATTCTAGAGAGCCTGAGCGCGAGCCACGAGG | |
ZmJAZ8-NF | CATTTCATTTGGAGAGGACCTCGAGATGGATCTGTTGGAGCGGAAT | ZmJAZ8-YFPN |
ZmJAZ8-NR | AACTTTTGCTCCATTCTAGACTGGGCCTTGCCCTCCGCTGTCAC | |
ZmJAZ8-NF | CATTTCATTTGGAGAGGACCTCGAGATGGATCTGTTGGAGCGGAAT | ZmJAZ8-YFPC |
ZmJAZ8-CR | CATCGTATGGGTACATTCTAGACTGGGCCTTGCCCTCCGCTGTCAC | |
ZmJAZ12-NF | CATTTCATTTGGAGAGGACCTCGAGATGGCGGCGTCCGCGAGGCC | ZmJAZ12-YFPN |
ZmJAZ12-NR | AACTTTTGCTCCATTCTAGAGCTCAGGTTCAGGTTGGACTTGAC | |
ZmJAZ12-NF | CATTTCATTTGGAGAGGACCTCGAGATGGCGGCGTCCGCGAGGCC | ZmJAZ12-YFPC |
ZmJAZ12-CR | CATCGTATGGGTACATTCTAGAGCTCAGGTTCAGGTTGGACTTGAC |
Table 2 Primers used for gene cloning
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 扩增产物 Product |
---|---|---|
ZmMYC2-F | ATGAACCTGTGGACGGAC | ZmMYC2-ORF |
ZmMYC2-R | TTACCTGCCCATGGCAGACCCG | |
ZmJAZ4-F | ATGGCCGCCTCCGGGAACAACA | ZmJAZ4-ORF |
ZmJAZ4-R | TCAGAGCCTGAGCGCGAGCCACGA | |
ZmJAZ8-F | ATGGATCTGTTGGAGCGGAATATT | ZmJAZ8-ORF |
ZmJAZ8-R | TTACTGGGCCTTGCCCTCCGCTGT | |
ZmJAZ12-F | ATGGCGGCGTCCGCGAGGCCCG | ZmJAZ12-ORF |
ZmJAZ12-R | CTAGCTCAGGTTCAGGTTGGACTT | |
ZmMYC2-GF | TCATTTGGAGAGGACCTCGAGATGAACCTGTGGACGGACGACAA | ZmMYC2-GFP |
ZmMYC2-GR | CCTCGCCCTTGCTCACCATTCTAGACCTGCCCATGGCAGACCCGGGCT | |
ZmJAZ4-GF | ATTTCATTTGGAGAGGACCTCGAGATGGCCGCCTCCGGGAACAACA | ZmJAZ4-GFP |
ZmJAZ4-GR | CCTCGCCCTTGCTCACCATTCTAGAGAGCCTGAGCGCGAGCCAC | |
ZmJAZ8-GF | ATTTCATTTGGAGAGGACCTCGAGATGGATCTGTTGGAGCGGAATA | ZmJAZ8- GFP |
ZmJAZ8-GR | CCTCGCCCTTGCTCACCATTCTAGACTGGGCCTTGCCCTCCGCT | |
ZmJAZ12-GF | ATTTCATTTGGAGAGGACCTCGAGATGGCGGCGTCCGCGAGGCC | ZmJAZ12-GFP |
ZmJAZ12-GR | CCTCGCCCTTGCTCACCATCTAGAGCTCAGGTTCAGGTTGGACT | |
ZmMYC2-NF | ATTTGGAGAGGACCTCGAGATGAACCTGTGGACGGAC | ZmMYC2-YFPN |
ZmMYC2-NR | CTTTTGCTCCATTCTAGACCTGCCCATGGCAGACCC | |
ZmMYC2-NF | ATTTGGAGAGGACCTCGAGATGAACCTGTGGACGGAC | ZmMYC2-YFPC |
ZmMYC2-CR | GTATGGGTACATTCTAGACCTGCCCATGGCAGACCC | |
ZmJAZ4-NF | CATTTCATTTGGAGAGGACCTCGAGATGGCCGCCTCCGGGAACAA | ZmJAZ4-YFPN |
ZmJAZ4-NR | AACTTTTGCTCCATTCTAGAGAGCCTGAGCGCGAGCCACGAGG | |
ZmJAZ4-NF | CATTTCATTTGGAGAGGACCTCGAGATGGCCGCCTCCGGGAACAA | ZmJAZ4-YFPC |
ZmJAZ4-CR | CATCGTATGGGTACATTCTAGAGAGCCTGAGCGCGAGCCACGAGG | |
ZmJAZ8-NF | CATTTCATTTGGAGAGGACCTCGAGATGGATCTGTTGGAGCGGAAT | ZmJAZ8-YFPN |
ZmJAZ8-NR | AACTTTTGCTCCATTCTAGACTGGGCCTTGCCCTCCGCTGTCAC | |
ZmJAZ8-NF | CATTTCATTTGGAGAGGACCTCGAGATGGATCTGTTGGAGCGGAAT | ZmJAZ8-YFPC |
ZmJAZ8-CR | CATCGTATGGGTACATTCTAGACTGGGCCTTGCCCTCCGCTGTCAC | |
ZmJAZ12-NF | CATTTCATTTGGAGAGGACCTCGAGATGGCGGCGTCCGCGAGGCC | ZmJAZ12-YFPN |
ZmJAZ12-NR | AACTTTTGCTCCATTCTAGAGCTCAGGTTCAGGTTGGACTTGAC | |
ZmJAZ12-NF | CATTTCATTTGGAGAGGACCTCGAGATGGCGGCGTCCGCGAGGCC | ZmJAZ12-YFPC |
ZmJAZ12-CR | CATCGTATGGGTACATTCTAGAGCTCAGGTTCAGGTTGGACTTGAC |
Fig.2 Vectors for subcellular localization and enzyme digestion identification A:Schematic diagram of vectors for subcellular localization,including control GFP vector and cell nucleus localization marker vector. B:Enzyme digestion identification of Xho I + Xba I in subcellular localization vectors. M1:100 bp DNA ladder. M2:1000 bp DNA ladder. Lane 1:pZmMYC2-GFP,lane 2:pZmJAZ4-GFP,lane 3:pZmJAZ8-GFP,and lane 4:pZmJAZ12-GFP
Fig.3 Vectors for BiFC assay and enzyme digestion identification A:Schematic diagram of vectors for BiFC assay. B:Enzyme digestion identification of BiFC vectors by Xho I + Xba I. M1:100 bp DNA ladder. M2:1 000 bp DNA ladder. Lane 1:pZmMYC2-YFPN,lane 2:pZmMYC2-YFPC,lane 3:pZmJAZ4-YFPN,lane 4:pZmJAZ4-YFPC,lane 5:pZmJAZ8-YFPN,lane 6:pZmJAZ8-YFPC,lane 7:pZmJAZ12-YFPN,and lane 8:pZmJAZ12-YFPC
Fig. 4 Subcellular localization of ZmJAZ4,ZmJAZ8,ZmJAZ12 and ZmMYC2 The ORFs of ZmJAZ4、ZmJAZ8、ZmJAZ12 and ZmMYC2 were fused at the N terminal of GFP. GFP indicates the target protein fluorescence channel. DsRed indicates fluorescence channel for a nuclear localization maker. Chl indicates the chloroplast spontaneous fluorescence channel. Bright indicates the bright field. The Merge indicates channel combined the GFP,DsRed,Chl and the bright field. Scale bar = 10 μm
Fig. 5 BiFC assay to verify interactions between ZmJAZs with ZmMYC2 ZmMYC2 and 3 to-be-verified genes were constructed into the N-terminal and C-terminal of YFP for BiFC assay. YFP indicates fluorescence channel triggered by BiFC interaction. The DsRed indicates fluorescence channel for nuclear localization maker. Chl indicates the chloroplast spontaneous fluorescence channel. Bright indicates the bright field. Merge indicates channel combined the GFP,DsRed,Chl and the bright field. Scale bar = 10 μm
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