Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (10): 128-135.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0338
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HOU Rui-ze BAO Yue CHEN Qi-liang MAO Gui-ling WEI Bo-lin HOU Lei-ping LI Mei-lan()
Received:
2023-04-11
Online:
2023-10-26
Published:
2023-11-28
HOU Rui-ze BAO Yue CHEN Qi-liang MAO Gui-ling WEI Bo-lin HOU Lei-ping LI Mei-lan. Cloning,Expression and Functional Identification of PRR5 Gene in Pakchoi[J]. Biotechnology Bulletin, 2023, 39(10): 128-135.
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 引物用途 Primer purpose |
---|---|---|
C-Bra009768 | F:ATGGGAGAAGTGAGCGACGAAG | 基因克隆 |
R:TCATTGTGGAGCTTCTTGTGTTGAG | Gene clone | |
G-Bra009768 | F:GAGAACACGGGGGACTCTAGAATGGGAGAAGTGAGCGACGAAG | 基因克隆 Gene clone |
R:ATAAGGGACTGACCACCCGGGTCATTGTGGAGCTTCTTGTGTTGAG | ||
N-YZ | F:GACGTTCCAACCACGTCTTC | 菌液PCR引物 |
R:CCAGACTGAATGCCCACAGG | Bacterial liquid PCR primer |
Table 1 Primers of Bra009768
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 引物用途 Primer purpose |
---|---|---|
C-Bra009768 | F:ATGGGAGAAGTGAGCGACGAAG | 基因克隆 |
R:TCATTGTGGAGCTTCTTGTGTTGAG | Gene clone | |
G-Bra009768 | F:GAGAACACGGGGGACTCTAGAATGGGAGAAGTGAGCGACGAAG | 基因克隆 Gene clone |
R:ATAAGGGACTGACCACCCGGGTCATTGTGGAGCTTCTTGTGTTGAG | ||
N-YZ | F:GACGTTCCAACCACGTCTTC | 菌液PCR引物 |
R:CCAGACTGAATGCCCACAGG | Bacterial liquid PCR primer |
引物名称 Gene name | 引物序列 Primer sequence(5'-3') | 引物用途 Primer purpose |
---|---|---|
ACTIN | F:GTTGCTATCCAGGCTGTTCT | 大白菜内参引物 |
R:AGCGTGAGGAAGAGCATAAC | Internal primers in Brassica pekinensis | |
Bra009768 | F:AGTTACAGAGTCGCTGCT | 荧光定量引物 |
R:TATTAACCGAGTCCTGTGTTG | Fluorescent quantitative primer | |
ACT11 | F:CACACTGGAGTGATGGTTGG | 拟南芥内参引物 |
R:ATTGGCCTTGGGGTTAAGAG | The internal reference gene of Arabidopsis |
Table 2 Primers of real time quantitative PCR
引物名称 Gene name | 引物序列 Primer sequence(5'-3') | 引物用途 Primer purpose |
---|---|---|
ACTIN | F:GTTGCTATCCAGGCTGTTCT | 大白菜内参引物 |
R:AGCGTGAGGAAGAGCATAAC | Internal primers in Brassica pekinensis | |
Bra009768 | F:AGTTACAGAGTCGCTGCT | 荧光定量引物 |
R:TATTAACCGAGTCCTGTGTTG | Fluorescent quantitative primer | |
ACT11 | F:CACACTGGAGTGATGGTTGG | 拟南芥内参引物 |
R:ATTGGCCTTGGGGTTAAGAG | The internal reference gene of Arabidopsis |
Fig. 1 Cloning of Bra009768 and detection results of its broth A: Cloning of Bra009768(M: DNA marker; 1-3: Bra009768). B: PCR detection of bacterial liquid(M: DNA marker; 1-12: Bra009768; 13-14: negative control)
Fig. 2 Bioinformatics analysis of Bra009768 A: Multiple alignment of Bra009768 amino acid sequences. B: Phylogenetic tree of Bra009768 homologous proteins. C, D: Prediction of secondary and tertiary structures of Bra009768 proteins
Fig. 4 Selection of transgenic Arabidopsis thaliana resistant plants A: Screening of resistant plants in T1 generation. B: Screening of resistant plants in T2 generation
植株 Plant | 抽薹时间 Bolting time/d | 侧薹数 Number of side bolting | 株高 Plant height/cm | 茎粗 Stem thick/mm |
---|---|---|---|---|
Col-1 | 13.75±0.82 | 11.50±0.31 | 25.90±0.88 | 0.45±0.04 |
T-1 | 12.50±2.38 | 11.25±2.12 | 25.75±3.13 | 0.54±0.12 |
Col-2 | 17.20±1.25 | 7.50±0.21 | 22.30±0.91 | 0.53±0.02 |
T-2 | 15.72±0.78 | 8.10±0.13 | 29.80±0.53 | 0.94±0.06 |
Table 3 Phenotypic statistics of T1 and T2 transgenic plants
植株 Plant | 抽薹时间 Bolting time/d | 侧薹数 Number of side bolting | 株高 Plant height/cm | 茎粗 Stem thick/mm |
---|---|---|---|---|
Col-1 | 13.75±0.82 | 11.50±0.31 | 25.90±0.88 | 0.45±0.04 |
T-1 | 12.50±2.38 | 11.25±2.12 | 25.75±3.13 | 0.54±0.12 |
Col-2 | 17.20±1.25 | 7.50±0.21 | 22.30±0.91 | 0.53±0.02 |
T-2 | 15.72±0.78 | 8.10±0.13 | 29.80±0.53 | 0.94±0.06 |
Fig. 5 Phenotypic observation of transgenic plants and RT-qPCR validation of BrcPRR5 A: Vegetative growth period. B: Bolting stage. C: Budding stage. D-E: Mature stage(left: wild type(Col); right: transgenic plants). F: RT-qPCR detection of transgenic plants
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