Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (10): 136-147.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0339
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CHEN Hao-ting1(), ZHANG Yu-jing1, LIU Jie1, DAI Ze-min1, LIU Wei1, SHI Yu1, ZHANG Yi1(), LI Tian-lai1,2()
Received:
2023-04-11
Online:
2023-10-26
Published:
2023-11-28
Contact:
ZHANG Yi, LI Tian-lai
E-mail:746852776@qq.com;harmony1228@163.com;ltl@syau.edu.cn
CHEN Hao-ting, ZHANG Yu-jing, LIU Jie, DAI Ze-min, LIU Wei, SHI Yu, ZHANG Yi, LI Tian-lai. Functional Analysis of WRKY6 Gene in Tomato Under Low-phosphorus Stress[J]. Biotechnology Bulletin, 2023, 39(10): 136-147.
引物名称 Primer name | 引物序列 Primer sequence(5'-3') |
---|---|
SlWRKY6-F | CAACCACCCATTACCACCAG |
SlWRKY6-R | GTTTGGATTTTGGGCTGTG |
LePT1;1-F | TTGGTGGTGATTATCCCCTTTC |
LePT1;1-R | GGCAGTTTCAGGCATCTTCATA |
LePT1;2-F | AAAATGGGACGAAAAAAGGTG |
LePT1;2-R | GATGGTAGCGGACAAAGGGT |
LePT1;3-F | TTCGATTTTGGCTTGGTTTTG |
LePT1;3-R | GTGCTGTCTCAGGCATCTTCA |
Actin-F | ACCACTGAGCACAATGTTACCG |
Actin-R | GTCCTCTTCCAGCCATCCA |
JC-WRKY6-F | ATGACGCACAATCCCACTATC |
JC-WRKY6-R | CTACCGTTGACTGATCACTTCC |
JC-RNAiWRKY6-F | GGAAGGTGGCTCCTACAAATG |
JC-RNAiWRKY6-R | CTTCCGATGAAGAGCGAGAATG |
Table 1 Table of primers
引物名称 Primer name | 引物序列 Primer sequence(5'-3') |
---|---|
SlWRKY6-F | CAACCACCCATTACCACCAG |
SlWRKY6-R | GTTTGGATTTTGGGCTGTG |
LePT1;1-F | TTGGTGGTGATTATCCCCTTTC |
LePT1;1-R | GGCAGTTTCAGGCATCTTCATA |
LePT1;2-F | AAAATGGGACGAAAAAAGGTG |
LePT1;2-R | GATGGTAGCGGACAAAGGGT |
LePT1;3-F | TTCGATTTTGGCTTGGTTTTG |
LePT1;3-R | GTGCTGTCTCAGGCATCTTCA |
Actin-F | ACCACTGAGCACAATGTTACCG |
Actin-R | GTCCTCTTCCAGCCATCCA |
JC-WRKY6-F | ATGACGCACAATCCCACTATC |
JC-WRKY6-R | CTACCGTTGACTGATCACTTCC |
JC-RNAiWRKY6-F | GGAAGGTGGCTCCTACAAATG |
JC-RNAiWRKY6-R | CTTCCGATGAAGAGCGAGAATG |
Fig. 1 Genetic transformation in tomato and PCR validation in the tomato with the T2 generation A, E: Co-culture. B, F: Screening and differentiation. C, G: Rooting. D, H: PCR identification of T2 generation tomato. M: DNA marker DL 2000. P: Plasmid groups. CK: Negative control. WT: Controlled plant. D(1-7)and H(1-8): Positive strains of overexpressing and interfering strains, respectively
Fig. 2 Expressions of WRKY6 in RNAi-WRKY6 and OE-WRKY6 in T2 generation transgenic tomato plants detected by RT-qPCR Different letters indicate significant differences among different treatments(P < 0.05). The same below
Fig. 3 Phenotypes of transgenic tomato plant RNAi-WRKY6 and OE-WRKY6 under low phosphorus conditions RNAi-WRKY6 from left to right is RNAi-WRKY6-1, RNAi-WRKY6-2; OE-WRKY6 from left to right is OE-WRKY6-1, OE-WRKY6-2
处理 Treatment | 地上鲜重 Abovegro und fresh weight/g | 地下鲜重 Undergro- und fresh weight/g | 地上干重 Abovegr- ound dry weight/g | 地下干重 Underground dry weight/g | 总鲜重 Total fresh weight/g | 总干重 Total dry weight/g |
---|---|---|---|---|---|---|
WT | 1.88±0.25b | 0.95±0.08b | 0.22±0.05b | 0.08±0.01b | 2.83±0.18b | 0.30±0.05b |
RNAi-SlWRKY6-1 | 1.71±0.34b | 1.35±0.16b | 0.20±0.04b | 0.08±0.01b | 3.06±0.49b | 0.28±0.05b |
RNAi-SlWRKY6-2 | 1.68+0.33b | 1.34+0.14b | 0.20+0.04b | 0.09+0.01b | 3.02+0.45b | 0.28+0.05b |
OE-SlWRKY6-1 | 3.38±0.08a | 1.94±0.14a | 0.46±0.04a | 0.14±0.01a | 5.33±0.19a | 0.60±0.05a |
OE-SlWRKY6-2 | 3.40+014a | 1.93+0.13a | 0.45+0.05a | 0.14+0.01a | 5.33+0.22a | 0.59+0.06a |
Table 2 Biomass of transgenic tomato plants RNAi-WRKY6 and OE-WRKY6 under low phosphorus conditions
处理 Treatment | 地上鲜重 Abovegro und fresh weight/g | 地下鲜重 Undergro- und fresh weight/g | 地上干重 Abovegr- ound dry weight/g | 地下干重 Underground dry weight/g | 总鲜重 Total fresh weight/g | 总干重 Total dry weight/g |
---|---|---|---|---|---|---|
WT | 1.88±0.25b | 0.95±0.08b | 0.22±0.05b | 0.08±0.01b | 2.83±0.18b | 0.30±0.05b |
RNAi-SlWRKY6-1 | 1.71±0.34b | 1.35±0.16b | 0.20±0.04b | 0.08±0.01b | 3.06±0.49b | 0.28±0.05b |
RNAi-SlWRKY6-2 | 1.68+0.33b | 1.34+0.14b | 0.20+0.04b | 0.09+0.01b | 3.02+0.45b | 0.28+0.05b |
OE-SlWRKY6-1 | 3.38±0.08a | 1.94±0.14a | 0.46±0.04a | 0.14±0.01a | 5.33±0.19a | 0.60±0.05a |
OE-SlWRKY6-2 | 3.40+014a | 1.93+0.13a | 0.45+0.05a | 0.14+0.01a | 5.33+0.22a | 0.59+0.06a |
处理 Treatment | 草酸Oxalic aid/(mg·g-1) | 苹果酸Malic acid/(mg·g-1) | 柠檬酸Citric acid/(μg·g-1) | 琥珀酸Succinic acid/(μg·g-1) | |||||
---|---|---|---|---|---|---|---|---|---|
0 d | 18 d | 0 d | 18 d | 0 d | 18 d | 0 d | 18 d | ||
WT | 12.91±0.35a | 7.47±1.00b | 6.86±0.13ab | 1.69±0.78a | 117.11±15.46a | 11.11±0.93a | 248.86±22.46a | 161.85±77.35b | |
RNAi-WRKY6-1 | 13.14±0.17a | 3.17±0.06c | 6.51±0.32b | 0.71±0.17b | 80.13±16.67b | 7.67±0.81b | 185.79±44.50bc | 78.35±10.86c | |
RNAi-WRKY6-2 | 13.13±0.23a | 3.07±0.06c | 6.41±0.06b | 0.61±0.04b | 77.02±10.20b | 6.93±0.35b | 180.53±28.81c | 70.30±3.98c | |
OE-WRKY6-1 | 13.14±0.29a | 9.22±0.90a | 5.90±0.48a | 2.56±0.25a | 110.70±29.50a | 11.22±2.33a | 229.93±32.63ab | 294.19±20.52a | |
OE-WRKY6-2 | 12.74±0.21a | 9.25±0.32a | 5.67±0.32ab | 2.48±0.09a | 98.16±12.68ab | 10.92±1.92a | 233.98±17.91ab | 293.02±1.09a |
Table 3 Determination of organic acid content in the roots of transgenic tomato plant RNAi-WRKY6 and OE-WRKY6 under low phosphorus condition
处理 Treatment | 草酸Oxalic aid/(mg·g-1) | 苹果酸Malic acid/(mg·g-1) | 柠檬酸Citric acid/(μg·g-1) | 琥珀酸Succinic acid/(μg·g-1) | |||||
---|---|---|---|---|---|---|---|---|---|
0 d | 18 d | 0 d | 18 d | 0 d | 18 d | 0 d | 18 d | ||
WT | 12.91±0.35a | 7.47±1.00b | 6.86±0.13ab | 1.69±0.78a | 117.11±15.46a | 11.11±0.93a | 248.86±22.46a | 161.85±77.35b | |
RNAi-WRKY6-1 | 13.14±0.17a | 3.17±0.06c | 6.51±0.32b | 0.71±0.17b | 80.13±16.67b | 7.67±0.81b | 185.79±44.50bc | 78.35±10.86c | |
RNAi-WRKY6-2 | 13.13±0.23a | 3.07±0.06c | 6.41±0.06b | 0.61±0.04b | 77.02±10.20b | 6.93±0.35b | 180.53±28.81c | 70.30±3.98c | |
OE-WRKY6-1 | 13.14±0.29a | 9.22±0.90a | 5.90±0.48a | 2.56±0.25a | 110.70±29.50a | 11.22±2.33a | 229.93±32.63ab | 294.19±20.52a | |
OE-WRKY6-2 | 12.74±0.21a | 9.25±0.32a | 5.67±0.32ab | 2.48±0.09a | 98.16±12.68ab | 10.92±1.92a | 233.98±17.91ab | 293.02±1.09a |
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