生物技术通报 ›› 2023, Vol. 39 ›› Issue (10): 136-147.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0339
陈浩婷1(), 张玉静1, 刘洁1, 代泽敏1, 刘伟1, 石玉1, 张毅1(), 李天来1,2()
收稿日期:
2023-04-11
出版日期:
2023-10-26
发布日期:
2023-11-28
通讯作者:
张毅,男,博士,教授,研究方向:设施蔬菜栽培与生理;E-mail: harmony1228@163.com;作者简介:
陈浩婷,女,博士研究生,研究方向:设施蔬菜栽培与生理;E-mail: 746852776@qq.com
基金资助:
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
Published:
2023-10-26
Online:
2023-11-28
摘要:
本研究旨在分析番茄转录因子WRKY6在低磷胁迫应答中的作用,为研究番茄耐低磷分子机制及挖掘提高番茄低磷耐受性和磷素利用率的基因资源奠定理论基础。试验以野生型番茄Ailsa Craig为材料,以其cDNA为模板克隆基因SlWRKY6,利用根癌农杆菌介导转化法构建RNAi-SlWRKY6和OE-SlWRKY6转基因番茄株系,并对野生型、RNAi-SlWRKY6和OE-SlWRKY63种不同基因型的番茄植株进行低磷胁迫处理,在处理第18天对叶片与根系进行表型观察和生理生化指标测定。表型观察结果显示,与RNAi-SlWRKY6转基因番茄相比,OE-SlWRKY6转基因番茄植株较矮壮,叶片数量较多,受低磷胁迫影响较小,表现出较强的耐低磷性。生理生化指标分析结果表明,与野生型相比,OE-SlWRKY6转基因番茄根系与叶片的有机磷和总磷含量显著升高,根系酸性磷酸酶活性和部分有机酸含量显著增加,磷转运体表达量显著降低。而RNAi-SlWRKY6转基因番茄受到低磷胁迫后,这些指标的变化趋势与过表达株系相反。低磷胁迫后,LePT1的相对表达量在OE-SlWRKY6植株根系中呈下降趋势,与RNAi-SlWRKY6植株中变化相反。在野生型、RNAi-SlWRKY6和OE-SlWRKY6植株根系中LePT2的相对表达量均呈整体上升趋势,而LePT3则呈整体下降趋势。由上述可知,SlWRKY6能够响应低磷胁迫,其表达量高低与番茄植株的低磷耐受性呈正相关,且可能影响磷转运体基因对低磷的响应。该研究结果可为进一步揭示番茄WRKY转录因子家族的功能及其对低磷胁迫的响应机制提供理论依据。
陈浩婷, 张玉静, 刘洁, 代泽敏, 刘伟, 石玉, 张毅, 李天来. 低磷胁迫下番茄转录因子WRKY6功能分析[J]. 生物技术通报, 2023, 39(10): 136-147.
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 |
表1 引物列表
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 |
图1 番茄的遗传转化和T2代番茄的 PCR 验证 A、E:共培养;B、F:筛选及分化;C、G:生根;D、H:T2代番茄 PCR 鉴定;M:DNA marker DL2000;P:质粒对照;CK:阴性对照;WT:对照植株;D(1-7)和H(1-8):分别是过表达株系和干扰株系的阳性苗
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
图2 RT-qPCR检测T2代转基因番茄植株RNAi-WRKY6和OE-WRKY6中WRKY6的表达量 不同小写字母表示不同处理间差异显著(P<0.05)。下同
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
图3 低磷条件下转基因番茄植株RNAi-WRKY6和OE-WRKY6的表型观察RNAi-WRKY6从左往右依次为RNAi-WRKY6-1、RNAi-WRKY6-2;OE-WRKY6从左往右依次为OE-WRKY6-1、OE-WRKY6-2
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 |
表2 低磷条件下转基因番茄植株RNAi-WRKY6和OE-WRKY6生物量的测定
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 |
图4 低磷条件下转基因番茄植株RNAi-WRKY6和OE-WRKY6无机磷含量的测定
Fig. 4 Determination of inorganic phosphorus content in transgenic tomato plants RNAi-WRKY6 and OE-WRKY6 under low phosphorus condition
图5 低磷条件下转基因番茄植株RNAi-WRKY6和OE-WRKY6酸性磷酸酶活性的测定
Fig 5 Determination of acid phosphatase activity in transgenic tomato plant RNAi-WRKY6 and OE-WRKY6 under low phosphorus condition
图6 低磷条件下转基因番茄植株RNAi-WRKY6和OE-WRKY6全磷含量的测定
Fig. 6 Determination of total phosphorus content of RNAi-WRKY6 and OE-WRKY6 transgenic tomato plants 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 |
表3 低磷条件下转基因番茄植株RNAi-WRKY6和OE-WRKY6根系有机酸含量的测定
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 |
图7 RNAi-WRKY6和OE-WRKY6转基因番茄根系中磷转运体表达量的测定
Fig. 7 Determination of phosphorus transporter expressions in the roots of transgenic tomato plant RNAi-WRKY6 and OE-WRKY6
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