低磷胁迫下番茄转录因子WRKY6功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0339

摘要/Abstract

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。

关键词: 番茄, 转录因子, SlWRKY6, 低磷胁迫, 功能分析, 磷含量, 酸性磷酸酶

Abstract:

The purpose of this study is to analyze the role of tomato transcription factor SlWRKY6 in response to low-phosphorus stress, and to lay a theoretical foundation for studying the molecular mechanism of low-phosphorus tolerance in tomato and exploring tomato germplasm resources of improving low-phosphorus tolerance and phosphorus utilization. Wild-type tomato Ailsa craig was used as the material and its cDNA was used as the template to clone SlWRKY6, and the RNAi-SlWRKY6 and OE-SlWRKY6 transgenic tomato plant lines were constructed by agrobacterium tumefacien-mediated transformation. Tomato plants with three different genotypes of wild type, RNAi-SlWRKY6 and OE-SlWRKY6, were subjected to low phosphorus. On the day 18 of low-phosphorus treatment, the results of phenotype and physiological and biochemical indexes in the leaves and roots were determined. Phenotypic observation showed that OE-SlWRKY6 transgenic tomato plants were of thicker and shorter after low phosphorus treatment while compared with RNAi-SlWRKY6 transgenic tomato plants, they had a higher number of leaves, and were less affected by low-phosphorus stress, and showed stronger low-phosphorus tolerance. The analysis results of physiological and biochemical indexes showed that the organic phosphorus and total phosphorus content in the roots and leaves of transgenic tomato plant OE-SlWRKY6 significantly increased compared with wild type, while the acid phosphatase activity and some organic acid contents in the roots significantly increased. The expressions of phosphorus transporters also significantly reduced. However, the trend of changes in these indicators in the transgenic tomato plant RNAi-SlWRKY6 under low-phosphorus stress was opposite to that in transgenic tomato plant OE-SlWRKY6. After low-phosphorus stress, the relative expressions of LePT1 decreased in the roots of OE-SlWRKY6 plants, which was opposite to that of RNAi-SlWRKY6 plants. The relative expressions of LePT2 in the roots of wild type, RNAi-SlWRKY6 and OE-SlWRKY6 plants showed an overall increasing trend, while the relative expressions of LePT3 showed an overall decreasing trend. According to the above results, SlWRKY6 responded to low-phosphorus stress and its expression was positively correlated with the low-phosphorus tolerance of tomato plants, and might affect the response of phosphorus transporter genes to low phosphorus. The results of this study may provide certain theoretical basis for further revealing the function of tomato WRKY transcription factor family and its response mechanism to low-phosphorus stress.

Key words: tomato, transcription factors, SlWRKY6, low-phosphorus stress, functional analysis, phosphorus content, acid phosphatase

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.

图/表 10

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

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

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）
处理 Treatment	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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