Functional Analysis of WRKY6 Gene in Tomato Under Low-phosphorus Stress

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

Abstract

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

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.

Figures/Tables 10

References 35

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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

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

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	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）
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