Analysis of Cold-, Heat-, and ABA-responsive <i>cis</i> Elements in Tomato <i>SlNAC1</i> Promoter

doi:10.13523/j.cb.2305017

China Biotechnology

2023, Vol. 43

Issue (11): 16-26 DOI: 10.13523/j.cb.2305017

Analysis of Cold-, Heat-, and ABA-responsive cis Elements in Tomato SlNAC1 Promoter

LI De-xin^1,²,YUE Lu³,TAO Xiang⁴,LIAO Hai¹,ZHOU Jia-yu^1,^**(

),HUANG Wei-zao^2,^**(

)

1 School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China
2 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
3 College of Resources and Environment, Aba Teachers University, Chengdu 623002, China
4 College of Life Science, Sichuan Normal University, Chengdu 610101, China

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Abstract

Tomato transcription factor SlNAC1 has been reported to regulate several biotic and abiotic stress responses, but its upstream regulatory transcription factors remain unknown, which limits our understanding of its molecular mechanism of stress responses. We constructed series of 5'-deleted SlNAC1 promoters (2 039 bp, 1 508 bp, 1 373 bp and 777 bp upstream of start codon) -driven GUS transgenic Nicotiana benthamiana and analyzed quantitatively their GUS activity under cold, heat and ABA treatment conditions in order to identify cold-, heat-, and ABA-responsive cis elements. Our results show that the GUS activity of 2 039 bp promoter-driven transgenic Nicotiana benthamiana increased much higher than that of any other transgenic Nicotiana benthamiana and wild-type Nicotiana benthamiana after cold and heat treatment, while the GUS activity of 1 508 bp promoter-driven transgenic Nicotiana benthamiana increased much higher than that of any other transgenic Nicotiana benthamiana and wild-type Nicotiana benthamiana after ABA treatment. These results indicate that both cold-responsive and heat-responsive cis elements were located in the region from -2 039 bp to -1 508 bp, and ABA-responsive cis element(s) was(were) located in the region from -1 508 bp to -777 bp. According to cis elements' prediction of SlNAC1 promoter, there were only one cold/heat/drought/salt-responsive cis element DRE/CRT in the region from -2 039 bp to -1 508 bp and only one ABA-responsive cis element ABRE in the region from -1 508 bp to -777 bp. Therefore, these two cis elements will be used as candidates for subsequent site-directed mutagenesis validation and screening of the upstream regulatory transcription factor of SlNAC1.

Key words： Transcription factor SlNAC1 Promoter cis element Stress-responsive

Received: 11 May 2023 Published: 01 December 2023

ZTFLH:

Q789

Corresponding Authors: **Jia-yu ZHOU,Wei-zao HUANG E-mail: spinezhou@home.swjtu.edu.cn;huangwz@cib.ac.cn

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	LI De-xin
	YUE Lu
	TAO Xiang
	LIAO Hai
	ZHOU Jia-yu
	HUANG Wei-zao

Cite this article:

LI De-xin, YUE Lu, TAO Xiang, LIAO Hai, ZHOU Jia-yu, HUANG Wei-zao. Analysis of Cold-, Heat-, and ABA-responsive cis Elements in Tomato SlNAC1 Promoter. China Biotechnology, 2023, 43(11): 16-26.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2305017 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I11/16

Table 1 Information of qRT-PCR primers

Table 2 Information of primers

Fig.1 Relative expression level of SlNAC1 under cold, heat and ABA treatments Asterisk represents significant difference (^** P<0.01, ^*** P<0.001, Student’s t test)

Fig.2 Prediction of stress-responsive cis elements in SlNAC1 promoter Red vertical lines show the 5'-positions of series of 5'-deleted SlNAC1 promoters; Stress-responsive cis-elements are underlined. W-box: The recognition site for WRKY transcription factors with the core sequence of TGAC; GT1GMSCAM4: The recognition site for GT transcription factors with the core sequence of GAAAAA; MYBCORE: The recognition site for MYB transcription factors with the core sequence of CNGTTR; DRE/CRT: The recognition site for DREB/CBF transcription factors with the core sequence of RCCGAC; ABRE: The recognition site for MYB/MYC transcription factors with the core sequence of PyACGTG(G/T)C; CuRE: The copper- and oxygen-responsive element with the core sequence of GTAC; MYCCONSENSUSAT: The low-temperature-responsive element with the core sequence of CANNTG; MYB1AT: The dehydration-responsive element with the core sequence of WAACCA; MYB1LEPR: The pathogen-response element with the core sequence of GTTAGTT; LTRE: The low-temperature-responsive element with the core sequence of CCGAAA; CRTDREHVCBF2: The low-temperature-responsive element with the core sequence of GTCGAC

Fig.3 Schematic diagram of GUS expression constructs driven by series of 5'-deleted SlNAC1 promoters and their T-DNA insertion sites on chromosome LB: Left border of T-DNA; RB: Right border of T-DNA

Fig. 4 GUS histochemical staining and GUS activity under cold treatment A. GUS histochemical staining under cold treatment B. Quantitative analysis of GUS activity under cold treatment. SlNAC1Pro1: Transgenic Nicotiana benthamiana plants driven by the 2 039 bp promoter; SlNAC1Pro2: Transgenic Nicotiana benthamiana plants driven by the 1 508 bp promoter; SlNAC1Pro3: Transgenic Nicotiana benthamiana plants driven by the 1 373 bp promoter; SlNAC1Pro4: Transgenic Nicotiana benthamiana plants driven by the 777 bp promoter. The scale is 2 mm. Asterisk represents significant difference. ^*** P< 0.001, Student’s t test

Fig.5 GUS histochemical staining and GUS activity under heat treatment A. GUS histochemical staining under heat treatment B. Quantitative analysis of GUS activity under heat treatment. SlNAC1Pro1: Transgenic Nicotiana benthamiana plants driven by the 2 039 bp promoter; SlNAC1Pro2: Transgenic Nicotiana benthamiana plants driven by the 1 508 bp promoter; SlNAC1Pro3: Transgenic Nicotiana benthamiana plants driven by the 1 373 bp promoter; SlNAC1Pro4: Transgenic Nicotiana benthamiana plants driven by the 777 bp promoter. The scale is 2 mm. Asterisk represents significant difference. ^*** P<0.001, Student’s t test

Fig.6 GUS histochemical staining and GUS activity under ABA treatment A. GUS histochemical staining under ABA treatment B. Quantitative analysis of GUS activity under ABA treatment. SlNAC1Pro1: Transgenic Nicotiana benthamiana plants driven by the 2 039 bp promoter; SlNAC1Pro2: Transgenic Nicotiana benthamiana plants driven by the 1 508 bp promoter; SlNAC1Pro3: Transgenic Nicotiana benthamiana plants driven by the 1 373 bp promoter; SlNAC1Pro4: Transgenic Nicotiana benthamiana plants driven by the 777 bp promoter. The scale is 2 mm. Asterisk represents significant difference. ^*** P<0.001, Student’s t test


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