Expression and promoter sequence analysis of NaD1 gene in Nicotiana alata

doi:10.3969/j.issn.1004-1524.2022.02.03

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (2): 232-239.DOI: 10.3969/j.issn.1004-1524.2022.02.03

• Crop Science • Previous Articles Next Articles

Expression and promoter sequence analysis of NaD1 gene in Nicotiana alata

GONG Weiwei¹(), ZHAO Yichen¹^,^*(), LUO Xianlin², YANG Lingling³, ZHAO Degang⁴

1. Key Laboratory of Mountain Plant Resources Protection and Germplasm Innovation, Ministry of Education, College of Tea, Guizhou University, Guiyang 550025, China
2. Key Laboratory of Mountain Plant Resources Protection and Germplasm Innovation, Ministry of Education, School of Pharmacy, Guizhou University, Guiyang 550025, China
3. Key Laboratory of Mountain Plant Resource Conservation and Germplasm Innovation, Ministry of Education, College of Life And Science, Guizhou University, Guiyang 550025, China
4. Guiyang Branch, National DUS Center, Guizhou Academy of Agricultural Sciences, Guiyang 550006, China

Received:2020-09-08 Online:2022-02-25 Published:2022-03-02
Contact: ZHAO Yichen

Abstract

Abstract:

Nicotiala alata defensin 1 (NaD1) is a plant defensin in the flowers of Nicotiana alata, which has defense activity against many pathogenic fungi and plays an important role in the natural defense immune system. In this study, the expression levels of NaD1 gene in roots, stems, leaves, flowers and leaves at root extension stage, vigorous growth stage and mature stage were detected by qRT-PCR. The results showed that the expression level of NaD1 gene in flowers was significantly higher than that in other tissues, and the expression of NaD1 gene in leaves firstly increased and then decreased during the growth and development of plants. In addition, 644 bp upstream of NaD1 gene 5' end was cloned by thermal asymmetric cross PCR. The transcription initiation site and motif element of NaD1 gene were analyzed by BDGP and Plant CARE online software. It was found that the promoter contained basic promoter elements and other cis acting elements, such as light response element box-Ⅰ, ethylene response element ERE and fungal induction response element TGACG-motif. It was found that NaD1 gene expression increased significantly with the increase of dark treatment time, and decreased significantly with the increase of light treatment time, which was corresponded to the light response element box-Ⅰ in the promoter. Two plant expression vectors pCAMBIA1391z-pNaD1-644∶∶GUS and pCAMBIA1391z-pNaD1-310∶∶GUS were constructed by 5' promoter deletion analysis. Transgenic tobacco containing -644-1 bp and -310-1 bp promoter fragments were obtained by genetic transformation. GUS staining showed that the core region of NaD1 gene promoter was -310-1 bp, it laid a foundation for further study on the function and transcriptional regulation mechanism of NaD1 gene.

Key words: Nicotiala alata, NaD1 gene, defensin, promoter, expression analysis

CLC Number:

S572

GONG Weiwei, ZHAO Yichen, LUO Xianlin, YANG Lingling, ZHAO Degang. Expression and promoter sequence analysis of NaD1 gene in Nicotiana alata[J]. Acta Agriculturae Zhejiangensis, 2022, 34(2): 232-239.

Figures/Tables 6

References 23

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引物名称 Primer name	引物序列 Primer sequence(5'-3')
Na-NaD1-D-F	ATGGCTCGCTCCTTGTGCT
Na-NaD1-D-R	TGCTTGAAGAAGAGATAATGGATAACTAA
N-actin-F	TGAGATGCACCACGAAGCTC
N-actin-R	CCAACATTGTCACCAGGAAGTG
Na-sp1	AGCTTTTCTGCATGGTGGTTTGG
Na-sp2	GAGACAAACCATAGGCAACAAAGAGC
Na-sp3	GAAGCACAAGGAGCGAGCCATAGT
N-actin-F	TGAGATGCACCACGAAGCTC
N-actin-R	CCAACATTGTCACCAGGAAGTG
NaD1-F-60	CTATGAGGTGCAAGCTAGAGAATG
NaD1-R-177	GGCACCTTCTGAGGATTTTGCTA

引物名称 Primer name	引物序列 Primer sequence(5'-3')
Na-NaD1-D-F	ATGGCTCGCTCCTTGTGCT
Na-NaD1-D-R	TGCTTGAAGAAGAGATAATGGATAACTAA
N-actin-F	TGAGATGCACCACGAAGCTC
N-actin-R	CCAACATTGTCACCAGGAAGTG
Na-sp1	AGCTTTTCTGCATGGTGGTTTGG
Na-sp2	GAGACAAACCATAGGCAACAAAGAGC
Na-sp3	GAAGCACAAGGAGCGAGCCATAGT
N-actin-F	TGAGATGCACCACGAAGCTC
N-actin-R	CCAACATTGTCACCAGGAAGTG
NaD1-F-60	CTATGAGGTGCAAGCTAGAGAATG
NaD1-R-177	GGCACCTTCTGAGGATTTTGCTA

功能预测 Putative function	元件名称 Element name	核心序列 Core sequence	数量 Number
转录起始点-30 核心启动子元件Transcription start point-30 core promoter element	TATA-box	CCTATAAATA,TATA	3
启动子和增强子的调控元件Regulatory elements for promoters and enhancers	CAAT-box	CAAT,CAAAT	2
高转录水平顺式作用元件High transcription level cis-acting element	5'UTR Py-rich stretch	TTTCTACTCT	1
光响应元件Light-responsive element	Box Ⅰ	TTTCAAA	1
光响应的部分元件Some components respond to light	Ⅰ-box	GGAAAAGGTG	1
防卫和胁迫响应顺式作用元件Defense and stress response cis-acting elements	TC-rich repeats	ATTTTTTTCA	1
干旱诱导相关的 MYB结合位点MYB binding sites related to drought induction	MBS	TAACTG	1
乙烯响应元件Ethylene response element	ERE	ATTTCAAA	1
茉莉酸甲酯响应元件Methyl jasmonate response element	TGACG-motif	TGACG	1
醇溶蛋白代谢有关的顺式作用调控元件	O2-site	GGAAAAGGTG	1
cis-acting regulatory elements related to gliadin metabolism

功能预测 Putative function	元件名称 Element name	核心序列 Core sequence	数量 Number
转录起始点-30 核心启动子元件Transcription start point-30 core promoter element	TATA-box	CCTATAAATA,TATA	3
启动子和增强子的调控元件Regulatory elements for promoters and enhancers	CAAT-box	CAAT,CAAAT	2
高转录水平顺式作用元件High transcription level cis-acting element	5'UTR Py-rich stretch	TTTCTACTCT	1
光响应元件Light-responsive element	Box Ⅰ	TTTCAAA	1
光响应的部分元件Some components respond to light	Ⅰ-box	GGAAAAGGTG	1
防卫和胁迫响应顺式作用元件Defense and stress response cis-acting elements	TC-rich repeats	ATTTTTTTCA	1
干旱诱导相关的 MYB结合位点MYB binding sites related to drought induction	MBS	TAACTG	1
乙烯响应元件Ethylene response element	ERE	ATTTCAAA	1
茉莉酸甲酯响应元件Methyl jasmonate response element	TGACG-motif	TGACG	1
醇溶蛋白代谢有关的顺式作用调控元件	O2-site	GGAAAAGGTG	1
cis-acting regulatory elements related to gliadin metabolism

Expression and promoter sequence analysis of NaD1 gene in Nicotiana alata

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