花烟草NaD1基因的表达及其启动子序列分析

doi:10.3969/j.issn.1004-1524.2022.02.03

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (2): 232-239.DOI: 10.3969/j.issn.1004-1524.2022.02.03

花烟草NaD1基因的表达及其启动子序列分析

龚伟伟¹(), 赵懿琛¹^,^*(), 罗显麟², 杨玲玲³, 赵德刚⁴

1.贵州大学茶学院,山地植物资源保护与种质创新教育部重点实验室,贵州贵阳 550025
2.贵州大学药学院,山地植物资源保护与种质创新教育部重点实验室,贵州贵阳 550025
3.贵州大学生命与科学学院,山地植物资源保护与种质创新教育部重点实验室,贵州贵阳 550025
4.贵州省农业科学院国家DUS中心贵阳分中心,贵州贵阳 550006

收稿日期:2020-09-08 出版日期:2022-02-25 发布日期:2022-03-02
通讯作者: 赵懿琛
作者简介:赵懿琛,E-mail: yczhao@gzu.edu.cn
龚伟伟(1991—),女,河南周口人,硕士研究生,主要从事生物工程研究, E-mail: 18838933792@163.com
基金资助:
中国烟草总公司贵州省公司科技项目(201608);贵州省高层次创新型人次培养(黔科合人才〔2016〕4003号)

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

摘要/Abstract

摘要：

花烟草(Nicotiala alata)的花中存在的植物防御素Nicotiala alata defensin 1(NaD1),对多种致病性真菌具有防御活性,在天然防御免疫系统中发挥着重要作用。本研究分别对花烟草根、茎、叶、花组织部分以及伸根期、旺长期、成熟期发育阶段叶片中NaD1基因的表达量进行qRT-PCR定量检测,结果显示,NaD1基因在花中表达量显著高于其他组织,在叶片中的表达量随着植物的生长发育先增加后降低。此外,通过热不对称交错PCR克隆得到NaD1基因5'端上游644 bp的片段,利用BDGP和Plant CARE在线软件分析启动子的转录起始位点及motif元件,发现其含有基本启动子元件和其他顺式作用元件,如光反应元件BOX-Ⅰ、乙烯反应元件ERE以及真菌诱导响应元件TGACG-motif等。通过对花烟草进行光胁迫处理,发现NaD1基因在花中的表达量随着暗处理时间的增长而显著增加,随着光处理时间的增长而显著降低,这与其启动子中存在的光反应元件BOX-Ⅰ相对应。通过启动子5'端缺失分析,构建了pCAMBIA1391z-pNaD1-644∶∶GUS与pCAMBIA1391z-pNaD1-310∶∶GUS两个植物表达载体,利用遗传转化获得包含-644~-1 bp和-310~-1 bp启动子片段的转基因烟草,GUS染色结果表明,NaD1基因启动子的核心区域在-310~-1 bp中,为进一步研究NaD1基因的功能和转录调控机制奠定了基础。

关键词: 花烟草, NaD1基因, 防御素, 启动子, 表达分析

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

中图分类号:

S572

龚伟伟, 赵懿琛, 罗显麟, 杨玲玲, 赵德刚. 花烟草NaD1基因的表达及其启动子序列分析[J]. 浙江农业学报, 2022, 34(2): 232-239.

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.

图/表 6

表1 引物序列

Table 1 The sequences of primers

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

图1 NaD1基因在花烟草不同组织器官(A)和不同生长发育阶段的叶片(B)中的表达量不同数据间没有相同小写字母表示差异显著(P<0.05)。下同。

Fig.1 Expression of NaD1 gene in different tissues and leaves at different growth and development stages of Nicotiana alata The bars with different letters show the significant difference at the level of 0. 05. The same as below.

图2 NaD1基因5'端调控区顺式作用元件

Fig.2 Analysis of cis-acting elements in regulatory sequence in 5' terminal of NaD1 gene

表2 NaD1基因5'端调控区顺式作用元件

Table 2 The cis-acting elements in regulatory sequence in 5' terminal of NaD1 gene

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

图3 不同光照处理(A)和黑暗处理(B)下NaD1基因在花烟草花中的表达量

Fig.3 Expression of NaD1 gene in Nicotiana alata under different light treatment conditions

图4 NaD1基因启动子转录活性验证 A,重组质粒双酶切琼脂糖凝胶电泳图:1,DL2000 Marker;2,pNaD1-644;3,pNaD1-310;4,15 000 Marker;B,抗性幼苗叶片GUS组织化学染色:1,野生型;2,pCAMBIA1391z-pNaD1-310∶∶GUS;3,pCAMBIA1391z-pNaD1-644∶∶GUS。

Fig.4 Verification of transcriptional activity of the NaD1 gene promoter A, Double enzyme digestion agarose gel electrophoresis of recombinant plasmid: 1, DL2000 Marker; 2, pNaD1-644; 3, pNaD1-310; 4, 15 000 Marker; B, GUS histochemical staining on the leaves of resistant seedlings: 1, Wild type; 2, pCAMBIA1391z-pNaD1-310∶∶GUS; 3, pCAMBIA1391z-pNaD1-644∶∶GUS.

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花烟草NaD1基因的表达及其启动子序列分析

Expression and promoter sequence analysis of NaD1 gene in Nicotiana alata

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