芝麻NAC转录因子基因SiNAC77的克隆及耐盐功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0096

生物技术通报 ›› 2023, Vol. 39 ›› Issue (11): 308-317.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0096

芝麻NAC转录因子基因SiNAC77的克隆及耐盐功能分析

张玉娟¹(), 黎冬华², 宫慧慧¹, 崔新晓¹, 高春华¹, 张秀荣¹, 游均²(), 赵军胜¹()

1.山东省农业科学院经济作物研究所，济南 250100
2.中国农业科学院油料作物研究所农业农村部油料作物生物学与遗传育种重点实验室，武汉 430062

收稿日期:2023-02-08 出版日期:2023-11-26 发布日期:2023-12-20
通讯作者: 赵军胜，男，博士，研究员，研究方向：经济作物遗传育种；E-mail: zhaojunshengsd@163.com；
游均，男，博士，副研究员，研究方向：芝麻抗逆与品质遗传改良；E-mail: junyou@caas.cn
作者简介:张玉娟，女，博士，助理研究员，研究方向：植物逆境分子生物学；E-mail: 723949246@qq.com
基金资助:
山东省科技型中小企业创新能力提升工程项目(2021TSGC1209);山东省农业科学院农业科技创新工程项目(CXGC2023A45);山东省农业科学院农业科技创新工程项目(CXGC2022B01);山东省农业科学院农业科技创新工程项目(CXGC2022F18)

Cloning and Salt-tolerance Analysis of NAC Transcription Factor SiNAC77 from Sesamum indicum L.

ZHANG Yu-juan¹(), LI Dong-hua², GONG Hui-hui¹, CUI Xin-xiao¹, GAO Chun-hua¹, ZHANG Xiu-rong¹, YOU Jun²(), ZHAO Jun-sheng¹()

1. Institute of Industrial Crops, Shandong Academy of Agricultural Sciences, Jinan 250100
2. Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture and Rural Affairs, Wuhan 430062

Received:2023-02-08 Published:2023-11-26 Online:2023-12-20

摘要/Abstract

摘要：

NAC转录因子在植物生长发育及盐胁迫响应过程中具有重要的调控作用。芝麻SiNAC77参与盐胁迫响应过程。克隆芝麻SiNAC77并分析其耐盐功能，为芝麻耐盐育种提供分子基础和基因资源。克隆SiNAC77的全长CDS序列，利用生物信息学软件对其基因序列和氨基酸序列特征进行分析。构建SiNAC77过表达载体，利用农杆菌介导法转化拟南芥。对过表达株系的耐盐表型和生理生化指标进行分析。结果表明，成功克隆了长度为1 008 bp的SiNAC77 CDS序列，编码335个氨基酸，相对分子量为135.93 kD，预测等电点为4.91，含有33个潜在的磷酸化位点；启动子区域含有MBS、STRE、ARE、ABRE和TCA等多个非生物胁迫及激素响应元件。成功构建了SiNAC77过表达载体，并转化拟南芥，获得12个独立的转基因阳性株系。在盐胁迫下，与野生型拟南芥相比，过表达株系的种子发芽率、初生根长度、鲜重均显著提高，转基因株系中Na⁺/K⁺和相对MDA含量显著降低，相对SOD和POD抗氧化酶活性则显著增强。过表达芝麻SiNAC77可以增强抗氧化酶活性，减少离子毒害和氧化损伤，提高转基因植株的耐盐性。

关键词: 芝麻, 盐胁迫, NAC转录因子, 耐盐基因, 功能分析

Abstract:

NAC transcription factors play an important role in the regulation of plant growth, development, and responses to salt stress. The gene SiNAC77 in Sesamum indicum L. is involved in the response process to salt stress. Here, SiNAC77 was cloned to investigate its salt-tolerant function stress, which may provide a theoretical reference and valuable gene resource for breeding salt-tolerant sesame（Sesamum indicum L.）plants. First, the coding sequence（CDS）of SiNAC77 was cloned from Luzhi No.1, and its gene sequence and amino acid sequence characteristics were analyzed using bioinformatics tools. Subsequently, an SiNAC77-overexpressed vector was constructed and transformed containing SiNAC77 was constructed and the transformation of Arabidopsis was conducted using Agrobacterium tumefaciens. Finally, the salt-tolerant genotypes, physiological and biochemical indexes of the transgenic plants were analyzed under saline stress. As results, the CDS region of SiNAC77, 1 008 bp in length, was cloned successfully. The SiNAC77 gene encoded a protein containing 335 amino acids, with a molecular weight of 135.93 kD, an isoelectric point of 4.91 and 33 potential phosphorylation sites. The SiNAC77 promoter contained MBS, STRE, ARE, ABRE and TCA elements, functionally known to be involved in abiotic stress and phytohormone responses. The SiNAC77 overexpression vector was constructed and transformed successfully into Arabidopsis, and 12 independent transgenic lines were generated. Compared with wild-type Arabidopsis under salt stress, the transgenic lines had significantly higher seed germination rates, longer primary roots, higher fresh weights, significantly lower Na⁺/K⁺ ratios and MDA contents and higher SOD and POD antioxidant enzyme activities. In summary, the overexpression of SiNAC77 improved the salt tolerance in transgenic plants by enhancing the activities of antioxidant enzymes and reducing ionic toxicity and oxidative damage.

Key words: sesame, salt stress, NAC transcription factor, salt-tolerant gene, functional analysis

张玉娟, 黎冬华, 宫慧慧, 崔新晓, 高春华, 张秀荣, 游均, 赵军胜. 芝麻NAC转录因子基因SiNAC77的克隆及耐盐功能分析[J]. 生物技术通报, 2023, 39(11): 308-317.

ZHANG Yu-juan, LI Dong-hua, GONG Hui-hui, CUI Xin-xiao, GAO Chun-hua, ZHANG Xiu-rong, YOU Jun, ZHAO Jun-sheng. Cloning and Salt-tolerance Analysis of NAC Transcription Factor SiNAC77 from Sesamum indicum L.[J]. Biotechnology Bulletin, 2023, 39(11): 308-317.

图/表 7

表1 本研究所用引物序列

Table 1 Primer sequence used in this study

引物名称Primer name	引物序列Primer sequence（5'-3'）	用途Purpose
NAC77-BamH I-F	tgtctagactcgagggatccATGGAACAGTTGGCCGGTTTTG	基因克隆Gene clone
NAC77-BamH I-R	agcctgcagccatggggatccTCAGTAGTTCCACAAGCAGTC	基因克隆Gene clone
35s-F	GACGCACAATCCCACTATCC	转基因植株检测Transgenic plant detection
NAC77-R	TCAGTAGTTCCACAAGCAGTC	转基因植株检测Transgenic plant detection
Actin-F	CGGCGAGTCATCGTCATCAATGT	拟南芥内参引物Internal primer of Arabidopsis thaliana
Actin-R	GTTCGGGATTACGGTGGTGGGA	拟南芥内参引物Internal primer of Arabidopsis thaliana
NAC77-Fq	CTCCACTCACGGACTTGTCA	荧光定量PCR引物Fluorescent quantitative PCR primer
NAC77-Rq	AGGAAAGGAGGGTTGGATTG	荧光定量PCR引物Fluorescent quantitative PCR primer

图1 SiNAC77启动子顺式元件预测

Fig. 1 Cis-elements prediction of SiNAC77 promoter

图2 SiNAC77蛋白序列NAM结构域

Fig. 2 NAM domain of SiNAC77 protein sequence

图3 SiNAC77蛋白序列磷酸化位点预测

Fig. 3 Phosphorylation site prediction of SiNAC77 protein sequence

图4 转基因拟南芥植株的鉴定 A：卡那霉素筛选T1转基因苗；B：T1转基因植株的PCR鉴定，1-12为转基因植株，“-”为纯水空白对照，“+”为质粒对照，WT为野生型植株对照，M为DL2000 marker；C：T2转基因拟南芥中SiNAC77的表达量检测，OE-1、OE-2和OE-3分别来自T1“4、5、8”转基因拟南芥家系。*表示在P<0.05水平上差异显著，**表示在P<0.01水平上差异显著。下同

Fig. 4 Identification of transgenic Arabidopsis plants A: Identification of T1 transgenic Arabidopsis plants by kanamycin. B: Identification of T1 transgenic Arabidopsis plants, 1-12 indicate transgenic lines, “-” indicates pure water blank control, “+” indicates plasmid control, WT indicates wild type of Arabidopsis plants control, and M indicates DL2000 marker. C: Expression of SiNAC77in transgenic Arabidopsis. OE-1, OE-2, and OE-3 were obtained from T1（4, 5 and 8）transgenic plants. * indicate significant difference at P<0.05 level; **indicates significant difference at P<0.01 level. The same below

图5 转基因拟南芥的耐盐性鉴定 A：盐胁迫对转基因拟南芥种子发芽率的影响；B：盐胁迫对转基因拟南芥初生根根长的影响；C：盐胁迫对转基因拟南芥生物积累量的影响；D：盐胁迫下野生型和转基因拟南芥植株的表型

Fig. 5 Resistance characteristics of transgenic Arabidopsis plants to salt stress A: Seed germination of transgenic Arabidopsis under salt stress. B: The elongation of the primary root of transgenic Arabidopsis under salt stress. C: Fresh weight of transgenic Arabidopsis under salt stress. D: Phenotypes of wild type and transgenic Arabidopsis plants under salt stress

图6 盐胁迫下转基因拟南芥相关生理指标测定

Fig. 6 Determination of physiological indexes in transgenic Arabidopsis plants under salt stress

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芝麻NAC转录因子基因SiNAC77的克隆及耐盐功能分析

Cloning and Salt-tolerance Analysis of NAC Transcription Factor SiNAC77 from Sesamum indicum L.

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