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

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

Abstract

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

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.

Figures/Tables 7

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

Fig. 1 Cis-elements prediction of SiNAC77 promoter

Fig. 2 NAM domain of SiNAC77 protein sequence

Fig. 3 Phosphorylation site prediction of SiNAC77 protein sequence

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

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

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

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