Cloning of Gene CtbHLH128 in Safflower and Response Function Regulating Drought Stress

doi:10.13560/j.cnki.biotech.bull.1985.2025-0059

Abstract

Abstract:

Objective Basic helix-loop-helix (bHLH) transcription factors represent one of the largest families of transcription factors in eukaryotes, and their members play crucial roles in plant growth, development, and stress responses. This study enriches the research on the function of the bHLH transcription factor family genes and lays an important foundation for analyzing the molecular regulatory mechanism of safflower (Carthamus tinctorius L.) seed development. Method Based on the complete CDS sequence of CtbHLH128 in the genome, primers were designed using Primer 6.0 software to clone and obtain the correct sequence of the gene, and bioinformatics was used to analyze the number of amino acids encoded by the gene, conserved domains, and phylogenetic tree. The CtbHLH128 fragment was constructed into a plant expression vector and transformed into Arabidopsis thaliana by Agrobacterium infection. Herbicide screening and PCR were used to identify positive strains. Drought stress was applied to the positive strains at the seedling and germination stages to explore the role of this gene in drought stress. Result The CtbHLH128 consists of 948 bp nucleotides, encoding 315 amino acids. The molecular weight of its encoded protein is 33 742.32 Da, and the theoretical isoelectric point is 9.02. CD-Search analysis results revealed that its C-terminus contained a typical HLH conserved domain located at amino acid residues 249-301. Phylogenetic tree revealed high similarity with bHLH128 gene in sunflower and lettuce. The CtbHLH128 gene was overexpressed in Arabidopsis, and three T3 positive strains were obtained through resistance screening and PCR identification. The positive strains were subjected to drought stress-related phenotypic analysis. The results revealed that transgenic Arabidopsis plants showed greater sensitivity to drought conditions at the seedling stage, but enhanced resistance to drought at the germination stage. Conclusion This suggests that the CtbHLH128 gene may possess potential functions in the responses to drought stress and seed germination.

Key words: safflower, transcription factor, CtbHLH128, gene cloning, sequence analysis, overexpression, drought stress

LI Kai-jie, WU Yao, LI Dan-dan. Cloning of Gene CtbHLH128 in Safflower and Response Function Regulating Drought Stress[J]. Biotechnology Bulletin, 2025, 41(8): 234-241.

Figures/Tables 7

Table 1 Information of primers in experiement

名称 Primer name	核苷酸序列 Nucleotide sequence （5′‒3′）	功能/基因名 Function/Gene name
PF1	ATGTATCCGAACTCCAATTCCTCT	Sequence clone （CtbHLH128）
PR1	TCATCTTGGTTTGCATCCACACGAA	Sequence clone （CtbHLH128）
PF2	atttggagaggacagggtaccATGTATCCGAACTCCAATTCCTCT	Recombination （CtbHLH128）
PR2	cttgctcaccatggtactagtTCAAACCTTAGACCGTCCACCACCA	Recombination （CtbHLH128）
PF3	GGTAATATCCGGAAACCTCCTCGGA	PCR identification （CtbHLH128）
PR3	GCGGGCGAGACCTAATCCTCCGCCT	PCR identification （CtbHLH128）
PF4	TAGATTCTTCTCACCTCCGC	semi-quantitative PCR （CtbHLH128）
PR4	GCTTGGCGCGAATTTTACAAG	semi-quantitative PCR （CtbHLH128）
PF5	GAACGGGAAATTGTCCGTG	semi-quantitative PCR （Atactin）
PR5	TGAACAATCGATGGACCTGAC	semi-quantitative PCR （Atactin）

Fig. 1 Agarose gel electrophoresis of transcription factors1: Marker; 2: target strip

Fig. 2 Phylogenetic analysis of CtbHLH128A: Phylogenetic tree. B: Multiple-sequence alignment and analysis of the bHLH motif of amino acid sequences between CtbHLH128 and the closely related species. Amt: Amaranthus tricolor; Brd: Brachypodium distachyon; Brn: Brassica napus; Caa: Capsicum annuum; Cis: Citrus sinensis; Cus: Cucumis sativus; Eug: Eucalyptus grandis; Glm: Glycine max; Goh: Gossypium hirsutum; Hea: Helianthus annuus; Hov: Hordeum vulgare; Jur: Juglans regia; Kln: Klebsormidium nitens; Las: Lactuca sativa; Mae: Manihot esculenta; Met: Medicago truncatula; Nen: Nelumbo nucifera; Nit: Nicotiana tabacum; Ors: Oryza sativa; Php: Physcomitrium patens; Pot: Populus trichocarpa; Ric: Ricinus communis; Sei: Setaria italica; Sly: Solanum lycopersicum; Stu: Solanum tuberosum; Zma: Zea mays; Sbi: Sorghum bicolor; Ct: Carthamus tinctorius

Fig. 3 Identification of positive transfected A. thalianaA: PCR identification results of the positive seedlings genomic DNA. B: Results of the semi-quantitative PCR between transgenic positive seedlings and control seedlings. #28-1, #29-1 and #32-4 are homozygous T3 positive lines transferred CtbHLH128 to Arabidopsis thaliana, and WT is wild-type. The same below

Fig. 4 Morphological diagram of overexpressed and wild-type Arabidopsis under drought stress during seedling stageOEbHLH#26, OEbHLH#7, OEbHLH#23, OEbHLH#3 and OEbHLH#36 indicate the different positive T2 lines. WT (col-gl) refers to wild type. The observation was conducted at 14 d after drought stress

Fig. 5 Germination rates of transgenic Arabidopsis and wild Arabidopsis seeds under 15% PEG simulated drought conditions** P<0.01. The same below

Fig. 6 Germination rates of transgenic Arabidopsis and wild Arabidopsis (WT)seeds under ABA conditions

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