红花CtbHLH128基因克隆及调控干旱胁迫应答功能研究

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

摘要/Abstract

摘要：

目的碱性螺旋‒环‒螺旋（basic helix-loop-helix, bHLH）转录因子广泛参与植物生长、发育和胁迫响应，是真核生物最大的转录因子家族之一。课题组前期在红花转录组数据中发现一个可能参与红花干旱及脱水负调控响应基因CtbHLH128，深入探究该基因参与干旱胁迫应答的功能情况，为红花生物育种等奠定重要基础。方法基于基因组中CtbHLH128的完整CDS序列，采用Primer 6.0软件设计引物，克隆并获得该基因正确序列，并采用生物信息学分析该基因编码氨基酸数目、保守结构域及系统发育树等；将CtbHLH128片段构建至植物表达载体，采用农杆菌侵染法转化拟南芥，并采用除草剂筛选及PCR等方法鉴定阳性株系，对阳性株系采用苗期和萌发期干旱胁迫以探究该基因在干旱胁迫中的作用。结果 CtbHLH128含948 bp核苷酸，编码315个氨基酸，其编码蛋白分子量为33 742.32 Da，理论等电点为9.02；CD-Search分析结果揭示，其C端含典型的HLH保守结构域，位于第249-301位氨基酸残基处；系统发育树揭示其与向日葵和生菜中bHLH128基因相似度较高；将CtbHLH128基因在拟南芥中过表达，经过抗性筛选和PCR鉴定共获得3个T₃代阳性株系，对阳性株系进行干旱胁迫相关的表型分析，结果揭示转基因拟南芥植株在苗期对干旱条件表现出更强的敏感性，但在萌发期对干旱的抗性增强。结论 CtbHLH128基因在干旱胁迫响应及种子萌发过程中具有重要功能。

关键词: 红花, 转录因子, CtbHLH128, 基因克隆, 序列分析, 过表达, 干旱胁迫

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

李开杰, 吴瑶, 李丹丹. 红花CtbHLH128基因克隆及调控干旱胁迫应答功能研究[J]. 生物技术通报, 2025, 41(8): 234-241.

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.

图/表 7

表1 试验中所用引物的信息

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）

图1 转录因子的琼脂糖凝胶电泳1：Marker；2：目的条带

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

图2 CtbHLH128系统进化分析A：系统发育树；B：CtbHLH128与相近物种同源基因氨基酸序列的多序列比对分析；Amt：苋；Brd：二穗短柄草；Brn：油菜；Caa：辣椒；Cis：甜橙；Cus：黄瓜；Eug：大桉；Glm：大豆；Goh：棉花；Hea：向日葵；Hov：大麦；Jur：胡桃；Kln：克里藻；Las：生菜；Mae：木薯；Met：苜蓿；Nen：莲；Nit：烟草；Ors：水稻；Php：小立碗藓；Pot：毛果杨；Ric：蓖麻；Sei：梁；Sly：番茄；Stu：马铃薯；Zma：玉米；Sbi：高粱；Ct：红花

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

图3 拟南芥阳性苗的鉴定A：阳性苗基因组DNA的PCR鉴定结果；B：转基因阳性苗与野生型对照的半定量PCR检测结果。#28-1、#29-1和#32-4为纯合T3代阳性转基因拟南芥株系，WT为野生型拟南芥，下同

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

图4 苗期干旱胁迫下过表达和野生型拟南芥的形态图OEbHLH#26、OEbHLH#7、OEbHLH#23、OEbHLH#3和OEbHLH#36表示转基因T2代株系；WT（col-gl）为野生型；干旱处理14 d

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

图5 过表达与野生型拟南芥种子在15% PEG模拟干旱条件下的萌发率** P<0.01，下同

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

图6 转基因拟南芥与拟南芥（WT）种子在ABA处理下的发芽率

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

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