陆地棉<i>GhbHLH149-like</i>基因耐盐功能验证

doi:10.7668/hbnxb.20196467

摘要/Abstract

摘要：

为深入研究棉花bHLH转录因子在盐胁迫响应中的功能,利用农杆菌介导的方法,将河北省农林科学院棉花研究所基因工程课题组前期鉴定的棉花bHLH转录因子GhbHLH149-like 转化野生型拟南芥,初步验证了GhbHLH149-like转录因子在拟南芥中的功能。蛋白质结构预测显示,GhbHLH149-like蛋白的二级结构主要由α-螺旋和无规则卷曲组成,是一种非跨膜的亲水性蛋白,实时荧光定量PCR结果表明,转基因拟南芥GhbHLH149-like基因的表达量比野生型拟南芥显著升高,表型和生理指标测定结果显示,在盐胁迫条件下,转基因拟南芥种子萌发率和根长均显著高于野生型拟南芥,转基因拟南芥幼苗长势明显优于野生型拟南芥,叶片中过氧化氢(H₂O₂)及超氧阴离子( ${\mathrm{O}}_{2}^{-}$)的含量显著低于野生型,超氧化物歧化酶(SOD)活性显著高于野生型植株。综上所述,GhbHLH149-like基因有可能通过参与拟南芥活性氧清除,从而显著提高其耐盐性,为深入解析棉花GhbHLH149-like基因在盐胁迫应答中的分子机制奠定了理论基础。

关键词: 陆地棉, GhbHLH149-like, 蛋白序列分析, 盐胁迫, 功能验证

Abstract:

To further investigate the function of cotton basic helix-loop-helix(bHLH)transcription factors in salt stress response,we introduced the cotton bHLH transcription factor GhbHLH149-like(previously identified by our team)into wild-type Arabidopsis thaliana via Agrobacterium-mediated transformation,and verified the function of GhbHLH149-like transcription factor in Arabidopsis.Protein structure prediction indicated that the secondary structure of GhbHLH149-like protein is mainly composed of α-helice and random coil,and it was a non-transmembrane hydrophilic protein.Quantitative Real-time PCR(qRT-PCR)results showed that the expression level of the GhbHLH149-like gene in transgenic Arabidopsis was significantly higher than wild-type.The results of phenotypic and physiological index assays indicated that the seed germination rate and root length of transgenic seedlings were significantly higher under salt stress,and the growth performance of transgenic seedlings was markedly better.Meanwhile,the contents of hydrogen peroxide(H₂O₂)and superoxide anion(O₂^-)were significantly lower,whereas the superoxide dismutase(SOD)activity was significantly higher in the transgenic lines.Taken together,GhbHLH149-like gene can significantly enhance the salt tolerance of Arabidopsis,presumably by participating in reactive oxygen species(ROS)scavenging.This study lays a theoretical foundation for the in-depth dissection of the molecular mechanism of cotton GhbHLH149-like gene in salt stress response.

Key words: Upland cotton, GhbHLH149-like, Protein sequence analysis, Salt stress, Function verification

中图分类号:

刘迪, 甄军波, 江萌萌, 刘琳琳, 冯晓晴, 迟吉娜. 陆地棉GhbHLH149-like基因耐盐功能验证[J]. 华北农学报, 2026, 41(3): 68-74. doi: 10.7668/hbnxb.20196467.

LIU Di, ZHEN Junbo, JIANG Mengmeng, LIU Linlin, FENG Xiaoqing, CHI Jina. Functional Verification of GhbHLH149-like Gene in Salt Tolerance in Upland Cotton[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(3): 68-74. doi: 10.7668/hbnxb.20196467.

http://www.hbnxb.net/CN/Y2026/V41/I3/68

图/表 7

表1 荧光定量PCR引物

Tab.1 Primer of qRT-PCR

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')
AtUBQ5-F	GACGCTTCATCTCGTCC
AtUBQ5-R	CCACAGGTTGCGTTAG
qGhbHLH149-like-F	ACAGGGTTCTCGCCGTTTT
qGhbHLH149-like-R	TATCGGGCTTCCTCAACCTG

图1 GhbHLH149-like蛋白的二级结构(A)和三维结构(B)预测

Fig.1 The secondary structure(A)and tertiary structure(B)prediction of GhbHLH149-like protein

图2 GhbHLH149-like氨基酸序列分析 A.磷酸化位点预测;B.蛋白质跨膜结构预测;C.蛋白亲/疏水性预测。

Fig.2 Amino acid sequence analysis of GhbHLH149-like A.Phosphorylation site prediction;B.Protein transmembrane domain prediction;C.Protein hydrophilicity/hydrophobicity prediction.

图3 拟南芥转基因株系中GhbHLH149-like的表达水平 A.转基因阳性植株检测:M.D2000 plus;CK-.阴性对照,CK+.阳性对照;B.转基因阳性植株基因表达量检测。不同小写字母表示差异显著(P<0.05)。图4-6同。

Fig.3 Expression analysis of GhbHLH149-like in Arabidopsis thaliana transgenic lines A.Detection of transgenic positive plants:M.D2000 plus;CK-.Negative control;CK+.Positive control;B.Detection of gene expression levels in transgenic positive plants.Different lowercase letters indicated significant difference(P<0.05).The same as Fig.4-6.

图4 盐胁迫下拟南芥萌发率分析 A.转基因株系与野生型的种子萌发试验;B.拟南芥萌发率分析。

Fig.4 Analysis of germination rate of Arabidopsis thaliana under salt stress A.Seed germination assay between transgenic lines and wild type;B.Analysis of germination rate.

图5 盐胁迫下拟南芥根长分析 A.转基因株系与野生型的根伸长试验;B.拟南芥根长分析。

Fig.5 Analysis of root length of Arabidopsis thaliana under salt stress A.Root elongation assay between transgenic lines and wild type;B.Quantitative analysis of root length.

图6 拟南芥在盐胁迫下的表型及生理指标分析 A.盐胁迫下拟南芥表型;B-D.生理指标分析。

Fig.6 Phenotypes and physiological indices analysis of Arabidopsis thaliana under salt stress A.Phenotypes of Arabidopsis thaliana under salt stress;B-D.Physiological indices analysis.

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陆地棉GhbHLH149-like基因耐盐功能验证

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陆地棉GhbHLH149-like基因耐盐功能验证

Functional Verification of GhbHLH149-like Gene in Salt Tolerance in Upland Cotton

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