大豆转录因子GmMYB069的克隆与耐盐功能鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2025-0348

摘要/Abstract

摘要：

目的 MYB（v-MYB avian myeloblastosis viral oncogene homolog）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。大豆GmMYB069参与盐胁迫响应过程。克隆大豆GmMYB069并分析其耐盐功能，为大豆耐盐育种提供分子基础和基因资源。方法克隆GmMYB069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GmMYB069过表达载体，利用农杆菌介导法转化大豆。对过表达株系的耐盐表型和生理生化指标进行分析。结果成功克隆了长度为1 059 bp的GmMYB069 CDS序列，编码352个氨基酸，相对分子量为39 309.26 Da，预测等电点为6.76；其蛋白为不稳定的亲水性蛋白，多肽链中无规则卷曲占比最大，没有跨膜结构与信号肽，与密花豆的亲缘关系最近，与蔓花生的亲缘关系最远。GmMYB069在根中的表达量较高，其次为茎、叶。在盐胁迫逆境条件下，GmMYB069基因在胁迫前期表达量呈上升趋势，可达到峰值，随着胁迫时间的延长，其表达量下降，表明其在早期可以响应盐胁迫。成功构建了GmMYB069过表达载体，并进行大豆根毛转化，获得过表达嵌合株系。在盐胁迫下，与空载植株相比，过表达株系的相对表达量显著提高，过表达嵌合植株叶片中抗氧化酶（SOD、POD）升高，MDA减少，Na⁺、K⁺含量在过表达嵌合植株根中无显著差异，Na⁺含量在过表达嵌合植株叶片中显著降低，K⁺则相反。结论过表达大豆GmMYB069可以增强抗氧化酶活性，阻止Na⁺从根向叶的运输，减少植物的盐害，提高转基因植株的耐盐性。

关键词: 大豆, GmMYB069, 基因克隆, 盐胁迫, 生物信息学分析, 功能分析

Abstract:

Objective MYB (v-MYB avian myoblastosis viral oncogene homolog) transcription factors play an important regulatory role in plant growth, development, and salt stress response. Soybean GmMYB069 is involved in the salt stress response process. Cloning soybean GmMYB069 and analyzing its salt tolerance function may provide molecular basis and genetic resources for soybean salt tolerance breeding. Method The full-length CDS sequence of GmMYB069 was cloned and its gene and amino acid sequence characteristics were analyzed using bioinformatics software. GmMYB069 overexpression vector was transferred and soybean was transformed using Agrobacterium mediated method. The salt-tolerant phenotype and physiological and biochemical indicators of overexpressed strains were analyzed. Result A 1 059 bp GmMYB069 CDS sequence was successfully cloned, encoding 352 amino acids with a relative molecular weight of 39 309.26 Da and a predicted isoelectric point of 6.76. Its protein was an unstable hydrophilic protein, with the largest proportion of irregular curls in the polypeptide chain. It lacked transmembrane structures and signal peptides and has the closest genetic relationship with Sophora alopecuroides and the farthest genetic relationship with Crassulaceae. GmMYB069 had a higher expression in the roots, followed by the stems and leaves. Under salt stress conditions, the expression of GmMYB069 gene showed an upward trend in the early stage of stress, reaching a peak. With the prolongation of stress time, its expression decreased, indicating that it can respond to salt stress in the early stage. The GmMYB069 overexpression vector was successfully constructed and transformed it into soybean root hairs, and an overexpressed chimeric strain was obtained. Under salt stress, compared with the empty load plants, the relative expression of overexpressing lines significantly increased. The antioxidant enzymes (SOD, POD) in the leaves of overexpressed chimeric plants increased, MDA decreased, and the Na⁺ and K⁺ content did not show significant differences in the roots of overexpressed chimeric plants. The Na⁺ content significantly decreased in the leaves of overexpressed chimeric plants, while the opposite was true for K⁺. Conclusion The overexpression of soybean GmMYB069 may enhance antioxidant enzyme activity, prevent Na⁺ transport from roots to leaves, reduce plant salt damage, and improve the tolerance of transgenic plants to salt.

Key words: soybean, GmMYB069, gene cloning, salt stress, bioinformatics analysis, functional analysis

杨悦, 李昌宁, 孙晓露, 李雅迁, 杜维俊, 王利祥, 王敏, 姬月梅. 大豆转录因子GmMYB069的克隆与耐盐功能鉴定[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0348.

YANG Yue, LI Chang-ning, SUN Xiao-lu, LI Ya-qian, DU Wei-jun, WANG Li-xiang, WANG Min, JI Yue-mei. Cloning and Identification of Salt Tolerance Function of Soybean Transcription Factor GmMYB069[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0348.

图/表 11

表1 GmMYB069引物序列

Table 1 GmMYB069 primer sequence

引物名称 Primer name	引物序列 Primer sequence（5′-3′）
GmMYB069-F	ATGGGAAGGTCACCGTGTTG
GmMYB069-R	CCACAAAATTGGTGACGCAGAA
qRT-GmCYP2-F	CGGGACCAGTGTGCTTCTTCA
qRT-GmCYP2-R	CCCCTCCACTACAAAGGCTCG
qRT-GmMYB069-F	GCCTCATGTGGTGTTGAGTG
qRT-GmMYB069-R	TACAGCCACTGTCCATTTGGT
GmMYB069-pUBi-F	TTGATGTGATTACAGTCTAGAATGGGAAGGTCACCGTGTTG
GmMYB069-pUBi-R	GTTAATTAACCCGCTGGTACCCTACCACAAAATTGGTGACGCA

图1 MYB家族成员在两种材料中的表达分析

Fig. 1 Expression analysis of MYB family members in two materials

图2 大豆GmMYB069基因在盐胁迫下的定量分析

Fig. 2 Quantitative analysis of soybean (Glycine max) GmMYB069 gene under salt stress

图3 GmMYB069基因克隆

Fig. 3 GmMYB069 gene cloning

图4 GmMYB069基因结构

Fig. 4 GmMYB069 gene structure

图5 GmMYB069蛋白特性分析A. GmMYB069蛋白疏水性预测图；B. GmMYB069蛋白跨膜结构域预测图；C. GmMYB069蛋白的信号肽分析（横坐标表示氨基酸的位置，纵坐标表示信号肽的概率）；D. GmMYB069蛋白的保守结构域；E. GmMYB069蛋白质三级结构

Fig. 5 Characterization of GmMYB069 proteinA. GmMYB069 protein hydrophobicity prediction map. B. GmMYB069 protein transmembrane domain prediction diagram. C. Signal peptide analysis of GmMYB069 protein （The horizontal coordinate indicates the position of the amino acid， and the vertical coordinate indicates the probability of the signal peptide）. D. Conservative domains of GmMYB069 protein. E. Tertiary structure of GmMYB069 protein

图6 GmMYB069与其他物种MYB蛋白多重序列比对（A）以及系统发育树（B）

Fig. 6 Multiple sequence alignment of GmMYB069 with MYB proteins from other species (A) and phylogenetic tree (B)

图7 大豆时空表达模式图A. 大豆不同组织GmMYB069基因表达量；B. 盐胁迫下大豆W82不同时间段GmMYB069基因表达量；C. 在150和535两种材料的不同时间段GmMYB069基因表达量。所示数据为平均数±方差，显著性差异使用t检验分析，ns表示无显著差异，*P<0.05，**P<0.01，***P<0.001，下同

Fig. 7 Temporal and spatial expression pattern diagram of soybeanA. Expressions of GmMYB069 gene in different tissues of soybean. B. The expressions of GmMYB069 gene in soybean W82 under salt stress at different time periods. C. The expressions of GmMYB069 gene at different time periods in material 150 and 535. The data shown is mean±variance, and significant differences were analyzed using t-test. ns indicates no significant difference, *P<0.05, **P<0.01, and ***P<0.001, the same below

图8 盐胁迫和水处理下GmMYB069-OE和对照EV的表达量分析

Fig. 8 Expression analysis of GmMYB069-OE and control EV under salt stress and water treatment

图9 基因GmMYB069过表达表型分析A：水处理下GmMYB069-OE和对照EV的表型（OE-Control：水处理下的过表达嵌合植株）；B：盐处理下GmMYB069-OE和对照EV的表型（OE-Salt：盐处理下的过表达嵌合植株）；C：盐胁迫下GmMYB069-OE和对照EV地下部表型（OE-Salt：盐处理下的过表达嵌合植株）

Fig. 9 Phenotypic analysis of GmMYB069 OverexpressionA. Phenotypes of GmMYB069-OE and control EV under water treatment (OE-Control: Overexpressed chimeric plants under water treatment). B. Phenotypes of GmMYB069-OE and control EV under salt treatment (OE-Salt: Overexpressed chimeric plants under salt treatment). C. Subterranean phenotype of GmMYB069-OE and control EV under salt stress (OE-Salt: Overexpressed chimeric plants under salt treatment)

图10 盐胁迫下GmMYB069过表达嵌合植株的生理指标和Na+、K+含量A：GmMYB069-OE叶片SOD活性；B：GmMYB069-OE叶片POD活性；C：GmMYB069-OE叶片MDA含量；D：GmMYB069-OE叶片SPAD值；E：GmMYB069-OE植株在根中的Na+含量；F：GmMYB069-OE植株在叶中的Na+含量；G：GmMYB069-OE植株在根中的K+含量；H：GmMYB069-OE植株在叶中的K+含量

Fig. 10 Physiological indicators and Na+, K+ contents of GmMYB069 overexpressing chimeric plants under salt stressA: SOD activity in GmMYB069-OE leaves. B: POD activity in GmMYB069-OE leaves. C: MDA content in GmMYB069-OE leaves. D: SPAD value of GmMYB069-OE leaves. E: Na+ content in the roots of GmMYB069-OE plants. F: Na+ content in the leaves of GmMYB069-OE plants. G: K+ content in the roots of GmMYB069-OE plants. H: K+ content in the leaves of GmMYB069-OE plants

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