小麦镉胁迫响应基因TaMYB1的功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0672

摘要/Abstract

摘要：

【目的】MYB家族成员在植物生长发育、生物胁迫和非生物胁迫等方面发挥重要调控作用，然而，小麦MYB转录因子在重金属镉（Cd）胁迫中的生物学功能研究较少。挖掘小麦Cd胁迫应答相关基因，阐明其在Cd胁迫中的生物学功能，为耐/低Cd小麦品种的选育奠定基础。【方法】构建Cd胁迫小麦根系酵母cDNA文库，筛选获得Cd胁迫应答基因，利用荧光定量PCR技术检测Cd胁迫条件下小麦不同组织中Cd胁迫应答基因的相对表达量；利用病毒诱导的基因沉默对该基因进行功能验证，检测对照和沉默植株的Cd含量、叶绿素含量、丙二醛（MDA）含量、超氧化物歧化酶（SOD）和过氧化物酶（POD）活力等生理生化指标。此外，对Cd胁迫应答基因及其在小麦和其他物种中的同源基因进行生物信息学分析（系统进化关系、序列特征和表达谱）。【结果】Cd胁迫主要抑制小麦根系发育，在添加0.002 mol/L Cd的酵母培养基上筛选Cd胁迫小麦根系酵母cDNA文库获得18个耐Cd转化子，其中5个转化子编码TaMYB1蛋白，转化TaMYB1酵母菌株在高Cd培养基中生长良好，而对照则明显受到抑制。RT-qPCR结果表明，TaMYB1在小麦幼苗中响应Cd胁迫。与对照相比，TaMYB1沉默植株中TaMYB1表达量明显下降，且根系和叶片Cd含量显著低于对照植株，叶绿素含量、SOD、POD活性高于对照植株，MDA含量则低于对照植株。同时，生物信息学分析发现，TaMYB1属于1R-MYB家族成员，具有高度保守的MYB和CC结构域，其包含7个外显子和6个内含子，在拔节期中期的花序和成熟期的花序表达最高，在灌浆前期种子和灌浆中后期种子中表达量最低。【结论】TaMYB1在小麦响应Cd胁迫应答中发挥重要作用。

关键词: 小麦, 非生物胁迫, 镉, 酵母, 病毒诱导的基因沉默

Abstract:

【Objective】Members of the MYB family play key roles in diverse biological processes including regulation of plant growth and development, biotic and abiotic stress. However, there were few researches specifically focusing on the function of MYB transcription factors in the response to cadmium（Cd）stress in Triticum aestivum. Therefore, identifying Cd-responsive genes and illustrating their biological function may lay a foundation for wheat Cd-resistant or low-Cd breeding. 【Method】Firstly, the yeast cDNA library of wheat roots under Cd stress was constructed, and then the Cd-resistant gene was screened. Then, the expression patterns of Cd-resistant gene in different tissues under Cd stress were examined by quantitative real-time PCR（RT-qPCR）. The function of the gene was verified by virus-induced gene silencing, and physiological and biochemical indicators such as the Cd content, chlorophyll content, malondialdehyde（MDA）content, superoxide dismutase（SOD）and peroxidase（POD）activities of the control and gene-silenced plants were detected. Finally, the candidate Cd-resistant gene and its homologous genes in other plants were analyzed by evolutionary relationship, gene structure and expression profile in detail. 【Result】The results indicated that root development of wheat was mainly inhibited under Cd stress. Total 18 positive transformants were screened by cDNA library on a tolerance of 0.002 mol/L Cd, and five transformants encoded TaMYB1 protein, which grew well while the control yeast strains restrained in high Cd medium. Results of RT-qPCR indicated that TaMYB1 responded to Cd stress in the wheat. Compared to the control, TaMYB1 expression in TaMYB1-silencing wheat significantly decreased and the concentration of Cd in the roots and leaves as well MDA content were remarkably lower than that in the control plants, while the contents of chlorophyll and the activities of SOD and POD increased apparently in TaMYB1-silencing plants. Meanwhile, bioinformatics analysis found that TaMYB1 belonged to the 1R-MYB family with highly conserved MYB and CC domains, containing seven exons and six introns. TaMYB1 expressed the highest in the inflorescences of two nodes or internodes visible stage and maximum stem length reached stage, while the lowest in in pre-filling seeds and mid- and late-filling seeds. 【Conclusion】TaMYB1 plays a important role in wheat response to Cd stress.

Key words: Triticum aestivum, abiotic stress, cadmium, yeast, virus-induced gene silencing

张怡, 张心如, 张金珂, 胡利宗, 上官欣欣, 郑晓红, 胡娟娟, 张聪聪, 穆桂清, 李成伟. 小麦镉胁迫响应基因TaMYB1的功能分析[J]. 生物技术通报, 2024, 40(1): 194-206.

ZHANG Yi, ZHANG Xin-ru, ZHANG Jin-ke, HU Li-zong, SHANGGUAN Xin-xin, ZHENG Xiao-hong, HU Juan-juan, ZHANG Cong-cong, MU Gui-qing, LI Cheng-wei. Functional Analysis of TaMYB1 Gene in Wheat Under Cadmium Stress[J]. Biotechnology Bulletin, 2024, 40(1): 194-206.

图/表 11

表1 引物序列

Table 1 Primer sequences

引物名称Primer name	引物序列Primer sequence（5'-3'）
TaMYB1-F	GTGAGTAAGGTTACCGAACTCCAAATGCAAATTGAG
TaMYB1-R	CGTGAGCTCGGTACCGGAGACGAGGCTAGCTCCGGC
TaMYB1-qPCR-F	AATTGAGGTCCAGAGACGACTG
TaMYB1-qPCR-R	GGTACTTCCCCTGGGCTTCG
TaActin-qPCR-F	CCAGGTATCGCTGACCGTAT
TaActin-qPCR-R	GCTGAGTGAGGCTAGGATGG

图1 Cd胁迫小麦表型检测 A：Cd胁迫小麦植株表型；B：Cd胁迫小麦植株根长；C：Cd胁迫小麦植株地上部长度；D：Cd胁迫小麦植株生物量。所有数据均重复3次以上，**代表在P=0.01水平上差异显著

Fig. 1 Phenotype analysis of wheat plant under Cd stress A: Phenotype of wheat plants under Cd stress. B: Root length of wheat plants under Cd stress. C: Shoot length of wheat plants under Cd stress. D: Total biomass of wheat plants under Cd stress. ** indicates significant difference from control plants at P=0.01

图2 Cd胁迫小麦根系的酵母双杂交文库质量检测 A：Cd胁迫小麦根系总RNA；B：利用SMART技术获得高质量cDNA； C：柱层析纯化后的cDNA；D, E：酵母文库插入片段PCR检测

Fig. 2 Quality detection of yeast two-hybrid library of wheat roots under Cd stress A: Total RNA of wheat roots under Cd stress. B: High-quality cDNA generated using SMART cDNA synthesis technology. C: Purified cDNA with column chromatography. D, E: PCR analysis of inserted clones from the yeast library

图3 转化子在添加不同Cd浓度的培养基上生长情况 A-C：在添加0.002、0.003和0.004 mol/L CdCl2的酵母培养基上筛选酵母文库的结果

Fig. 3 Transformants with the medium containing different cadmium concentration A-C: Refer to the screening results of yeast two-hybrid library in the yeast medium containing 0.002, 0.003 and 0.004 mol/L CdCl2

图4 酵母菌落在添加CdCl2的SD-Leu培养基中的生长曲线 A：酵母菌在正常条件下的生长曲线；B：酵母菌在高Cd培养基中的生长曲线

Fig. 4 Growth curves of yeast colonies in the SD-Leu medium added with CdCl2 A: Growth curves of yeast transformants growing in SD-Leu medium. B: Growth curves of yeast transformants growing in 0.004 mol/L CdCl2 medium

图5 TaMYB1响应Cd胁迫表达量分析 A：根系；B：地上部分。所有数据均重复3次以上，**代表在P=0.01水平上差异显著，下同

Fig. 5 Expression analysis of TaMYB1 under Cd stress A: Root. B: Shoot. ** indicates significant difference from control plants at P=0.01, the same below

图6 沉默TaMYB1基因增强小麦对Cd耐受 A：TaMYB1基因沉默和对照植株在正常条件和Cd胁迫后的表型； B：TaMYB1沉默植株中TaMYB1基因表达量检测；C：TaMYB1基因沉默和对照植株Cd胁迫后植株的Cd含量

Fig. 6 Silencing of TaMYB1 enhanced wheat tolerance to cadmium A: Phenotypes of TaMYB1 gene-silenced and control plants under normal conditions and cadmium stress. B: Relative expression level of TaMYB1 gene in gene-silenced plants. C: Cadmium content of TaMYB1 gene-silenced and control plants after cadmium stress

图7 TaMYB1沉默和对照植株在正常条件和Cd胁迫后的生理指标检测 A-D中数据均取自代表小麦叶片

Fig. 7 Physiological index detection of TaMYB1 gene-silenced and control plants under normal conditions and cadmium stress The data in A-D were all taken from wheat leaves

图8 TaMYB1及其他物种同源蛋白的系统进化关系

Fig. 8 Phylogenetic relationships of TaMYB1 and its homologous proteins in other species

图9 TaMYB1及其小麦同源基因的序列特征及系统进化关系 A：TaMYB1及其同源蛋白的系统进化分析；B：TaMYB1及其同源蛋白的保守基序；C：TaMYB1及其同源基因的结构；D：保守基序1的序列标识图；E：保守基序2的序列标识图

Fig. 9 Sequence features and phylogenetic relationships of TaMYB1 and its homologous genes in wheat A: Phylogenetic tree of TaMYB1 and its homologous proteins was analyzed. B: Conserved motifs of TaMYB1 and its homologous protein. C: Structures of TaMYB1 and its homologous genes. D: The sequence logo of the conserved motif 1. E: The sequence logo of the conserved motif 2

图10 TaMYB1及其小麦中同源基因的组织表达谱

Fig. 10 Tissue expression profile of TaMYB1 and its homologous genes in wheat

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