大豆转录因子NAC1耐低磷胁迫的功能研究

Function Analysis of the Soybean Transcription Factor NAC1 in Tolerance to Low Phosphorus

【目的】磷含量偏低是影响酸性土壤作物产量的重要因素。大豆(Glycine max)是重要的粮食和油料作物,也为喜磷作物,缺磷则影响其产量与品质。NAC(NAM,ATAF1/2,CUC2)转录因子家族参与多种植物对生物胁迫和非生物胁迫响应的调控,是否参与大豆低磷胁迫响应尚未深入研究。以耐低磷野生大豆BW69为材料,克隆获得耐低磷基因GsNAC1并对其表达特性及功能进行分析,为深入解析GsNAC1调控大豆低磷胁迫及其机制奠定基础。【方法】从野生大豆BW69克隆GsNAC1的全长序列,并通过生物信息学分析探究其编码氨基酸序列的特征。随后,利用实时荧光定量PCR技术(qRT-PCR)对其组织表达模式进行分析,并通过激光共聚焦显微镜观察其编码蛋白的亚细胞定位。此外,通过大豆遗传转化试验,获得转基因株系并进行表型分析。最后,通过转录组联合分析来鉴定转基因植株中与低磷胁迫相关的差异表达基因(differentially expressed genes,DEGs)。【结果】成功克隆获得GsNAC1,编码区全长876 bp,通过构建系统发育树发现GsNAC1与AtATAF1的序列相似性为62.46%,与Williams 82参考基因组的GmNAC1序列没有差异;进一步的亚细胞定位结果显示,GsNAC1定位于细胞核;基于qRT-PCR技术,发现GsNAC1在大豆的根、茎、叶、顶端、花和豆荚均有表达,在根部的相对表达量最高,且受到低pH和低磷诱导表达显著上调。通过水培法和土培法进行表型试验,在低磷处理下,与野生型(WT)相比,转基因株系鲜重根冠比、总根长、根表面积、根体积和磷含量均显著高于WT。结合转录组测序数据进行分析,发现GsNAC1可能通过促进GmALMT6、GmALMT27、GmPAP27和GmWRKY21等基因表达增强其对低磷胁迫的耐受性。【结论】GsNAC1受低pH和低磷诱导表达上调,过量表达GsNAC1可以显著增强大豆对低磷胁迫的耐受性,在低磷胁迫反应中起促进作用。GsNAC1可能通过调控下游基因表达增强大豆对酸性低磷胁迫的耐受性。

【Objective】The productivity of acid soil crops is severely impacted by the limited availability of phosphorus.Soybean(Glycine max)is an important grain and oil crop,known for its preference for phosphorus.Phosphorus deficiency significantly affect both the yield and quality of soybean.While the NAC(NAM,ATAF1/2,CUC2)transcription factor family has been recognized for its involvement in regulating plant responses to various biotic and abiotic stresses,its role in soybean under low phosphorus stress remains largely unexplored.In this study,we focused on the low-phosphorus-tolerant wild soybean variety BW69 as our material,with the objective of cloning and analyzing the expression patterns and functions of the low-phosphorus-tolerant gene GsNAC1.This investigation lays the foundation for a deeper understanding the mechanisms behind the regulation of GsNAC1 response to low phosphorus stress.【Method】The full-length sequence of GsNAC1 was cloned from BW69,and the characteristics of its encoded amino acid sequence were explored by bioinformatics analysis.In addition,the tissue expression patterns of GsNAC1 were examined through quantitative real-time PCR(qRT-PCR).The subcellular localization of GsNAC1 was observed using laser confocal microscopy.Furthermore,soybean genetic transformation experiments were conducted for further phenotype analysis,and RNA-seq analysis was performed to identify differentially expressed genes(DEGs)related to low phosphorus stress.【Result】The GsNAC1 gene was successfully cloned,with a full-length coding region of 876 bp.Phylogenetic analysis showed a 62.46%sequence similarity between GsNAC1 and AtATAF1,and no difference was observed with the GmNAC1 sequence from the Williams 82 reference genome.Subcellular localization experiments further revealed that GsNAC1 was localized in the nucleus.Using qRT-PCR,it was discovered that GsNAC1 is expressed in roots,stems,leaves,apes,flowers and pods,with the highest relative expression level found in the roots.Notably,GsNAC1 exhibited significant upregulation in response to low pH and low phosphorus conditions.To assess the phenotypic effects,we performed experiments using both hydroponic and soil cultivation methods under low phosphorus conditions.The transgenic lines showed notable increases in root/shoot ratio,total root length,root surface area,root volume,and phosphorus content compared to the wild type(WT).Transcriptome analysis revealed that GsNAC1 may enhance tolerance to low phosphorus stress by promoting the expression of genes such as GmALMT6,GmALMT27,GmPAP27,and GmWRKY21.【Conclusion】The expression of GsNAC1 was up-regulated by low pH and low phosphorus,and overexpression of GsNAC1 significantly enhanced the tolerance to low phosphorus stress in soybean,playing a promoting role in the response to low phosphorus stress.Besides,GsNAC1 may enhance the tolerance to low phosphorus stress in soybean by regulating the expression of downstream genes.