Table 5_Genome-wide identification and expression analysis of soybean bHLH transcription factor and its molecular mechanism on grain protein synthesis.doc

IntroductionSoybean seeds have a protein content of about 40% and are widely used due to their unique nutritional value. Research has found that drought and nitrogen fertilizer environments are conducive to the formation and accumulation of grain protein. Nitrogen is an essential element for soybean growth and development, and is converted into grain protein through a series of pathways such as the soybean root nodule system. The earliest report on the regulation of nodulation by bHLH transcription factors in leguminous plants was in 2011, but the network regulatory mechanism of their involvement in soybean protein synthesis is still unclear.
MethodsThis study we use BLASTP and HMMER to identified 296 soybean GmbHLH genes through whole-genome identification and systematic analysis and is classified into 29 subfamilies, by comprehensively analyzing complex biological issues such as gene structure, function, interactions, and regulatory networks.
ResultsThis study explores the life processes of soybean growth, development, metabolism, and resistance to adversity.The non-synonymous substitution rate/synonymous substitution rate (Ka/Ks) analysis indicates that most of the homologous genes have undergone purifying selection (Ka/Ks << 1). Cis-acting element analysis of the promoter revealed that this gene family plays an important role in stress response, growth and development, hormone regulation, and other processes. RNA-seq data and qRT-PCR experiments indicated that GmbHLH genes were diversely expressed in different organs/tissues, and many GmbHLH genes were found to be differentially expressed under salt, and drought stresses, suggesting their critical role in soybean resistance to abiotic stress.
DiscussionThe GmbHLH98 gene(LOC100778376), which is highly expressed under both drought and salt stress, was selected for functional validation. Molecular and agronomic trait analyses of positive transgenic offspring showed that the protein content of soybean seeds increased by 36.8%, indicating that drought and salt conditions promote protein synthesis. This study provides a theoretical basis for exploring the synergistic regulation of drought and salt response and protein synthesis by GmbHLH98 in the future.These results provide fundamental information about the soybean bHLH genes and will aid in their further functional elucidation and exploitation.

引言
大豆种子蛋白质含量约为40%，凭借其独特的营养价值被广泛应用。研究表明，干旱与氮肥环境有利于籽粒蛋白质的形成与积累。氮是大豆生长发育所必需的元素，可通过大豆根瘤系统等一系列通路转化为籽粒蛋白质。2011年首次报道了豆科植物中碱性螺旋-环-螺旋（basic Helix-Loop-Helix，bHLH）转录因子对结瘤的调控作用，但其参与大豆蛋白质合成的网络调控机制仍不明晰。

方法
本研究通过全基因组鉴定与系统分析，借助BLASTP与HMMER工具共鉴定得到296个大豆GmbHLH基因，并将其划分为29个亚家族；同时综合分析了基因结构、功能、互作关系及调控网络等复杂生物学问题。

结果
本研究解析了大豆生长、发育、代谢及抗逆等生命过程。非同义替换率/同义替换率（Ka/Ks）分析结果显示，大多数同源基因经历了纯化选择（Ka/Ks << 1）。启动子顺式作用元件分析表明，该基因家族在胁迫响应、生长发育、激素调控等过程中发挥重要作用。RNA测序（RNA-seq）数据与实时荧光定量PCR（qRT-PCR）实验结果显示，GmbHLH基因在不同器官/组织中呈现差异化表达；且在盐胁迫与干旱胁迫条件下，多个GmbHLH基因存在显著表达差异，表明其在大豆抗非生物胁迫过程中发挥关键作用。

讨论
本研究选取在干旱与盐胁迫下均高表达的GmbHLH98基因（LOC100778376）进行功能验证。对阳性转基因后代的分子与农艺性状分析结果表明，大豆种子的蛋白质含量提升了36.8%，证实干旱与盐胁迫可促进蛋白质合成。本研究为后续解析GmbHLH98协同调控干旱与盐胁迫响应及蛋白质合成的机制提供了理论依据。本研究结果为大豆bHLH基因家族提供了基础数据，将有助于后续对其功能的进一步解析与开发利用。