为研究大豆(Glycine max)株型发育形成的分子机制, 以大豆“Williams 82”为材料, 分别取长短日照下不同发育时期植株的顶芽和腋芽, 提取RNA后等量混合, 采用Gateway方法构建酵母双杂交核系统cDNA文库, 并对文库进行了转录本多样性分析。实验结果表明, 构建的文库库容为1.2×10⁷ CFU, 插入片段重组率达100%且平均长度大于1 000 bp, 包含29 170个基因, 符合建库的标准, 可用于后续酵母双杂交的筛选。以大豆开花期和株型重要调控因子SOC1a为诱饵, 构建pGBKT7-SOC1a诱饵蛋白表达载体, 经毒性和自激活活性检测后, 利用构建的大豆顶芽和腋芽cDNA文库进行筛选, 共获得32个阳性克隆。进一步通过DNA测序及BLAST比对和功能注释分析, 得到14个与SOC1a互作的候选蛋白。其中, 克隆了5个候选蛋白, 并将其编码基因构建到pGADT7载体上, 然后分别与pGBKT7-SOC1a进行一对一的回转验证, 实验结果显示其中2个候选蛋白与SOC1a发生互作。进一步通过免疫共沉淀和荧光素酶互补实验证明了SEP2蛋白和SOC1a蛋白的互作关系。综上, 本研究成功构建了大豆顶芽和腋芽酵母双杂交核系统cDNA文库, 鉴定到的与SOC1a互作的蛋白将为从分子水平探究SOC1a调控大豆株型发育的机制提供理论基础。

INTRODUCTION: The shoot apexes and axillary buds determine crop growth and yield potential, with their developmental states directly shaping shoot architecture. However, there are currently few cDNA libraries constructed for shoot apexes and/or axillary buds in soybean (Glycine max).

RATIONALE: By constructing cDNA libraries for shoot apexes and axillary buds, we can gain an in-depth understanding of the core mechanisms underlying plant architecture in soybean at the molecular level, thereby providing theoretical foundations and genetic resources for the design of high-yielding and well-adapted soybean varieties.

RESULTS: This study constructed a yeast two-hybrid (Y2H) nuclear system cDNA library using shoot apexes and axillary buds from the cultivar "Williams 82" grown under long-day and short-day conditions at different developmental stages. Equal amounts of RNA extracted from these tissues were pooled and subjected to cDNA library construction using the Gateway method, followed by transcript diversity analysis. The resultant library had a capacity of 1.2×10⁷ CFU, with 100% recombination rate and an average length exceeding 1000 bp of the inserted fragments, covering 29,170 genes. This cDNA library meets the library construction standards and is suitable for subsequent Y2H screening. Using the key floral transition and shoot architecture regulator SOC1a as a bait, we first tested the toxicity and self-activation of the recombinant pGBKT7-SOC1a and then performed library screening. A total of 50 positive clones were obtained, and after DNA sequencing, BLAST alignment, and functional annotation, 14 candidate interacted proteins were identified. Among them, five candidate proteins were cloned into pGADT7 vector and subjected to pairwise retransformation assays with pGBKT7-SOC1a, confirming physical interactions between two of these proteins and SOC1a. Furthermore, the interaction between SOC1a and one of the candidate proteins, SEP2, was demonstrated through co-immunoprecipitation and luciferase complementation imaging assays.

CONCLUSION: This study establishes a high-quality Y2H cDNA library for soybean meristematic tissues and identifies novel SOC1a-interacting proteins, providing critical molecular insights into SOC1a-mediated regulation of soybean shoot architecture development.

Screening of SOC1a-interacting proteins using a yeast cDNA library constructed from soybean shoot apexes and axillary buds (A), with interactions confirmed by yeast retransformation (B), co-immunoprecipitation (C), and luciferase complementation imaging (D) assays.