花生GRF基因家族的鉴定与分析

Identification and analysis of the GRF gene family in peanut

摘要:

花生(Arachis hypogaea L.)是重要的油料作物，广泛种植于热带和亚热带地区。生长调节因子(growth-regulating factors, GRFs)是调控植物生长和逆境应答的重要转录因子。为了提高花生的产量和抗逆性，研究其GRFs在生长发育和逆境胁迫中的作用至关重要。本研究通过生物信息学方法分析了AhGRF基因家族的理化性质、进化关系、染色体定位以及序列差异，通过实时荧光定量PCR (real-time fluorescence quantitative PCR, qRT-PCR)技术揭示了AhGRF基因家族在干旱和低温胁迫下的表达模式，构建亚细胞定位表达载体分析AhGRF2b和AhGRF3b蛋白在细胞中的定位，最后利用酵母双杂交技术分析AhGRF3b的互作蛋白。结果显示，花生中共鉴定出24个AhGRF基因，不均匀地分布在16条染色体上，编码氨基酸长度为268‒630 aa，相对分子量为29 842.27‒67 980.83 Da，该基因家族蛋白多数呈酸性且预测定位于细胞核；系统发育分析发现AhGRF基因家族成员可以分为6个簇；多重序列比对结果表明大多数AhGRF含有保守的QLQ和WRC结构域；干旱胁迫和低温胁迫处理下，多个AhGRF基因表达量显著上调，尤其是AhGRF2b和AhGRF3b基因，表明多个基因可响应干旱和低温胁迫；烟草瞬时表达实验表明，AhGRF2b定位在细胞核和细胞质中，AhGRF3b定位在细胞核中。此外，酵母双杂分析发现，AhGRF3b与过氧化氢酶蛋白AhCAT3可能存在互作，推测花生AhGRF基因可通过调控活性氧的清除来参与花生抗逆。本研究的结果为花生抗逆育种提供了一定的理论基础。

Abstract:

Peanut (Arachis hypogaea L.) is an important oilseed crop widely cultivated in tropical and subtropical regions. The growth-regulating factors (GRFs) are key transcription factors that regulate plant growth and responses to stress. To improve the peanut yield and stress tolerance, it is crucial to investigate the roles of GRFs in growth, development, and stress responses. In this study, we analyzed the physicochemical properties, evolutionary relationships, chromosomal localization, and sequence variations of the AhGRF gene family by bioinformatics methods. Using qRT-PCR, we revealed the expression patterns of AhGRF genes under drought and cold stress conditions. Subcellular localization expression vectors were constructed to determine the cellular distribution of AhGRF2b and AhGRF3b. Finally, yeast two-hybrid (Y2H) assays were performed to identify interacting proteins of AhGRF3b. The results revealed that twenty-four AhGRF genes were identified in peanut, which were unevenly distributed across 16 chromosomes. The deduced proteins ranged from 268 to 630 aa in length, with molecular weights spanning 29 842.27 to 67 980.83 Da. Most AhGRFs were acidic and predicted to be localized in the nucleus. Phylogenetic analysis classified the AhGRF family members into six distinct clades. Multiple sequence alignment demonstrated that the majority of AhGRF genes contained conserved QLQ and WRC domains. Under drought and cold stress conditions, several AhGRF genes, particularly AhGRF2b and AhGRF3b, exhibited significantly upregulated expression, which indicated their responsiveness to abiotic stresses. Transient expression in tobacco showed that AhGRF2b was localized in both the nucleus and cytoplasm, while AhGRF3b was localized in the nucleus. Furthermore, Y2H assays revealed that AhGRF3b may interact with AhCAT3 (catalase), suggesting that AhGRF genes may enhance stress tolerance by regulating reactive oxygen species scavenging. These findings provide a theoretical basis for improving the stress tolerance in peanut breeding programs.