背景:DELLA蛋白是调节植物发育和非生物胁迫反应中许多有趣的生物过程的关键因素。然而,大白菜中DELLA蛋白的功能和信息知之甚少。
方法:使用拟南芥中的5个DELLA基因序列作为探针,在大白菜数据库(Brassica数据库(BRAD))中通过Blast搜索鉴定了大白菜中的5个DELLA基因。国家生物技术信息中心(NCBI)ExPaSy,瑞士模型,DNAMAN,采用MEGA11、PlantCARE对大白菜DELLA基因家族进行鉴定和分析。通过定量实时聚合酶链反应(qRT-PCR)分析基因表达。通过BraA10gRGL3和酵母杂种的过表达和表型分析验证了BraA10gRGL3的功能。
结果:在这项研究中,基于芸苔属数据库鉴定并克隆了5个与拟南芥同源的BraDELLAs,即,BraA02gRGL1、BraA05gRGL2、BraA10gRGL3、BraA06gRGA和BraA09gRGA。所有布拉德拉都包含德拉,TVHYNP,和GRAS保守域。顺式元件分析表明,这5个DELLA基因的启动子区域均含有光响应元件,TCT图案,I-box,G-box,和框4,其与GA信令相关联。转录组分析结果证明,BraA02gRGL1、BraA05gRGL2和BraA10gRGL3在不同生长阶段的Y2中的表达均低于Y7,这与Y7表现出比Y2更强的胁迫耐受性的表型一致。值得强调的是,即使通过BraA10gRGL3-Y7在拟南芥中的过表达,与野生型(WT)拟南芥相比,叶片尺寸更小,鲜重更低:哥伦比亚,在BraA10gRGL3-Y7中观察到对非生物胁迫的更强响应。这表明BraA10gRGL3-Y7可以通过抑制植物的生长来提高植物的抗逆性。此外,酵母双杂交实验证实,BraA10gRGL3-Y7可以与BraA05gGID1a-Y7、BraA04gGID1b1、BraA09gGID1b3-Y2和BraA06gGID1c相互作用,而BraA10gRGL3-Y2不能与任何BraGID1相互作用。
结论:总的来说,BraDELLAs在植物发育和对非生物胁迫的响应中起着重要作用。BraA10gRGL3-Y2和BraA10gRGL3-Y7之间的氨基酸序列差异可能导致其蛋白质结合位点的变化,从而影响它们与BraGID1家族蛋白的相互作用。该系统分析为进一步研究大白菜DELLA基因功能特性奠定了基础。
BACKGROUND: DELLA protein is a crucial factor which played pivotal roles in regulating numerous intriguing biological processes in plant development and abiotic stress responses. However, little is known about the function and information of DELLA protein in Chinese cabbage.
METHODS: Using 5 DELLA gene sequences in Arabidopsis Thaliana as probes, 5 DELLA genes in Chinese cabbage were identified by Blast search in Chinese cabbage database (Brassica database (BRAD)). The National Center for Biotechnology Information (NCBI), ExPaSy, SWISS-MODEL, DNAMAN, MEGA 11, PlantCARE were used to identify and analyze the DELLA gene family of Chinese cabbage. Gene expression was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The function of BraA10gRGL3 was verified by overexpression and phenotypic analysis of BraA10gRGL3 and yeast hybrid.
RESULTS: In this
study, 5 BraDELLAs homologous to Arabidopsis thaliana were identified and cloned based on the Brassica database, namely, BraA02gRGL1, BraA05gRGL2, BraA10gRGL3, BraA06gRGA and BraA09gRGA. All BraDELLAs contain the DELLA, TVHYNP, and GRAS conserved domains. Cis-element analysis revealed that the promoter regions of these 5 DELLA genes all contain light-responsive elements, TCT motif, I-box, G-box, and box 4, which are associated with GA signaling. Transcriptome analysis results proved that the expression of BraA02gRGL1, BraA05gRGL2, and BraA10gRGL3 in Y2 at different growth stages were lower than them in Y7, which is consistent with the phenotype that Y7 exhibited stronger stress tolerance than Y2. It is worth emphasizing that even through the overexpression of BraA10gRGL3-Y7 in Arabidopsis resulted in smaller leaf size and lower fresh weight compared to the wild type (WT) Arabidopsis: Columbia, a stronger response to abiotic stresses was observed in BraA10gRGL3-Y7. It indicated that BraA10gRGL3-Y7 can improve the stress resistance of plants by inhibiting their growth. Moreover, the yeast two-hybrid experiment confirmed that BraA10gRGL3-Y7 can interacted with BraA05gGID1a-Y7, BraA04gGID1b1, BraA09gGID1b3-Y2, and BraA06gGID1c, whereas BraA10gRGL3-Y2 cannot interact with any BraGID1.
CONCLUSIONS: Collectively, BraDELLAs play important role in plant development and response to abiotic stress. The differences in amino acid sequences between BraA10gRGL3-Y2 and BraA10gRGL3-Y7 may result in variations in their protein binding sites, thus affecting their interaction with the BraGID1 family proteins. This systematic analysis lays the foundation for further
study of the functional characteristics of DELLA genes of Chinese cabbage.