不结球白菜开花相关基因BcGI的克隆、亚细胞定位及基因沉默功能验证

Cloning,subcellular localization and silencing function verification of BcGI gene involved in flowering pathway in non-heading Chinese cabbage

[目的]本文旨在揭示不结球白菜 GIGANTEA(GI)基因的功能,验证其与CONSTANS(CO)、FLOWERING LOCUS T(FT)基因之间的调控关系以及其在植物抽薹开花中的作用。[方法]以不结球白菜品种‘苏州青’为材料,提取RNA并反转录成cDNA,同源克隆 BcGI 基因,Gateway构建表达载体pEarleyGate101- BcGI -YFP,利用农杆菌介导法将表达载体注射入本氏烟草中,激光共聚焦显微镜观察其亚细胞定位。Gateway构建沉默载体 BcGI -RNAi,利用农杆菌介导法将沉默载体导入拟南芥中,观察野生型和转基因植株抽薹期的表型变化。采用RT-qPCR技术,检测 GI、CO、FT 基因在转基因和野生型拟南芥植株抽薹期的基因表达量。[结果]通过同源克隆,得到 BcGI 基因,其含有1个3 516 bp的开放阅读框(ORF),编码1 171个氨基酸。亚细胞定位结果显示,BcGI定位于细胞核中。RT-qPCR结果表明, BcGI 基因在沉默转基因植株中的相对表达量比野生型植株低;同时当 GI基因表达受到抑制时,CO、FT 基因在沉默转基因植株中的相对表达量也比野生型明显降低,沉默转基因植株与野生型相比表现为开花延迟且抽薹期莲座叶数明显增多。[结论] BcGI定位于细胞核,参与正向调控下游 CO、FT 基因的表达,进而影响不结球白菜光周期开花途径。

[Objectives] The paper aimed to explore the function of GI gene in Brassica campestris ssp. chinensis ,and verify its regulatory relationship with CO and FT genes,and its role in plant flowering.[Methods] The RNA was extracted from the non-heading Chinese cabbage‘Suzhouqing’and transcribed reversely into cDNA. The gene BcGI was obtained by homologous cloning method. Using Gateway method,we constructed the expression vector pEarleyGate101- BcGI -YFP. The expression vector was injected into Nicotiana benthamiana by Agrobacterium-mediated method,and its subcellular localization was observed by laser confocal microscopy. Also,we constructed the silencing vector BcGI -RNAi to explore the phenotypic changes of wild-type and silenced plants during the flowering period. RT-qPCR was used to detect the gene expression of GI,CO and FT in the flowering stage of transgenic and wild-type plants.[Results] By homologous cloning,we obtained BcGI including a 3 516 bp open reading frame(ORF)and encoding 1 171 amino acids. Subcellular localization analysis showed that BcGI was localized in the nucleus. The RT-qPCR results of wild-type and BcGI -RNAi silencing transgenic plants showed that the relative expression of BcGI gene in transgenic plants was lower than that in wild-type plants. When the expression of GI gene was inhibited,the relative expressions of CO and FT genes in transgenic plants were significantly lower than wild-type plants. The silenced plants showed late flowering compared with wild-type plants,and the number of rosette leaves significantly increased at blotting stage.[Conclusions] The BcGI was located in the nucleus and participated in the positive regulation of downstream CO and FT gene expression,which in turn affected the photoperiod flowering pathway of non- heading Chinese cabbage.