WANG Jianjun,XU Yuanyuan,LIU Tongkun,et al.Cloning and function analysis of BrbHLH49 gene in purple tsai-tai[J].Journal of Nanjing Agricultural University,2021,44(3):421-427.[doi:10.7685/jnau.202008009]





Cloning and function analysis of BrbHLH49 gene in purple tsai-tai
王建军 徐园园 刘同坤 侯喜林
南京农业大学作物遗传与种质创新国家重点实验室/农业农村部华东地区园艺作物生物学 与种质创制重点实验室/园艺学院, 江苏 南京 210095
WANG Jianjun XU Yuanyuan LIU Tongkun HOU Xilin
State Key Laboratory of Crop Genetics and Germplasm Enhancement/Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crop in East China, Ministry of Agriculture and Rural Affairs/College of Horiticulture, Nanjing Agricultural University, Nanjing 210095, China
purple tsai-taiBrbHLH49 genefunction analysisanthocyanin
[目的] 本文旨在研究紫菜薹BrbHLH49基因在花青素合成中的功能。[方法] 以紫菜薹为材料,通过同源克隆获得BrbHLH49基因全长序列,并与其他物种中的同源序列进行进化树分析;构建pEarlyGate101-BrbHLH49-YFP载体并转化导入烟草叶片中,研究BrbHLH49蛋白的亚细胞定位;采用荧光定量PCR (RT-qPCR)技术分析BrbHLH49基因在紫菜薹和‘四九菜心’(‘CX-49’)不同组织中的表达情况,同时分析野生型和35S:BrbHLH49-YFP转基因拟南芥中花青素合成相关基因的表达。[结果] BrbHLH49基因含有1 431 bp开放阅读框,编码476个氨基酸,其编码的蛋白定位于细胞核中。在进化过程中BrbHLH49编码的氨基酸序列与甘蓝型油菜和野甘蓝(原变种)同源性最高,亲缘关系最近。RT-qPCR结果表明,紫菜薹的薹和叶中BrbHLH49相对表达量均显著高于‘CX-49’,且BrbHLH49转基因拟南芥植株中花青素合成相关基因的表达量显著高于野生型。[结论] BrbHLH49蛋白定位于细胞核中。BrbHLH49基因在紫菜薹的薹和叶中高表达,且拟南芥中过表达该基因能显著提高花青素的合成。
[Objectives] The purpose of this study was to explore the function of BrbHLH49 gene in anthocyanin synthesis of purple tsai-tai. [Methods] Using purple tsai-tai as the material,the full-length BrbHLH49 was cloned by homology cloning method. The amino acid sequence alignment and evolution analysis were performed with other species. To study the subcellular localization of BrbHLH49 protein,the vector pEarlyGate101-BrbHLH49-YFP was constructed,and transformed into the agrobacterium,and then injected into tobacco leaves. Moreover,RT-qPCR was used to analyze the expression of BrbHLH49 gene in different tissues of purple tsai-tai and ‘CX-49’. And the relative expression levels of genes related to anthocyanin synthesis in wild-type and 35S:BrbHLH49-YFP transgenic Arabidopsis thaliana were analyzed by RT-qPCR. [Results] The open reading frame length of BrbHLH49 gene was 1 431 bp,encoding 476 amino acids. Subcellular localization results showed that BrbHLH49 protein was expressed in the nucleus. During plants evolution,the amino acid sequence of BrbHLH49 encoded had a close relationship with those of Brassica napus and Brassica oleracea var. oleracea. RT-qPCR results showed that the relative expression levels of BrbHLH49 in the stalks and leaves of purple tsai-tai were significantly higher than those of ‘CX-49’. Meanwhile,the expression levels of the genes related to anthocyanin synthesis in the transgenic A.thaliana plants were significantly higher than those of the wild-type. [Conclusions] BrbHLH49 was located in the nucleus. The BrbHLH49 gene was highly expressed in the stalks and leaves of purple tsai-tai. And overexpression of BrbHLH49 gene in A.thaliana could significantly increase the synthesis of anthocyanin.


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更新日期/Last Update: 1900-01-01