Journal of Nuclear Agricultural Sciences ›› 2019, Vol. 33 ›› Issue (9): 1677-1685.DOI: 10.11869/j.issn.100-8551.2019.09.1677

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Cloning and Expression Analysis of UFGT From Spine Grape (Vitis davidii Foëx.) Callus

LAI Chengchun1,2,4,5,*, PAN Hong1,3, HUANG Xiangui1,2, FAN Lihua1,2, LAI Zhongxiong3, DUAN Changqing4, LIU Wenhui5   

  1. 1 Institute of Agricultural Engineering and Technology, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003;
    2 Fujian Key Laboratory of Agricultural Product (Food) Processing, Fuzhou, Fujian 350003;
    3 Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002;
    4 College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083;
    5 Beijing Huiyuan Food & Beverage Co., Ltd., Beijing 101305;
  • Received:2018-05-17 Revised:2018-09-11 Online:2019-09-09 Published:2019-07-23

刺葡萄愈伤组织UFGT基因克隆及表达分析

赖呈纯1,2,4,5,*, 潘红1,3, 黄贤贵1,2, 范丽华1,2, 赖钟雄3, 段长青4, 刘文慧5   

  1. 1 福建省农业科学院农业工程技术研究所,福建 福州 350003;
    2 福建省农产品(食品)加工重点实验室,福建 福州 350003;
    3 福建农林大学园艺植物生物工程研究所,福建 福州 350002;
    4 中国农业大学 食品科学与营养工程学院,北京 100083;
    5 北京汇源食品饮料有限公司,北京 101305
  • 通讯作者: *同第一作者。
  • 作者简介:赖呈纯,男,副研究员,主要从事园艺植物生物技术与植物细胞代谢工程研究。E-mail:lccisland@163.com
  • 基金资助:

    福建省自然科学基金(2016J01126)

Abstract:

In order to reveal the function of UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) in regulating anthocyanin biosynthesis in spine grape (Vitis davidii Foëx.) callus at the cellular level, the gene encoding VdUFGT was isolated from spine grape callus by using RT-PCR with RACE technology. Its sequence was analyzed by bioinformatics and the expression profiles were investigated by real-time quantitative PCR (RT-qPCR). The results showed that the open reading frame (ORF) of cDNA and DNA of VdUFGT are 1 371 bp and 1 448 bp in length, respectively. It contains two exons and one intron, encoding 456 amino acid residues. The VdUFGT protein, an unstable and hydrophilic protein with negative charges, is a member of the UDPGT superfamily, including a UDPGT domain and a UDP: flavonoid glycosyltransferase YjiC feature regions. Six Vitis plants are clustered into the same group according to the phylogenetic tree, which constructed by proteins encoded from UFGT homologous genes. RT-qPCR analysis showed that the VdUFGT transcription level of red callus of spine grape was significantly higher than that of white callus, which was above 79 times when they were cultured for 25 days. During the continuous culture of spine grape callus, the VdUFGT expression level changed greatly in red callus, two peaks occurred at the middle stage of rapid-growth and the beginning of senescence, respectively. While the expression level of VdUFGT in white callus of spine grape changed not as wells as in red callus, it always maintained at a very low level. These results suggested that VdUFGT is the major gene involved in the regulation of anthocyanin biosynthesis in cell cultures of spine grape callus, and its regulatory role was mainly worked at the middle stage of rapid-growth and the beginning of senescence. This result would lay a foundation for further studying of the regulatory mechanism of anthocyanin biosynthesis in spine grape cells.

Key words: spine grape(Vitis davidii Foëx.), callus, flavonoid 3-O-glucosyltransferase gene(UFGT), gene cloning, gene expression

摘要:

为从细胞水平上揭示刺葡萄3-O-类黄酮葡萄糖基转移酶(UFGT)基因调控花青素合成的功能,以刺葡萄愈伤组织为试验材料,根据刺葡萄愈伤组织转录组UFGT序列片段,利用RT-PCR结合RACE技术克隆得到VdUFGT基因,并对其进行生物信息学和表达特性分析。结果表明,VdUFGT基因cDNA和DNA开放阅读框(ORF)分别为1 371 bp和1 448 bp,包含2个外显子和1个内含子,编码456个氨基酸,为带负电荷的不稳定的亲水性蛋白,具有一个UDPGT结构域,是UDPGT超家族成员,包含UDP-黄酮糖基转移酶特征区域。由UFGT同源基因编码蛋白所构建的系统发育树,与植物进化的关系相一致,6个葡萄属植物聚为一支。RT-qPCR分析表明,刺葡萄红色愈伤组织VdUFGT转录水平极显著高于白色愈伤组织,培养25 d的2个细胞培养物差异可达79倍;在刺葡萄愈伤组织连续培养过程中,红色愈伤组织中VdUFGT转录水平变化幅度较大,在愈伤组织快速生长中期和衰老初期分别出现峰值,而刺葡萄白色愈伤组织VdUFGT转录水平与红色愈伤组织相比变化幅度不大,且始终维持在一个较低的水平。说明VdUFGT对刺葡萄红色愈伤组织细胞培养物中的花青素生物合成有重要的调控作用,这种调控作用主要发生在刺葡萄愈伤组织细胞快速生长中期和细胞衰老初期。本研究结果为进一步阐明VdUFGT调控刺葡萄细胞花青素合成的机制奠定了理论基础。

关键词: 刺葡萄, 愈伤组织, 3-O-类黄酮葡萄糖基转移酶基因(UFGT), 基因克隆, 基因表达