调控葡萄酒石酸生物合成VvbHLH79基因克隆及表达分析
CSTR:
作者:
作者单位:

云南农业大学

作者简介:

通讯作者:

中图分类号:

基金项目:

国家自然科学基金项目(面上项目,重点项目,重大项目)


Cloning and Expression Analysis of the VvbHLH79 Gene Involved in Regulating Grape Tartaric Acid Biosynthesis
Author:
Affiliation:

Fund Project:

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    摘要:

    【目的】葡萄是典型的酒石酸积累型果实,酒石酸不仅影响葡萄果实的味道,还决定葡萄酒的色泽、口感、微生物稳定性和陈酿潜力,但调控酒石酸生物合成的转录因子及机制尚不明确。【方法】bHLH转录因子参与调控植物的生长发育、次生代谢和抗逆性等多种生物学过程,课题组前期筛选到1条与酒石酸生物合成相关的bHLH转录因子。研究以酿酒葡萄‘琼瑶浆’、‘马瑟兰’为试验材料,采用PCR方法从中克隆到与酒石酸含量相关的VvbHLH79转录因子,对其进行生物信息学和亚细胞定位分析;利用HPLC和qRT-PCR技术测定葡萄果实在不同发育时期的酒石酸含量和VvbHLH79的表达量;并构建植物表达载体,使用农杆菌介导的果梗侵染法瞬时转化葡萄幼果果实之后检测VvbHLH79的表达量和酒石酸含量,以明确VvbHLH79基因在葡萄果实中对酒石酸含量的作用,为进一步探究VvbHLH79在调控葡萄酒石酸生物合成的分子机制奠定基础。【结果】结果表明:(1)通过RNA-seq测序筛选获得1个葡萄VvbHLH79基因,该基因CDS序列全长831 bp,编码277个氨基酸,相对分子量为66.22kD,理论等电点为5.15,属于无信号肽和跨膜结构的不稳定亲水性蛋白;氨基酸序列第147-232位含有bHLH-SF保守结构域,属于bHLH转录因子家族;亚细胞定位在细胞核。(2)同源序列比对和系统进化树结果显示,葡萄VvbHLH79编码蛋白与河岸葡萄中的bHLH89编码的蛋白(登录号:XP_034699340.1)序列一致性最高,达到99.64%。(3)qRT-PCR分析表明,VvbHLH79随着‘琼瑶浆’果实的发育,相对表达量在降低,从座果期到成熟期降低了67.25%左右;HPLC测定结果显示,酒石酸的积累主要在葡萄果实转色之前,转色之后酒石酸含量就快速下降。(4)VvbHLH79在低酒石酸葡萄果实中瞬时过表达后使酒石酸含量显著升高,而将VvbHLH79在高酒石酸葡萄果实中沉默后酒石酸含量显著下降,证实了VvbHLH79基因正向调控酒石酸含量。【结论】研究推测,VvbHLH79转录因子可能在葡萄酒石酸生物合成通路中发挥作用。

    Abstract:

    【Objective】 Grapes are typical accumulators of tartaric acid, which not only affects the taste of grape berries but also determines the color, texture, microbial stability, and aging potential of wine. 【Methods】 The bHLH transcription factors are involved in the regulation of various biological processes in plants, including growth and development, secondary metabolism, and stress responses. Our research group has previously screened a bHLH transcription factor related to tartric acid biosynthesis. In this study, using ‘Traminer’, ‘Marselan’ wine grape as experimental material, we cloned the tartaric acid content related transcription factor VvbHLH79 by PCR. We performed bioinformatic and subcellular localization analyses of VvbHLH79. Furthermore, we measured the tartaric acid content and VvbHLH79 expression levels in grape berries at different developmental stages using HPLC and qRT-PCR techniques. We also constructed a plant expression vector and the expression and tartaric acid content of VvbHLH79 were detected after transient transformation of young grape berries using Agrobacterium-mediated fruiting pedicle infection, in order to clarify the role of the VvbHLH79 gene on the content of tartaric acid in grape berries.These results provide a foundation for further exploration of the molecular mechanisms underlying the regulation of tartaric acid biosynthesis by VvbHLH79. 【Results】 The results showed: (1) A grapevine VvbHLH79 gene was obtained by RNA-seq sequencing, with a full-length CDS sequence of 831 bp, encoding 277 amino acids, a relative molecular weight of 66.22 kD, and a theoretical isoelectric point of 5.15. It belongs to the class of unstable hydrophilic proteins without signal peptides and transmembrane structures. Amino acid residues 147-232 contain the bHLH-SF conserved domain, classifying it within the bHLH transcription factor family. The protein is localized in the cell nucleus. (2) Homologous sequence alignment and phylogenetic tree results showed that the grapevine VvbHLH79-encoded protein had the highest sequence similarity with the bHLH89-encoded protein in riverbank grape (Accession number: XP_034699340.1), with a sequence identity of 99.64%. (3) qRT-PCR analysis revealed a gradual decrease in VvbHLH79 relative expression levels during the development of ‘Traminer’ grapes fruits, decreasing by approximately 67.25% from veraison to harvest. HPLC results indicated that the accumulation of tartaric acid occurred mainly before the color change of the grapes, with a rapid decrease in tartaric acid content after the color change. (4) Transient overexpression of VvbHLH79 in low tartaric acid grape berries resulted in a significant increase in tartaric acid content, whereas silencing of VvbHLH79 in high tartaric acid grape berries resulted in a significant decrease in tartaric acid content, confirming that the VvbHLH79 gene positively regulates tartaric acid content. 【Conclusion】 This study suggests that the VvbHLH79 transcription factor may play a role in the tartaric acid biosynthesis pathway in grape berries.

    参考文献
    相似文献
    引证文献
引用本文

杨环齐.调控葡萄酒石酸生物合成VvbHLH79基因克隆及表达分析[J].西北植物学报,2024,44(4):602-611

复制
分享
相关视频

文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:2023-09-09
  • 最后修改日期:2023-11-27
  • 录用日期:2023-12-04
  • 在线发布日期: 2024-03-28
  • 出版日期:
文章二维码