冬枣cDNA三框表达文库构建及ZjRWD40基因上游调控因子的筛选

Construction of three-frame expression library of Zizyphus jujuba cv. Dongzao cDNA and screening of upstream regulator factors of ZjRWD40 gene

关键词:  冬枣, RWD40基因, cDNA文库, 酵母单杂交, 顺式作用元件

Key words:  Zizyphus jujuba cv. Dongzao, RWD40 gene, cDNA library, yeast one-hybrid, cis-acting element

摘要:

冬枣是重要的以食代药鲜食水果,RWD40通过调节DNA甲基化水平影响下游基因表达和调控果实发育,进而影响果实风味品质。RWD40蛋白是参与甲基化调控途径的关键蛋白,为了揭示冬枣中何种上游蛋白调控RWD40基因的表达,该研究利用酵母单杂交技术初步筛选冬枣ZjRWD40基因的上游候选调控因子。结果表明:(1)冬枣cDNA三框表达文库的滴度为4×10⁹ CFU·mL^-1,重组率为100%。(2)从冬枣ZjRWD40基因家族启动子区筛选了应激防御元件ABRE、MBS和TGACG-motif,分别以其诱饵序列构建诱饵载体,命名为Bait1-ABRE、Bait2-MBS和Bait3-TGACG-motif。(3)酵母单杂交筛选上游调控因子的结果显示,Bait1-ABRE具有自激活现象,Bait2-MBS和Bait3-TGACG-motif诱饵载体初步调取11条基因序列,其中5条与植物抗逆反应直接相关,它们可能通过与MBS元件和TGACG-motif元件互作来调控冬枣ZjRWD40基因的表达,进而调控冬枣的DNA甲基化水平。该研究结果为揭示ZjRWD40基因调控冬枣果实发育分子网络机制提供一定的参考,同时为冬枣抗逆育种奠定理论基础。

Abstract:

Zizyphus jujuba cv. Dongzao, a late-maturing fresh-eating jujube variety unique to China, features tender flesh, abundant juice and minimal residues. It is not only nutritionally rich but also has the value of serving as a medicinal substitute, making it highly popular among consumers. DNA methylation, as an important epigenetic modification method, plays a core role in plants' response to stress and the process of fruit development. RWD40 protein is a key protein involved in the methylation regulation pathway. It affects the expression of a series of downstream genes by regulating the DNA methylation level, and thus has a profound impact on the development process of fruits, the synthesis and accumulation of flavor substances, etc., ultimately affecting the fruit quality. In order to reveal which upstream protein regulates the expression of RWD40 gene remains unclear, this study employed the yeast one-hybrid technique to preliminarily screen for upstream candidate regulatory factors of the ZjRWD40 gene. The results were as follows:(1)The titer of the three-frame expression library of Z. jujuba cv. Dongzao cDNA reached 4×10⁹ CFU·mL^-1, and the recombination rate was 100%.(2)Stress-defense elements ABRE, MBS, and TGACG-motif were identified from the promoter region of the ZjRWD40 gene family, and bait vectors Bait1-ABRE, Bait2-MBS, and Bait3-TGACG-motif were constructed through bait sequences, respectively.(3)The results of screening upstream regulatory factors through yeast one-hybrid showed that Bait1-ABRE exhibited a self-activation. A total of eleven gene sequences were initially retrieved from the Bait2-MBS and Bait3-TGACG-motif bait vectors. Among them, five gene sequences were directly related to plant stress-resistance responses. The proteins encoded by these genes might regulated the expression of the ZjRWD40 gene through interactions with the MBS element and the TGACG-motif element, thus affecting its DNA methylation level. This study provides certain reference for the molecular network mechanism by which the ZjRWD40 gene regulates the fruit development of Z. jujuba cv. Dongzao, and also lays a theoretical foundation for the drought-resistant breeding of Z. jujuba cv. Dongzao. Future research will focus on using transcriptome sequencing technology to identify the downstream regulatory genes of ZjRWD40, aiming to further clarify its molecular pathway in the stress regulation of Z. jujuba cv. Dongzao.