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| 冬枣cDNA三框表达文库构建及ZjRWD40基因上游调控因子的筛选 |
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王嘉琪1, 王惠冉1, 王佳伟1,3, 周军1,2, 任玉锋1,2, 徐文娣1,2, 张琨1, 乔帅1, 张智凯1
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1. 北方民族大学 生物科学与工程学院,银川 750021；2. 宁夏经济林遗传改良创新团队,银川 750021；3.甘肃连城国家级自然保护区管理局,兰州 730333
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| 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，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。 |
| 关键词: 冬枣(Zizyphus jujuba cv. ‘Dong zao’)，RWD40基因，cDNA文库，酵母单杂交，顺式作用元件 |
| DOI：10.11931/guihaia.gxzw202409014 |
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| 基金项目:国家自然科学基金（32060669）；经济林遗传改良创新基金项目（2022QCXTD04）；第六批宁夏青年科技人才托举工程；北方民族大学研究生创新项目（YCX24138）。 |
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| Construction of three-frame expression library of Zizyphus jujuba cv. ‘Dong zao’ cDNA and screening of upstream regulator factors of ZjRWD40 gene |
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WANG Jiaqi1, WANG Huiran1, WANG Jiawei1,3, ZHOU Jun1,2, REN Yufeng1,2, XU Wendi1,2, ZHANG Kun1, QIAO Shuai1,ZHANG Zhikai1
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1. School of Biological Science and Engineering, North Minzu University, Yinchuan 750021, China；2. Innovation Team for Genetic Improvement of Economic Forest, Yinchuan 750021, China；3. Gansu Liancheng National Nature Reserve Administration, Lanzhou 730333, China
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| Abstract: |
| Zizyphus jujuba cv. ‘Dong zao’, 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. The 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. However, the molecular mechanism regulating the expression of the 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 Zizyphus jujuba cv. ‘Dong zao’ 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 respectively. (3) The results of screening upstream regulatory factors through yeast one-hybrid showed that Bait1-ABRE exhibited a self-activation. A total of 11 gene sequences were initially retrieved from the Bait2-MBS and Bait3-TGACG-motif bait vectors. Among them, 5 sequences were directly related to plant stress-resistance responses. The proteins encoded by these genes may regulate the expression of the ZjRWD40 gene through interactions with the MBS element and the TGACG-motif element, thus affecting its growth, development, and fruit quality under stress environments. This study provides important insights into the molecular network mechanism by which the ZjRWD40 gene regulates the fruit development of Zizyphus jujuba cv. ‘Dong zao’, and also lays a theoretical foundation for the stress-resistant breeding of Zizyphus jujuba cv. ‘Dong zao’. 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 Zizyphus jujuba cv. ‘Dong zao’. |
| Key words: Zizyphus jujuba cv. ‘Dong zao’, RWD40 gene, cDNA library, Yeast one-hybrid, cis-acting original |
