铁皮石斛<i>AP</i>3-3基因及启动子的克隆与分析

王义琴; 孙博; 何玲; 师凯辉; 黄鑫; 王冬梅; 陈宇; 臧睿; 和凤美

doi:10.19303/j.issn.1008-0384.2022.005.005

铁皮石斛AP3-3基因及启动子的克隆与分析

Cloning and Analyzing of AP3-3 and Its Promoter from Dendrobium officinale

摘要

摘要:
目的 AP3-3属于MADS-box基因家族B类基因，参与兰科植物花被和唇瓣的形成，克隆该基因并进行启动子分析可以进一步研究该基因的生物学功能及其启动子调控作用机制。
方法使用RT-PCR和常规PCR技术克隆铁皮石斛DoAP3-3基因及其启动子序列，进行生物信息学分析，构建启动子缺失片段与GUS基因融合表达载体，农杆菌介导转化铁皮石斛原球茎，进行瞬时表达。
结果 DoAP3-3基因cDNA长度为675 bp，编码蛋白质的分子式为C₁₁₂₉H₁₈₀₃N₃₃₃O₃₄₇S₁₂，分子量25.98 kDa，pI为 8.71，不稳定性指数40.14，GRAVY为−0.823，不存在跨膜区域，亚细胞定位预测得分为细胞核87.0%、线粒体8.7%、细胞质4.3%。启动子片段长度1885 bp，顺式作用元件含有大量的光响应元件等；3个启动子片段均可驱动GUS基因表达，表达强度为−1885～0 bp＞−1604～0 bp＞−750～0 bp。
结论 DoAP3-3蛋白具有碱性、亲水性和不稳定性，无跨膜结构域，亚细胞定位于细胞核中。DoAP3-3启动子可能受光照、植物激素、MYB转录蛋白等多个因素调控，具备启动活性，且随启动子缺失长度减少呈现增加趋势。

Abstract:
Objective AP3-3 of class B gene of MADS box family that involves in the formation of perianth and labellum of Dendrobium officinale was cloned to study the biological function, and the promoter analyzed to decipher the regulation mechanism.
Method Sequences of AP3-3 and promoter of D. officinale were cloned by RT-PCR and conventional PCR, and bioinformatics analyzed. A fusion expression vector of promoter-deleted fragments and GUS gene was constructed. Agrobacterium tumefaciens-mediated transformation was used to transform protocorm of D. officinale for the transient expression.
Result The cDNA length of DoAP3-3 was 675 bp with an encoded formula of C₁₁₂₉H₁₈₀₃N₃₃₃O₃₄₇S₁₂, a molecular weight of 25.98 kDa, a PI of 8.71, an instability index of 40.14, and GRAVY of −0.823. There was no transmembrane region detected in the protein. The predicted score of subcellular localization was 87.0% in nucleus, 8.7% in mitochondria, and 4.3% in cytoplasm. The 1885 bp promoter fragment had a cis acting element containing a significant number of photo-responsive elements among others. The 3 promoter fragments could drive GUS with an order of expression intensity of −885–0 bp＞−1 604–0 bp＞−750–0 bp.
Conclusion The predicted DoAP3-3 was an alkali, hydrophilic, and unstable protein with no transmembrane domain and a subcellular localization in the nucleus. The DoAP3-3 promoter might be regulated by light, plant hormones, MYB transcription protein, etc., and exhibited activities that increased with decreasing deletion length.

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