高颖 杨亚苓 李慕紫 贺丽霞 李慧

摘要： AP2/ERF（APETALA2/ethylene-responsive factor）是一个庞大的转录因子超家族，其蛋白质中均含有1段或2段由60～70个氨基酸残基组成的结构域，该家族基因广泛存在于植物体内，在植物生长发育，生物及非生物胁迫响应，植物次级代谢中均发挥着重要的作用。本试验利用前期的青花菜转录组数据，采用RT-PCR 技术从青花菜中分离克隆WRI4基因，利用生物信息学分析软件对其进行基因结构及特征分析。结果表明，青花菜WRI4转录因子基因共编码308个氨基酸，WRI4具有2个AP2/ERF结构域，属于AP2/ERF家族成员。WRI4蛋白为亲水性蛋白，无跨膜结构，二级结构以无规则卷曲和α-螺旋，β-转角为主。氨基酸序列比对及进化树分析结果表明，青花菜WRI4转录因子与白菜、油菜相应的转录因子有较高的相似性。采用同源重组技术构建了WRI4过表达载体，为进一步深入探究该转录因子的功能，培育青花菜新品种奠定了基础。

关键词： 青花菜;WRI4;AP2/ERF;同源重组克隆

中图分类号： S635.3;Q785 文献标识码： A 文章编号： 1000-4440（2021）03-0710-08

Cloning， bioinformatic analysis and expression vector construction of broccoli WRI4 gene

GAO Ying1， YANG Ya-ling1， LI Mu-zi1， HE Li-xia2， LI Hui1

（1.College of Horticulture and Landscape Architecture， Tianjin Agricultural University， Tianjin 300384， China;2.College of Life Sciences， Nankai University， Tianjin 300071， China）

Abstract： AP2/ERF （APETALA2/ethylene-responsive factor） is a large superfamily of transcription factors， there is one or two domains composed of 60-70 amino acid residues in its proteins. The family genes widely exist in plants and play important roles in plant growth and development， responses to biological and abiotic stresses and botanic secondary metabolism. WRI4 gene was isolated and cloned from broccoli by RT-PCR technique based on the previous data of transcriptome in broccoli， then the structure and characteristics of WRI4 gene were analyzed by bioinformatic analysis software. The results showed that， 308 amino acids were encoded by WRI4 transcription factor gene in broccoli， and there were two AP2/ERF domains in WRI4 transcription factor， which belonged to AP2/ERF family member. The WRI4 protein was hydrophilic and had no transmembrane structure， its main secondary structures were random coil， α-helix and β-rotation angle. Results of amino acid sequence alignment and evolutionary tree analysis showed that， WRI4 transcription factors in broccoli had high similarity with the corresponding transcription factors in cabbage and rape. Overexpression vector of WRI4 was constructed by homologous recombination technique， which provided basis for further exploring on its functions and cultivating new varieties of broccoli.

Key words： broccoli;WRI4;AP2/ERF;homologous recombination cloning

高盐、干旱、极端温度等不利因素对大多数植物的生长均有抑制作用。面对逆境胁迫，植物已经进化出复杂的系统来调节对压力信号的适应[1]。AP2/ERF（APETALA2/ethylene-responsive factor）属于植物中特有的一类转录因子超家族。其廣泛参与植物的生物与非生物胁迫，并在一些信号交叉途径中作为中间因子参与调节[2]。目前已有研究结果表明，AP2/ERF转录因子家族对植物适应非生物逆境、植物生长发育、花器官形态建成以及种子发育有重要影响[3]。

AP2/ERF家族均含有1个或2个DNA结合域，其保守域由60～70个氨基酸组成[4]。……