青花菜BoDof5.3基因的克隆及渍水胁迫表达特征分析

2020-02-22 01:51裴徐梨荆赞革徐境唐征宋波
江苏农业学报 2020年6期
关键词:基因克隆

裴徐梨 荆赞革 徐境 唐征 宋波

摘要:从青花菜中克隆得到一个Dof基因,命名为BoDof5.3。序列分析结果表明:该基因ORF全长774 bp,编码257个氨基酸,编码的蛋白质为亲水性蛋白质。青花菜BoDof5.3蛋白具有典型的Zf-Dof结构域,其二级结构以无规则卷曲为主。系统进化分析结果表明,青花菜BoDof5.3蛋白与甘蓝、花椰菜Dof蛋白亲缘关系最近,在进化树上聚为一组, 同属植物的Dof蛋白具有较近的亲缘关系。qRT-PCR分析结果显示,在渍水胁迫0 d到2 d时,BoDof5.3基因表达量呈下降趋势,6 d时表达量升高为对照的1.3倍,推测该基因可能参与青花菜渍水胁迫的响应。

关键词:青花菜;Dof基因;基因克隆;渍水胁迫;表达特征

中图分类号:S635.3文献标识码:A文章编号:1000-4440(2020)06-1498-05

Abstract:In this study, a Dof gene named BoDof5.3 was cloned from broccoli. Sequence analysis results showed that this gene was 774 bp in length, which encoded 257 amino acids. Furthermore, the protein encoded by this gene was a hydrophilic protein. The BoDof5.3 contained a typical Zf-Dof domain, and its main secondary structure was the random coil. Results of phylogenetic analysis indicated that the BoDof5.3 protein of broccoli had the highest homology with the Dof protein of cabbage and cauliflower, and they were clustered into a group on phylogenetic tree. Furthermore, the Dof protein in the same genus had close relationship. The results of qRT-PCR showed that the expression level of BoDof5.3 decreased from 0 d to 2 d under waterlogging stress, and the expression level at six days of waterlogging stress was 1.3 times as much as that of control. It is speculated that BoDof5.3 gene may be involved in the response of broccoli to waterlogging stress.

Key words:Brassica oleracea var. italica;Dof gene;gene clone;waterlogging stress;expression feature

Dof(DNA binding with one finger)是植物特有的一类转录因子,其在N-末端具有长度为52 aa的Dof结构域,可与DNA结合,同时还可进行蛋白-蛋白互作。C-末端的可变结构域可在不同的代谢途径中产生多样的调控功能[1-2]。目前Dof基因已在拟南芥、水稻、大豆、大麦、红花、玉米等多种植物中成功克隆[3-8]。

前人研究发现,Dof基因广泛参与碳氮代谢、开花、花和花粉發育、保卫细胞特异基因的调控、防御反应等多种生物学过程。在柽柳中,20个ThDof基因参与了胁迫应答。过表达ThDof16基因可明显提高植株抗干旱和高盐胁迫 [9]。在盐胁迫条件下,过表达红花CtDof1基因可增强转基因拟南芥对盐胁迫的抗性[7]。Hennig等在研究拟南芥Dof蛋白OBP1时发现,该蛋白可通过调控谷胱甘肽S-转移酶6的表达来对植物激素和胁迫信号作出响应[10]。

青花菜(Brassica oleracea var.italica)与花椰菜、甘蓝同属甘蓝类蔬菜,以小花蕾和嫩花茎为产品,营养丰富。其喜温暖湿润,但不耐涝。浙江省地处东部沿海,每年8-9月常受台风和特大暴雨影响,青花菜育苗和定植期间易遭淹水而大面积死苗,造成严重经济损失。前期试验结果显示BoDof5.3基因在高温和盐胁迫条件下表达量可发生较大变化。因此,本试验通过克隆青花菜BoDof5.3基因,并对其特性、同源进化树以及在渍水胁迫下的表达特征进行分析,以期为研究该基因的功能及其参与渍水胁迫的应答机制提供基础。

1材料与方法

1.1试验材料

以青花菜WN12-95为材料,种植于光照培养箱内,培养条件为25 ℃/16 h(白天)~18 ℃/8 h(晚上)。待植株长至5叶期时开始对其进行渍水胁迫处理,处理时间为0 d、2 d和6 d,每个处理设3次生物学重复。

1.2RNA的提取和cDNA的合成

叶片经液氮冷冻后迅速研磨成粉末,用于RNA的提取。总RNA提取使用TaKaRa Mini BEST Plant RNA Extraction Kit试剂盒(TaKaRa公司产品),cDNA的合成采用Prime Script 1st Strand cDNA Synthesis Kit 试剂盒(TaKaRa公司产品)。产物置于-80 ℃的超低温冰箱中保存备用。

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