大白菜转录因子基因<i>BrPIF5</i>功能验证及表达分析

doi:10.7668/hbnxb.20195034

摘要/Abstract

摘要：

大白菜叶球的抱合方式是决定商品器官外观形状、口感和抗逆性的主要性状。为探究大白菜抱合方式形成的内在分子机理,以叠抱和舒心大白菜为试验材料,克隆得到转录因子BrPIF5基因的全长序列,并进行生物信息学分析,构建植物过表达载体以及利用农杆菌介导转化到烟草,获得阳性转化植株,通过qRT-PCR检测了BrPIF5基因在烟草中的表达水平。结果表明,BrPIF5基因编码的蛋白为亲水性蛋白,具有连续且完整的634 bp开放阅读框,蛋白内共含有210个氨基酸,该蛋白由较多α螺旋结构和无规则卷曲构成,其中存在1个AP2/ERF结构域。BrPIF5蛋白与其余9个基因家族成员共含有1个1号保守基序,且位置相较于其他基因家族成员存在差异,位于蛋白序列前端。系统进化树显示,BrPIF5基因与家族内的SoPIF15、BhPIF1、BoPIF4、AtPIF4和BrPIF4家族成员具有较近的进化关系。通过农杆菌介导的遗传转化方法获得过表达BrPIF5烟草株系,烟草阳性转化株表现出叶片向内卷曲,通过qRT-PCR检测发现,该基因在叠抱类型大白菜中表达量高于舒心大白菜,阳性烟草植株的基因表达量高于对照。进一步证明了BrPIF5基因控制大白菜叶片向内卷曲,从而促进叶球叠抱类型的形成。

关键词: 大白菜, BrPIF5基因, 表达分析, 叶片发育, PIF基因家族

Abstract:

The folding mode of Chinese cabbage leaf ball is the main character that determines the appearance shape,taste and stress resistance of commercial organs.In order to explore the internal molecular mechanism of the formation of the folding mode of Chinese cabbage,we cloned the full length sequence of the transcription factor BrPIF5 gene from overlaping and outward-curling Chinese cabbage as experimental materials,and conducted bioinformatics analysis,constructed the plant overexpression vector,and used Agrobacterium to mediate the transformation into tobacco to obtain positive transformation plants.The expression level of BrPIF5 gene in tobacco was detected by qRT-PCR.The results showed that the protein encoded by BrPIF5 gene was a hydrophilic protein with a continuous and complete open reading frame of 634 bp,containing 210 amino acids.The protein was composed of more α-helical structure and random curl,including an AP2/ERF domain.BrPIF5 protein and the other 9 gene family members contained a conserved motif 1,and the position was different from that of other gene family members,which was located in the front of the protein sequence.Phylogenetic tree showed that BrPIF5 gene had close evolutionary relationship with SoPIF15,BhPIF1,BoPIF4,AtPIF4 and BrPIF4 family members.The tobacco strain with overexpression of BrPIF5 was obtained by Agrobacterum-mediated genetic transformation,and the leaves of the tobacco positive transformation strain showed inward curling.qRT-PCR showed that the expression level of BrPIF5 gene in the overlaping Chinese cabbage was higher than that in the outward-curling Chinese cabbage,and the gene expression level in the positive tobacco plants was higher than that in the control.It was further proved that BrPIF5 gene controlled the inward curling of Chinese cabbage leaves,thus promoting the formation of leaf ball folding type.

Key words: Brassica rapa ssp.pekinensis, BrPIF5 gene, Expression analysis, Leaf development, PIF gene family

孟川, 马小超, 吴芳, 王清凤, 马蕾, 王洪乐, 王明秋, 刘晓东. 大白菜转录因子基因BrPIF5功能验证及表达分析[J]. 华北农学报, 2024, 39(6): 18-26. doi: 10.7668/hbnxb.20195034.

MENG Chuan, MA Xiaochao, WU Fang, WANG Qingfeng, MA Lei, WANG Hongle, WANG Mingqiu, LIU Xiaodong. Functional Validation and Expression Analysis of BrPIF5 Transcription Factor Gene in Brassica rapa[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(6): 18-26. doi: 10.7668/hbnxb.20195034.

http://www.hbnxb.net/CN/Y2024/V39/I6/18

图/表 10

图1 叠抱类型与舒心类型白菜

Fig.1 Leafy head of overlapping type cabbage and outward-curling type cabbage

图2 BrPIF5蛋白二级结构预测

Fig.2 Secondary structure prediction of BrPIF5 protein

图3 BrPIF5蛋白三级结构预测

Fig.3 Tertiary structure prediction of BrPIF5 protein

图4 PIF基因家族成员蛋白的保守基序分析

Fig.4 Conserved motif analysis of the PIF gene family members proteins

图5 PIF基因家族成员基因序列分析

Fig.5 Gene sequence analysis of PIF gene family members

图6 BrPIF5和其他PIF基因家族成员的序列比对和系统进化分析

Fig.6 Sequence alignment and phylogenetic analysis of BrPIF5 and other PIF gene family members

图7 BrPIF5基因植物表达载体的建构和载体图谱 A.构建的植物表达载体图谱;B.菌落PCR验证:1.酶切条带,M.DNA分子量标记。

Fig.7 Construction of BrPIF5 gene plant expression vector and vector map A.The constructed plant expression vector map;B.PCR validation of colonies:1.Digestion bands,2.DNA Marker.

图8 转基因株系PCR鉴定 +.阳性对照;-.阴性对照;O.水对照。

Fig.8 PCR identification of transgenic lines +.The positive control;-.The negative control;O.The water control.

图9 转基因株系与野生型株系表型特征 A.野生型本氏烟草(正拍);B.野生型本氏烟草(俯拍);C.转基因本氏烟草(正拍);D.转基因本氏烟草(俯拍)。

Fig.9 Phenotypic traits of transgenic strains and wild-type strains A.Wild-type N.benthamiana(frontal shot);B.Wild-type N.benthamiana(overhead shot);C.Transgenic N.benthamiana(frontal shot);D.Transgenic Nicotiana benthamiana(overhead shot).

图10 BrPIF5基因在不同植株中的表达水平不同小写字母表示差异显著(P<0.05)。

Fig.10 The expression levels of the BrPIF5 gene in different plants Different lowercase letters indicate significant differences(P<0.05).

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大白菜转录因子基因BrPIF5功能验证及表达分析

Functional Validation and Expression Analysis of BrPIF5 Transcription Factor Gene in Brassica rapa

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