薄荷EβF合成酶基因的克隆及功能分析

doi:10.7668/hbnxb.20194727

摘要/Abstract

摘要：

EβF合成酶基因在植物体内合成[反]-β-法尼烯,用于抵御蚜虫侵害。为探明薄荷中该基因的表达模式和功能,从薄荷叶片中克隆得到1个新的EβF合成酶基因McβFS1,并对其进行生物信息学分析和表达模式分析。构建过表达载体,利用农杆菌介导的浸花法转化拟南芥,并进行蚜虫生物测定。结果表明,McβFS1基因CDS序列全长1 650 bp,编码549个氨基酸。编码蛋白分子质量为63.85 ku,等电点为5.23,无跨膜结构域。二级结构预测其α-螺旋、β-转角、延伸链结构和无规则卷曲所占比例分别为69.58%,3.10%,4.01%和23.32%。系统进化分析结果显示,该基因编码的氨基酸序列与胡椒薄荷的Q5W283.1序列相似度达91.47%,亲缘关系较近,但序列差异较大,是一个新的EβF合成酶基因。荧光定量PCR结果显示,McβFS1在根、茎、叶、花中均有表达,且在叶中的表达量显著高于根、茎中的表达。蚜虫的选择性分析表明,转基因拟南芥对蚜虫具有较强的趋避性。

关键词: 薄荷, EβF合成酶, 蚜虫, 生物信息学分析, 表达分析

Abstract:

EβF synthetase gene(EβFS)enables the production of(E)-β-farnesene in plants to control aphids.In order to identify the expression characteristics and the function of EβFS from Mentha canadensis,McβFS1 was isolated from mint leaves,and bioinformatics analysis and expression patterns were analyzed.McβFS1 over expression vector was constructed and then was transformed into wild-type Arabidopsis by Agrobacterium-mediated floral dip method.The transgenic Arabidopsis thaliana was subjected to aphids bioassays.The results showed that the coding region of McβFS1 was 1 650 bp,encoding 549 amino acids.The protein molecular weight was 63.85 ku,the isoelectric point was 5.23,and there was no transmembrane domain.The predicted secondary structure contained α-helix,β-turn,extended strand,and random coil,which had 69.58%,3.10%,4.01%,and 23.32%,respectively.Phylogenetic analysis revealed that McβFS1 had a close genetic relationship of 91.47% similarity with Mentha× piperita Q5W283.1.However,the sequence difference is significant,making it a new EβF synthase gene.The qRT-PCR results showed that McβFS1 was expressed in root,stem,leaf and flower,and the expression level in leaf was significantly higher than that in root and stem.The choice test suggested that transgenic Arabidopsis was higher resistant to aphids than wild type.

Key words: Mentha canadensis, EβF synthetase, Aphid, Bioinformatics, Expression analysis

刘迪, 甄军波, 刘琳琳, 冯晓晴, 迟吉娜. 薄荷EβF合成酶基因的克隆及功能分析[J]. 华北农学报, 2024, 39(3): 96-103. doi: 10.7668/hbnxb.20194727.

LIU Di, ZHEN Junbo, LIU Linlin, FENG Xiaoqing, CHI Jina. Cloning and Function Analysis of EβF Synthetase Gene from Mentha canadensis[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 96-103. doi: 10.7668/hbnxb.20194727.

http://www.hbnxb.net/CN/Y2024/V39/I3/96

图/表 10

表1 引物序列

Tab.1 Sequence of primers

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	作用 Function
MethaEβF-F	CACCATGGCTACAAACGGCGTCGT	基因克隆,载体构建
MethaEβF-R	TCAAAAGACTATGGCATCAACAA
qEβF-F	AACCCATGCTCGCAGTATCAACC	实时荧光定量PCR,转基因拟南芥检测
qEβF-R	TGTGGCAATCCAACGCAGTCAG
qActin-F	CCAGGAATTGCTGATAGGATGAG	内参基因
qActin-R	GCGCCACCACCTTAATCTTC
AtUBQ5-F	GACGCTTCATCTCGTCC
AtUBQ5-R	CCACAGGTTGCGTTAG

图1 EβF合成酶基因扩增 M.Trans2K DNA Marker;1,2.EβF基因PCR扩增。

Fig.1 EβF gene PCR amplification M.Trans2K DNA Marker;1,2.EβF gene PCR amplification.

图2 McβFS1蛋白的跨膜结构预测

Fig.2 Transmembrane structure prediction of McβFS1 protein

图3 McβFS1蛋白的二级结构和三级结构预测 A.二级结构;B.三级结构。

Fig.3 Secondary structure and tertiary structure prediction of McβFS1 protein A.Secondary structure;B.Tertiary structure.

图4 McβFS1氨基酸序列多重比对红线标注为DDxxD及RxR保守区域。胡椒薄荷.AAB95209.1;亚洲薄荷.AEA49038.1;夏枯草.AZB50512.1;香橙.AAK54279.1;黄花蒿.AAX39387.1;母菊.AIW60869.1。

Fig.4 Multiple alignments of the amino acid sequence of McβFS1 The DDxxD and RxR domain are marked in red.Mentha × piperita.AAB95209.1; Mentha asiatica.AEA49038.1;Prunella vulgaris.AZB50512.1;Citrus junos.AAK54279.1;Artemisia annua.AAX39387.1;Matricaria chamomilla.AIW60869.1.

图5 EβF同源蛋白系统进化分析

Fig.5 Phylogenetic analysis of EβF homologous protein

图6 McβFS1在不同器官的表达分析不同大写字母表示差异极显著(P<0.01)。图8同。

Fig.6 Expression analysis of McβFS1 in different organs Different capital letters indicate extremely significant differences (P<0.01).The same as Fig.8.

图7 McβFS1过表达载体示意图

Fig.7 Model of McβFS1 gene over-expression vector

图8 转基因拟南芥的筛选鉴定 A.抗卡纳拟南芥筛选;B.单拷贝拟南芥筛选;C.转基因拟南芥PCR鉴定;D.转基因拟南芥的实时定量PCR检测。

Fig.8 Identification of transgenic Arabidopsis A.Screening of Arabidopsis thaliana resistant to Kana;B.Screening of single copy transgenic Arabidopsis lines;C.PCR identification of transgenic Arabidopsis;D.qRT-PCR analysis of transgenic Arabidopsis thaliana plants.

图9 转基因拟南芥对蚜虫的选择性试验不同小写字母表示差异显著(P<0.05)。

Fig.9 Choice test of transgenic Arabidopsis to Aphids Different lowercase letters indicate significant differences(P<0.05).

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