Cloning and Function Analysis of EβF Synthetase Gene from Mentha canadensis

doi:10.7668/hbnxb.20194727

Abstract

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

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.

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Figures/Tables 10

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

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

Fig.2 Transmembrane structure prediction of McβFS1 protein

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

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.

Fig.5 Phylogenetic analysis of EβF homologous protein

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

Fig.7 Model of McβFS1 gene over-expression vector

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.

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

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