Cloning and Functional Analysis of the MbEGS Gene from Melaleuca bracteata

doi:10.16420/j.issn.0513-353x.2021-0562

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (8): 1747-1760.doi: 10.16420/j.issn.0513-353x.2021-0562

• Research Papers • Previous Articles Next Articles

Cloning and Functional Analysis of the MbEGS Gene from Melaleuca bracteata

QIU Ziwen¹^,³, LIU Linmin¹, LIN Yongsheng¹, LIN Xiaojie¹, LI Yongyu¹^,²^,^*(), WU Shaohua¹^,²^,^*(), YANG Chao¹^,²^,^*()

1. College of Horticulture,Fujian Agriculture and Forestry University,Fuzhou 350002,China
2. Institute of Natural Products of Horticultural Plants,Fujian Agriculture and Forestry University,Fuzhou 350002,China
3. Agricultural Science and Technology Development Center of Chaozhou in Guangdong Province,Chaozhou,Guangodng 521000,China

Received:2021-11-07 Revised:2022-03-24 Online:2022-08-25 Published:2022-09-05
Contact: LI Yongyu,WU Shaohua,YANG Chao E-mail:lilin3182@163.com;348279953@qq.com;55753827@qq.com

Abstract

Abstract:

Melaleuca bracteata is rich in essential oil,the main component of which is methyl eugenol with the content is as high as 86.5%. Eugenol is the precursor for the synthesis of methyl eugenol,but the biosynthetic pathway of essential oil is not clear. Based on the transcriptome databases of Melaleuca bracteata,two eugenol synthase genes（MbEGS1 and MbEGS2）were identified and cloned from Melaleuca bracteata. The coding sequences（CDS）of MbEGS1 and MbEGS2 were 963 and 972 bp in length,encoding 320 and 323 amino acids,respectively. Multiple sequence alignment and phylogenetic tree analysis showed that the deduced proteins MbEGS1 and MbEGS2 had the highest similarity （65.92% and 63.89%）with CbIGS1. qRT-PCR analysis showed that MbEGS1 and MbEGS2 were expressed in flowers,leaves and stems of Melaleuca bracteata. The highest expression level was observed in flowers,followed by leaves and stems,which was similar to the eugenol content in different tissues of Melaleuca bracteata,showing a significant positive correlation between the gene expression and eugenol content. MbEGS1 was expressed in roots of Melaleuca bracteata,and the expression level was higher than that in stems and lower than that in leaves. However,MbEGS2 was not expressed in roots. When MeJA was used to treat Melaleuca bracteata,MeJA could induce the expression of MbEGS1 and MbEGS2 and eugenol synthesis,which showed a trend of first increase and then decrease. Treatment with MeJA at 0.1 mmol · L^-1 had the most significant effect on gene expression,and their transcription levels were positively correlated with the eugenol content. Subcellular localization showed that MbEGS1 was mainly located in plant cell membrane and nucleus,and MbEGS2 was mainly located in plant cell membrane. Ectopic expression of MbEGS1 and MbEGS2 showed that overexpression of MbEGS1 and MbEGS2 promoted the synthesis of eugenol in Fragaria vesca（Yellow Wonder）. The results show that both MbEGS1 and MbEGS2 genes are involved in eugenol biosynthesis.

Key words: Melaleuca bracteata, eugenol synthase, gene cloning, expression analysis, functional identification

CLC Number:

S687

QIU Ziwen, LIU Linmin, LIN Yongsheng, LIN Xiaojie, LI Yongyu, WU Shaohua, YANG Chao. Cloning and Functional Analysis of the MbEGS Gene from Melaleuca bracteata[J]. Acta Horticulturae Sinica, 2022, 49(8): 1747-1760.

Figures/Tables 9

Table 1 Sequence of specific primers used in this study

引物用途 Primer use	引物名称 Primer name	引物序列（5′-3′） Primer sequence
基因克隆 Gene cloning	MbEGS1-F1	ATGGCAGGAGAGGCCGAGAAAA
	MbEGS1-R1	TCATTCGAAAGCAGCCCTAGCTG
	MbEGS2-F1	ATGACGATCATAAGCAGTAGTTGCA
	MbEGS2-R1	TCATTCCAATACAGCGCTTGC
实时荧光定量引物 qRT-PCR primer	MbEGS1-QF	TTCGGGAGTGAGGAGGATAA
	MbEGS1-QR	TGGGGATGAAGGAGGTTGTT
	MbEGS2-QF	CCCCACCTTTGTCTTCGCTC
	MbEGS2-QR	AACCTCTTTATGTTGCCCGC
	GAPDH-F	GAAGGAATACAAGCCCGACC
	GAPDH-R	GACCCTCAACAATGCCAAAT
亚细胞定位和瞬时过表达 Subcellular localization and transient overexpression	MbEGS1-YF1	ATGGCAGGAGAGGCCGAG
	MbEGS1-YR1	TCATTCGAAAGCAGCCCTAGC
	MbEGS2-YF2	ATGACGATCATAAGCAGTAGTTGC
	MbEGS2-YR2	TCATTCCAATACAGCGCTTG
	MbEGS1-YF2	AGTGGTCTCTGTCCAGTCCTATGGCAGGAGAGGCCGAG
	MbEGS1-YR2	GGTCTCAGCAGACCACAAGTTCATTCGAAAGCAGCCCTAGC
	MbEGS2-YF2	AGTGGTCTCTGTCCAGTCCTATGACGATCATAAGCAGTAGTTGC
	MbEGS2-YR2	GGTCTCAGCAGACCACAAGTTCATTCCAATACAGCGCTTG

Fig. 1 Eugenol content in different tissue（A）and the leaves（B）under different treatment of Melaleuca bracteata Different lowercase letters indicate significant differences（P < 0.05）. The same below.

Fig. 2 MbEGS1 and MbEGS2 cloning

Fig. 3 MbEGS1（A）and MbEGS2（B）and their deduced amino acid sequences

Fig. 4 Evolutionary tree based on MbEGS homologous protein of different species Mb：Melaleuca bracteata;Cb：Clarkia breweri;Ob：Ocimum basilicum;Ph：Petunia × hybrida;Tp：Thuja plicata;La：Lupinus albus;Lc：Linum corymbulosum;Lu：Linum usitatissimum;Fi：Forsythia × intermedia;At：Arabidopsis thaliana;Lp：Linum perenne;Zm：Zea mays;Ps：Pisum sativum;Ms：Medicago sativa;Ca：Cicer arietinum;Gm：Glycine max;Nt：Nicotiana tabacum;Ole：Olea europaea;Bp：Betula pendula;Pop：Populus trichocarpa;Pyrc：Pyrus communis;Pt：Pinus taeda;Gb：Ginkgo biloba.

Fig. 5 Analysis of transcriptional expression patterns of MbEGS in different tissues and MeJA treatments

Fig. 6 Subcellular localization of MbEGS1 and MbEGS2 in leaf cells of Nicotiana benthamiana

Fig. 7 Relative expression levels of MbEGS1 and MbEGS2 in transgenic strawberry fruit

Table 2 Content of eugenol in strawberry transgenic fruits

表达载体 Expression vector	丁香酚含量/（μg · g^-1） Eugenol content
pCam1304-MCS35S（对照Control）	0.235 ± 0.004 a
pCam1304-MCS35S-MbEGS1	0.263 ± 0.009 b
pCam1304-MCS35S-MbEGS2	0.327 ± 0.019 c

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Cloning and Functional Analysis of the MbEGS Gene from Melaleuca bracteata

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