Cloning and Expression Analysis of Powdery Mildew Responsive Gene McMLO1 in Momordica charantia

doi:10.7668/hbnxb.20191760

Abstract

Abstract:

MLO gene,acting as susceptibility factor,play an important role in regulating responses of host plants against powdery mildew.To determine the gene function of bitter gourd McMLO1 gene,cloning,bioinformatics and expression analysis were carried out in the present study.The results suggested that the full-length of McMLO1 was 4 019 bp,among which the CDS sequence was 1 707 bp,encoding 568 amino acids.ProtParam prediction indicated McMLO1,whose molecular weight and theoretical isoelectric point was 65.40 ku and 9.36,respectively,was an unstable hydrophilic protein which located in cell membrane.McMLO1 protein,harboring a conserved MLO domain of 477 amino acids,was consisted of random coil and alpha helix on secondary structure.Multiple sequence alignment and phylogenetic analysis revealed high homology between McMLO1 and cucumber MLO protein.qRT-PCR analysis suggested that the expression level of McMLO1 in leaf was much higher than that in other tissues.Moreover,the expression level of McMLO1 was significantly up-regulated and reached peak at 6 hours after inoculation with powdery mildew pathogen,indicating McMLO1 participate in the process in response to powdery mildew in bitter gourd.

Key words: Momordica charantia, Powdery mildew, McMLO1, Gene clone, Expression analysis

CHEN Longzheng, LIU Jing, LIU Zhiyang, XIA Pengfei, YUAN Xihan, NING Yu. Cloning and Expression Analysis of Powdery Mildew Responsive Gene McMLO1 in Momordica charantia[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 165-171. doi: 10.7668/hbnxb.20191760.

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Figures/Tables 9

Tab.1 The sequences of 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
CDS扩增	McMLO1a	F:ATGGATAAATCTGTAGAACAGCGTACT
CDS amplification		R:TCATTTGGCAAATGAGAAGTCG
基因全长扩增	McMLO1b	F:TTTGGAATTGTCCTGTTTTGC
Gene amplification		R:TAAGAGGGAAAAGAATTAACTTTCTT
荧光定量PCR	qMcMLO1	F:GTAGAACAGCGTACTTTGGAA
Real-time PCR		R:CCACTTTCCAGTGAGGTGAA
	18sRNA	F:ACCGAACCCATGTTCACTC
		R:CAAGCACGGAAGAAAATCGAA

Fig.1 Amplification of McMLO1 gene of bitter gourd
1,2,3.Gene cloning of McMLO1;M.2 000 bp Marker.

Fig.2 Prediction of McMLO1 transmembrane

Fig.3 Secondary structure prediction of McMLO1 protein
Blue .Alpha helix;Red.Extended strand;Green.Beta turn;Purple.Random coil.

Fig.4 cis-acting elements analysis on promoter region of McMLO1

Fig.5 Multiple sequences alignment of MLO domain from different species
TM.Transmembrane domains.

Fig.6 Phylogenetic analysis of MLO proteins from different species

Fig.7 Relative expression levels of McMLO1 in different tissues of bitter gourd
Different lowercase letters indicate significant differences at P<0.05.The same as Fig.8.

Fig.8 Expression analysis of McMLO1 in response to powdery mildew infection

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