苦瓜白粉病响应基因<i>McMLO1</i>的克隆及表达分析

doi:10.7668/hbnxb.20191760

摘要/Abstract

摘要：

MLO基因作为感病因子在调控寄主植物对白粉病的应答反应中发挥着重要作用。为明确苦瓜McMLO1基因在白粉病胁迫下的基因功能,以苦瓜自交系K24为材料对该基因进行了克隆、生物信息预测及表达分析。结果表明,McMLO1基因全长4 019 bp,包含15个外显子,其中CDS序列长为1 707 bp,编码568个氨基酸。ProtParam预测显示,McMLO1蛋白的分子质量为65.40 ku,理论等电点为9.36,属不稳定亲水蛋白,主要位于细胞膜上;McMLO1蛋白包含一个由477个氨基酸组成的MLO保守结构域,其二级结构主要由无规则卷曲和α-螺旋组成。氨基酸多重序列比对和系统进化分析表明,McMLO1与黄瓜MLO蛋白的同源性较高。qRT-PCR检测结果发现,McMLO1在叶片中的表达量显著高于根、茎、果实等组织。白粉病接种后,McMLO1基因的表达量显著提高,并在6 h达到峰值,推测该基因参与了苦瓜对白粉病侵染的早期应答反应。

关键词: 苦瓜, 白粉病, McMLO1, 基因克隆, 表达分析

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

陈龙正, 刘静, 刘之洋, 夏彭飞, 袁希汉, 宁宇. 苦瓜白粉病响应基因McMLO1的克隆及表达分析[J]. 华北农学报, 2022, 37(1): 165-171. doi: 10.7668/hbnxb.20191760.

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.

http://www.hbnxb.net/CN/Y2022/V37/I1/165

图/表 9

表1 本研究使用的引物

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

图1 苦瓜McMLO1基因的扩增
1,2,3.McMLO1基因克隆;M.2 000 bp Marker。

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

图2 McMLO1蛋白的跨膜结构预测

Fig.2 Prediction of McMLO1 transmembrane

图3 McMLO1蛋白的二级结构预测
蓝色.α-螺旋;红色.延伸链;绿色.β-转角;紫色.无规则卷曲。

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

图4 McMLO1基因启动子区域顺式作用元件分析

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

图5 不同物种的MLO蛋白保守结构域的多序列比对分析
TM.跨膜结构域。

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

图6 不同物种MLO蛋白的系统进化分析

Fig.6 Phylogenetic analysis of MLO proteins from different species

图7 McMLO1基因在苦瓜不同组织的相对表达水平
不同小写字母表示在P<0.05水平上差异显著。图8同。

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.

图8 McMLO1基因在白粉病胁迫下的相对表达水平

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

参考文献 30

[1]	Lu H Y,Lin B F.Wild bitter melon alleviates dextran sulphate sodium-induced murine colitis by suppressing inflammatory responses and enhancing intestinal regulatory T cells[J].Journal of Functional Foods,2016,23:590-600.doi:10.1016/j.jff.2016.03.014.
[2]	Raina K,Kumar D,Agarwal R.Promise of bitter melon(Momordica charantia)bioactives in cancer prevention and therapy[J].Seminars in Cancer Biology,2016,40/41:116-129.doi:10.1016/j.semcancer.2016.07.002.
[3]	田丽波.苦瓜遗传图谱构建及白粉病抗性的QTL定位[D].沈阳:沈阳农业大学,2015.
	Tian L B.Constructing of genetic maps and QTL mapping of powdery mildew resistance in bitter melon[D].Shenyang:Shenyang Agricultural University,2015.
[4]	Bidzinski P,Noir S,Shahi S,Reinstädler A,Gratkowska D M,Panstruga R.Physiological characterization and genetic modifiers of aberrant root thigmomorphogenesis in mutants of Arabidopsis thaliana MILDEW LOCUS O genes[J].Plant, Cell & Environment,2014,37(12):2738-2753.doi:10.1111/pce.12353.
[5]	Meng J G,Liang L,Jia P F,Wang Y C,Li H J,Yang W C.Integration of ovular signals and exocytosis of a Ca²+ channel by MLOs in pollen tube guidance[J].Nature Plants,2020,6(2):143-153.doi:10.1038/s41477-020-0599-1.
[6]	Lim C W,Lee S C.Functional roles of the pepper MLO protein gene,CaMLO2,in abscisic acid signaling and drought sensitivity[J].Plant Molecular Biology,2014,85(1/2):1-10.doi:10.1007/s11103-013-0155-8.
[7]	Acevedo-Garcia J,Kusch S,Panstruga R.Magical mystery tour:MLO proteins in plant immunity and beyond[J].The New Phytologist,2014,204(2):273-281.doi:10.1111/nph.12889.
[8]	Panstruga R.Serpentine plant MLO proteins as entry portals for powdery mildew fungi[J].Biochemical Society Transactions,2005,33(2):389-392.doi:10.1042/bst0330389.
[9]	Büschges R,Hollricher K,Panstruga R,Simons G,Wolter M,Frijters A,van Daelen R,van der Lee T,Diergaarde P,Groenendijk J,Töpsch S,Vos P,Salamini F,Schulze-Lefert P.The barley Mlo gene:A novel control element of plant pathogen resistance[J].Cell,1997,88(5):695-705.doi:10.1016/s0092-8674(00)81912-1.
[10]	Consonni C,Humphry M E,Hartmann H A,Livaja M,Durner J,Westphal L,Vogel J,Lipka V,Kemmerling B,Schulze-Lefert P,Somerville S C,Panstruga R.Conserved requirement for a plant host cell protein in powdery mildew pathogenesis[J].Nature Genetics,2006,38(6):716-720.doi:10.1038/ng1806.
[11]	Wang Y P,Cheng X,Shan Q W,Zhang Y,Liu J X,Gao C X,Qiu J L.Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew[J].Nature Biotechnology,2014,32(9):947-951.doi:10.1038/nbt.2969.
[12]	Pessina S,Angeli D,Martens S,Visser R G F,Bai Y L,Salamini F,Velasco R,Schouten H J,Malnoy M.The knock-down of the expression of MdMLO19 reduces susceptibility to powdery mildew(Podosphaera leucotricha)in apple(Malus domestica)[J].Plant Biotechnology Journal,2016,14(10):2033-2044.doi:10.1111/pbi.12562.
[13]	Matsumura H,Hsiao M C,Lin Y P,Toyoda A,Taniai N,Tarora K,Urasaki N,Anand S S,Dhillon N P S,Schafleitner R,Lee C R.Long-read bitter gourd(Momordica charantia)genome and the genomic architecture of nonclassic domestication[J].Proceedings of the National Academy of Sciences of the United States of America,2020,117(25):14543-14551.doi:10.1073/pnas.1921016117.
[14]	Livak K J,Schmittgen T D.Analysis of relative gene expression data using Real-time quantitative PCR and the 2^-ΔΔCT method[J].Methods (San Diego, Calif),2001,25(4):402-408.doi:10.1006/meth.2001.1262.
[15]	Dhillon N P S,Sanguansil S,Srimat S,Schafleitner R,Manjunath B,Agarwal P,Xiang Q,Masud M A T,Myint T,Hanh N T,Cuong T K,Balatero C H,Salutan-Bautista V,Pitrat M,Lebeda A,McCreight J D.Cucurbit powdery mildew-resistant bitter gourd breeding lines reveal four races of Podosphaera xanthii in Asia[J].HortScience,2018,53(3):337-341.doi:10.21273/HORTSCI12545-17.
[16]	Dhillon N P S,Sanguansil S,Srimat S,Laenoi S,Schafleitner R,Pitrat M,McCreight J D.Inheritance of resistance to cucurbit powdery mildew in bitter gourd[J].HortScience,2019,54(6):1013-1016.doi:10.21273/HORTSCI13906-19.
[17]	Kusch S,Panstruga R.Mlo-based resistance:An apparently universal "weapon" to defeat powdery mildew disease[J].Molecular Plant-Microbe Interactions,2017,30(3):179-189.doi:10.1094/MPMI-12-16-0255-CR.
[18]	Wan D Y,Guo Y,Cheng Y,Hu Y,Xiao S Y,Wang Y J,Wen Y Q.Crispr/Cas9-mediated mutagenesis of Vvmlo3 results in enhanced resistance to powdery mildew in grapevine(Vitis vinifera)[J].Horticulture Research,2020,7(1):116.doi:10.1038/s41438-020-0339-8.
[19]	Nekrasov V,Wang C M,Win J,Lanz C,Weigel D,Kamoun S.Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion[J].Scientific Reports,2017,7(1):482.doi:10.1038/s41598-017-00578-x.
[20]	Zheng Z,Nonomura T,Appiano M,Pavan S,Matsuda Y,Toyoda H,Wolters A M A,Visser R G F,Bai Y L.Loss of function in Mlo orthologs reduces susceptibility of pepper and tomato to powdery mildew disease caused by Leveillula taurica[J].PLoS One,2013,8(7):e70723.doi:10.1371/journal.pone.0070723.
[21]	Bai Y L,Pavan S,Zheng Z,Zappel N F,Reinstädler A,Lotti C,De Giovanni C,Ricciardi L,Lindhout P,Visser R G,Theres K,Panstruga R.Naturally occurring broad-spectrum powdery mildew resistance in a central American tomato accession is caused by loss of mlo function[J].Molecular Plant-Microbe Interactions,2008,21(1):30-39.doi:10.1094/MPMI-21-1-0030.
[22]	程鸿,孔维萍,何启伟,王晓巍.CmMLO2:一个与甜瓜白粉病感病相关的新基因[J].园艺学报,2013,40(3):540-548.doi:10.16420/j.issn.0513-353x.2013.03.018.
	Cheng H,Kong W P,He Q W,Wang X W.CmMLO2:A novel gene closely associated with the powdery mildew in melon[J].Acta Horticulturae Sinica,2013,40(3):540-548.
[23]	Devoto A,Piffanelli P,Nilsson I,Wallin E,Panstruga R,von Heijne G,Schulze-Lefert P.Topology,subcellular localization,and sequence diversity of the Mlo family in plants[J].The Journal of Biological Chemistry,1999,274(49):34993-35004.doi:10.1074/jbc.274.49.34993.
[24]	Iovieno P,Andolfo G,Schiavulli A,Catalano D,Ricciardi L,Frusciante L,Ercolano M R,Pavan S.Structure,evolution and functional inference on the Mildew Locus O(MLO)gene family in three cultivated Cucurbitaceae spp.[J].BMC Genomics,2015,16:1112.doi:10.1186/s12864-015-2325-3.
[25]	Bhat R A,Miklis M,Schmelzer E,Schulze-Lefert P,Panstruga R.Recruitment and interaction dynamics of plant penetration resistance components in a plasma membrane microdomain[J].Proceedings of the National Academy of Sciences,2005,102(8):3135-3140.doi:10.1073/pnas.0500012102.
[26]	邱显钦,包满珠,张颢,蹇洪英,王其刚,晏慧君,张婷,唐开学.野蔷薇(Rosa multiflora)抗白粉病基因RmMlo的克隆与表达分析[J].园艺学报,2011,38(10):1999-2004.doi:10.16420/j.issn.0513-353x.2011.10.022.
	Qiu X Q,Bao M Z,Zhang H,Jian H Y,Wang Q G,Yan H J,Zhang T,Tang K X.Cloning and expression analysis of resistance powdery mildew gene RmMlo in Rosa multiflora[J].Acta Horticulturae Sinica,2011,38(10):1999-2004.
[27]	Berg J A,Appiano M,Bijsterbosch G,Visser R G F,Schouten H J,Bai Y L.Functional characterization of cucumber(Cucumis sativus L.)clade V MLO genes[J].BMC Plant Biology,2017,17(1):80.doi:10.1186/s12870-017-1029-z.
[28]	Feechan A,Jermakow A M,Torregrosa L,Panstruga R,Dry I B.Identification of grapevine MLO gene candidates involved in susceptibility to powdery mildew[J].Functional Plant Biology,2008,35(12):1255-1266.doi:10.1071/FP08173.
[29]	Berg J A,Appiano M,Santillán Martínez M,Hermans F W K,Vriezen W H,Visser R G F,Bai Y L,Schouten H J.A transposable element insertion in the susceptibility gene CsaMLO8 results in hypocotyl resistance to powdery mildew in cucumber[J].BMC Plant Biology,2015,15:243.doi:10.1186/s12870-015-0635-x.
[30]	Qin B,Wang M,He H X,Xiao H X,Zhang Y,Wang L F.Identification and characterization of a potential candidate Mlo gene conferring susceptibility to powdery mildew in rubber tree[J].Phytopathology,2019,109(7):1236-1245.doi:10.1094/PHYTO-05-18-0171-R.

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苦瓜白粉病响应基因McMLO1的克隆及表达分析

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