Cloning and Functional Analysis of CmoMLO Genes in Chinese Pumpkin (Cucurbita moschata)

doi:10.11924/j.issn.1000-6850.casb2025-0954

Abstract

Abstract:

To analyze the structure of CmoMLO gene family members in clade V of Chinese pumpkin (Cucurbita moschata) and their relationship with powdery mildew resistance, this study utilized a highly powdery mildew-susceptible pumpkin line as material. Five MLO homologous genes from clade V of Chinese pumpkin (CmoMLO4, CmoMLO8, CmoMLO13, CmoMLO18, and CmoMLO19) were cloned using TA cloning. Additionally, pTRV2-VIGS silencing vectors for the five genes were constructed through homologous cloning, and changes in the expression levels of the relevant genes were detected. Complete open reading frame (ORF) sequences (1803-2067 bp) of the five CmoMLO genes were obtained. Sequence analysis revealed that the amino acid sequences of the cloned target genes showed ≥99% identity with the reference sequences in the Chinese pumpkin genome database, and all contained seven transmembrane domains with highly conserved transmembrane region amino acid sequences. Following Podosphaera xanthii inoculation for 24 h, the expression levels of CmoMLO8 and CmoMLO19 in the control plants were upregulated by 53.5-fold and 13.5-fold, respectively. After 24 h of inoculation, the target gene expression levels in pTRV2-CmoMLO8 and pTRV2-CmoMLO19 silenced plants were significantly reduced, with upregulation of only 8-fold and 3.7-fold, respectively. The CmoMLO8 and CmoMLO19 genes of clade V CmoMLO family are key genes involved in Chinese pumpkin response to powdery mildew infection.

Key words: Chinese pumpkin, powdery mildew, MLO gene, cloning, virus-induced gene silencing, functional analysis

CLC Number:

S642.1

LI Ke, DU Furong, ZHANG Mei, ZHAO Mingming, HU Xinyan, CHEN Xiaoguang. Cloning and Functional Analysis of CmoMLO Genes in Chinese Pumpkin (Cucurbita moschata)[J]. Chinese Agricultural Science Bulletin, 2026, 42(20): 35-43.

Figures/Tables 7

References 25

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试验	基因名称	引物序列(5′→3′)
TA克隆	CmoMLO4	F	CCTTCACTTCCAACCTCTCCC
	CmoMLO4	R	TCCCCTTCATTTACATCAACCAAA
	CmoMLO8	F	CGAAGACAGTGGAGCTTTGC
	CmoMLO8	R	TCAAACTTTTATGCCCCGTTTGT
	CmoMLO13	F	AGTAGCTCCGTAGTCCATCCA
	CmoMLO13	R	ACATCCACGAACTTCTAAACGA
	CmoMLO18	F	GCTTGAGAGCTTTGTTTCTGT
	CmoMLO18	R	GAACGTGCAAGAATGTGCTGT
	CmoMLO19	F	CGAATCTGCACTCTCTCTTT
	CmoMLO19	R	TCATACTCCATCGACTCTTC
同源克隆	PDS	F	gtgagtaaggttaccgaattcTGCTCGAGAGGAACGACGAG
	PDS	R	cgtgagctcggtaccggatccTATTGGGATCAGCCTAACATGTCTA
	MS-4	F	gtgagtaaggttaccgaattcATATCGCTGCTGCTGACGGT
	MS-4	R	cgtgagctcggtaccggatccGGCATTGACAGGGGAGACG
	MS-8	F	gtgagtaaggttaccgaattcCTTGCACGTTCTTGGCGTACA
	MS-8	R	cgtgagctcggtaccggatccACTTTTATGCCCCGTTTGTGTT
	MS-13	F	gtgagtaaggttaccgaattcGTTCTGGTTCAATCGCCCTCG
	MS-13	R	cgtgagctcggtaccggatccATGTGGTGAGTTGGAGTGGTGG
	MS-18	F	gtgagtaaggttaccgaattcCATCACCCACAGCACATTCTTG
	MS-18	R	cgtgagctcggtaccggatccTGCAAGAAGTGACAGATCACAGATG
	MS-19	F	gtgagtaaggttaccgaattcTGCTGCTGGGGTTCATTTCT
	MS-19	R	cgtgagctcggtaccggatccCACGGCGGAAAGTGGCAT
	pTRV2		TCAGGCGGTTCTTGTGTGTC (检测引物)
荧光定量	PDS	F	GAGTCAAGGTGCTGGATCGT
	PDS	R	CTAGACGTTCACCAGTCGCT
	CmoMLO4	F	AAAAACTGGCATCACACGGC
	CmoMLO4	R	GTGGGCGACTAGACATAGGC
	CmoMLO8	F	TGGAACAGAGCAGCGTACTTT
	CmoMLO8	R	CAAGAGCTGGCTTGTGCTTTT
	CmoMLO13	F	CGAGCACGTAGAGGATGTGG
	CmoMLO13	R	GGGGTGATCGAACCACGATT
	CmoMLO18	F	CGCTCCCACTCTATGCTCTG
	CmoMLO18	R	GCGGTGTTGCTTCATGTGTT
	CmoMLO19	F	AAAGTTCCGTTCGTGTCGGA
	CmoMLO19	R	TTTCCCACGACTTCCAGCTC
	18SRNA	F	AAACCTTACCAGCCCTTGAC
	18SRNA	R	CGCTCGTTATAGGACTTGACC

试验	基因名称	引物序列(5′→3′)
TA克隆	CmoMLO4	F	CCTTCACTTCCAACCTCTCCC
	CmoMLO4	R	TCCCCTTCATTTACATCAACCAAA
	CmoMLO8	F	CGAAGACAGTGGAGCTTTGC
	CmoMLO8	R	TCAAACTTTTATGCCCCGTTTGT
	CmoMLO13	F	AGTAGCTCCGTAGTCCATCCA
	CmoMLO13	R	ACATCCACGAACTTCTAAACGA
	CmoMLO18	F	GCTTGAGAGCTTTGTTTCTGT
	CmoMLO18	R	GAACGTGCAAGAATGTGCTGT
	CmoMLO19	F	CGAATCTGCACTCTCTCTTT
	CmoMLO19	R	TCATACTCCATCGACTCTTC
同源克隆	PDS	F	gtgagtaaggttaccgaattcTGCTCGAGAGGAACGACGAG
	PDS	R	cgtgagctcggtaccggatccTATTGGGATCAGCCTAACATGTCTA
	MS-4	F	gtgagtaaggttaccgaattcATATCGCTGCTGCTGACGGT
	MS-4	R	cgtgagctcggtaccggatccGGCATTGACAGGGGAGACG
	MS-8	F	gtgagtaaggttaccgaattcCTTGCACGTTCTTGGCGTACA
	MS-8	R	cgtgagctcggtaccggatccACTTTTATGCCCCGTTTGTGTT
	MS-13	F	gtgagtaaggttaccgaattcGTTCTGGTTCAATCGCCCTCG
	MS-13	R	cgtgagctcggtaccggatccATGTGGTGAGTTGGAGTGGTGG
	MS-18	F	gtgagtaaggttaccgaattcCATCACCCACAGCACATTCTTG
	MS-18	R	cgtgagctcggtaccggatccTGCAAGAAGTGACAGATCACAGATG
	MS-19	F	gtgagtaaggttaccgaattcTGCTGCTGGGGTTCATTTCT
	MS-19	R	cgtgagctcggtaccggatccCACGGCGGAAAGTGGCAT
	pTRV2		TCAGGCGGTTCTTGTGTGTC (检测引物)
荧光定量	PDS	F	GAGTCAAGGTGCTGGATCGT
	PDS	R	CTAGACGTTCACCAGTCGCT
	CmoMLO4	F	AAAAACTGGCATCACACGGC
	CmoMLO4	R	GTGGGCGACTAGACATAGGC
	CmoMLO8	F	TGGAACAGAGCAGCGTACTTT
	CmoMLO8	R	CAAGAGCTGGCTTGTGCTTTT
	CmoMLO13	F	CGAGCACGTAGAGGATGTGG
	CmoMLO13	R	GGGGTGATCGAACCACGATT
	CmoMLO18	F	CGCTCCCACTCTATGCTCTG
	CmoMLO18	R	GCGGTGTTGCTTCATGTGTT
	CmoMLO19	F	AAAGTTCCGTTCGTGTCGGA
	CmoMLO19	R	TTTCCCACGACTTCCAGCTC
	18SRNA	F	AAACCTTACCAGCCCTTGAC
	18SRNA	R	CGCTCGTTATAGGACTTGACC

基因名称	登录号(ID)	CDS序列		氨基酸序列
基因名称	登录号(ID)	一致性/%	相似性/%	一致性/%	相似性/%
CmoMLO4	CmoCh02G008830	98.87	98.87	100.00	100.00
CmoMLO8	CmoCh04G014330	99.67	99.67	99.30	99.30
CmoMLO13	CmoCh13G001880	99.89	99.89	99.66	99.83
CmoMLO18	CmoCh18G008900	99.79	99.79	99.83	99.83
CmoMLO19	CmoCh20G005110	99.45	99.45	99.50	99.50

基因名称	登录号(ID)	CDS序列		氨基酸序列
基因名称	登录号(ID)	一致性/%	相似性/%	一致性/%	相似性/%
CmoMLO4	CmoCh02G008830	98.87	98.87	100.00	100.00
CmoMLO8	CmoCh04G014330	99.67	99.67	99.30	99.30
CmoMLO13	CmoCh13G001880	99.89	99.89	99.66	99.83
CmoMLO18	CmoCh18G008900	99.79	99.79	99.83	99.83
CmoMLO19	CmoCh20G005110	99.45	99.45	99.50	99.50