中国南瓜CmoMLO基因的克隆及功能分析

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

摘要/Abstract

摘要：

为了解析中国南瓜第V分支CmoMLO基因家族成员的结构及其与白粉病抗性的关系，本研究以高感白粉病的南瓜品系为材料，利用TA克隆了中国南瓜第V分支的5个MLO同源基因(CmoMLO4、CmoMLO8、CmoMLO13、CmoMLO18、CmoMLO19)；并通过同源克隆的方法构建了5个基因的pTRV2-VIGS沉默载体，监测了相关基因的表达量变化。获得了5个CmoMLO基因完整的ORF(1803~2067 bp)序列，序列分析发现，克隆到的目的基因所编码的氨基酸序列与中国南瓜基因组数据库中的参考序列氨基酸一致性≥99%，且均含7个跨膜结构域，其跨膜区氨基酸序列高度保守。在接种白粉病菌24 h后，对照组中CmoMLO8与CmoMLO19的表达量分别显著上调表达了53.5倍与13.5倍。接种24 h后，经pTRV2-CmoMLO8与pTRV2-CmoMLO19沉默处理的植株中，目标基因的表达量显著降低，上调倍数仅为8倍和3.7倍。第V分支CmoMLO家族的CmoMLO8与CmoMLO19基因在中国南瓜响应白粉病菌侵染过程中扮演着关键角色。

关键词: 中国南瓜, 白粉病, MLO基因, 克隆, 病毒诱导基因沉默, 功能解析

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

中图分类号:

S642.1

李可, 杜付荣, 张梅, 赵明明, 胡新燕, 陈晓光. 中国南瓜CmoMLO基因的克隆及功能分析[J]. 中国农学通报, 2026, 42(20): 35-43.

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.

图/表 7

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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