一个烟草eIF4E1新等位基因的鉴定及共显性分子标记的开发与应用

doi:10.3969/j.issn.1004-1524.20250419

浙江农业学报 ›› 2026, Vol. 38 ›› Issue (7): 1327-1335.DOI: 10.3969/j.issn.1004-1524.20250419

一个烟草eIF4E1新等位基因的鉴定及共显性分子标记的开发与应用

王仁刚¹(), 林世锋¹^,^*(), 王自力¹, 余世洲¹, 张洁¹, 余婧¹, 龙明锦¹, 李莉²

¹ 贵州省烟草科学研究院烟草行业烟草分子遗传重点实验室, 贵州贵阳 550081
² 贵州师范大学生命科学学院, 贵州贵阳 550025

收稿日期:2025-02-18 出版日期:2026-07-25 发布日期:2026-08-20
作者简介:王仁刚,研究方向为烟草遗传育种与分子生物学研究。E-mail:rengangwang@126.com
通讯作者: *林世锋,E-mail: linshifeng1978@163.com
基金资助:
国家自然科学基金(32060342);中国烟草总公司贵州省公司科技项目(201901);中国烟草总公司贵州省公司科技项目(2023XM04)

Identification of a novel eIF4E1 allele in tobacco and the development and application of its codominant molecular marker

WANG Rengang¹(), LIN Shifeng¹^,^*(), WANG Zili¹, YU Shizhou¹, ZHANG Jie¹, YU Jing¹, LONG Mingjin¹, LI Li²

¹ Key Laboratory of Molecular Genetics of CNTC, Guizhou Academy of Tobacco Science, Guiyang 550081, China
² School of Life Sciences, Guizhou Normal University, Guiyang 550025, China

Received:2025-02-18 Published:2026-07-25 Online:2026-08-20

摘要/Abstract

摘要：

为发掘烟草马铃薯Y病毒隐性抗病基因eIF4E1的优异等位基因,以抗性晒烟品种盘县大柳叶为研究对象,通过等位性检测和序列分析确定其抗病基因与eIF4E1的等位关系,基于双向等位基因特异性PCR技术建立针对盘县大柳叶eIF4E1突变型等位基因的共显性分子标记。结果表明,盘县大柳叶的PVY抗性与eIF4E1突变有关,在eIF4E1基因编码区cDNA序列第267~268号位点发生双碱基缺失,导致移码突变并在第2外显子中产生提前终止密码子。针对盘县大柳叶eIF4E1新等位基因开发出一个Bi-PASA共显性分子标记,可以有效区分抗、感亲本及杂交后代的基因型。综上,在晒烟盘县大柳叶中首次发现一个双碱基缺失突变类型的eIF4E1新等位基因,并开发出一个理想的共显性分子标记,可用于烟草抗PVY分子标记辅助选择育种。

关键词: 晒烟, 马铃薯Y病毒, eIF4E1, 新等位基因, 共显性分子标记

Abstract:

To fully explore and effectively utilize the excellent alleles of the recessive resistance gene eIF4E1 against Potato virus Y (PVY) in tobacco, taking the PVY resistant sun-cured tobacco variety, Panxiandaliuye, as the research object, allelism test and sequence analysis were performed to finalize the allelic relationship of its resistance gene with eIF4E1 and the gene mutation type. A co-dominant molecular marker based on bi-directional PCR amplification of specific alleles (Bi-PASA) was developed for detecting the mutant eIF4E1 gene in Panxiandaliuye. The results showed that the resistance to PVY in Panxiandaliuye was referable to the mutation of eIF4E1 gene. There was a 2-bp deletion in position 267-268 in the coding region cDNA sequence of eIF4E1 gene in Panxiandaliuye, which caused a frame-shift mutation and resulted in a premature stop codon in exon 2. A codominant molecular marker was developed for the novel eIF4E1 mutant allele in Panxiandaliuye, which could effectively distinguish the genotypes of resistant and susceptible parents and their hybrid offsprings. In summary, a novel eIF4E1 allele with a two base pair deletion was discovered for the first time in the sun-cured tobacco Panxiandaliuye, and an ideal codominant molecular marker been developed for molecular marker-assisted selection for PVY resistance in tobacco.

Key words: sun-cured tobacco, Potato virus Y, eIF4E1, novel allele, codominant molecular marker

中图分类号:

S572

王仁刚, 林世锋, 王自力, 余世洲, 张洁, 余婧, 龙明锦, 李莉. 一个烟草eIF4E1新等位基因的鉴定及共显性分子标记的开发与应用[J]. 浙江农业学报, 2026, 38(7): 1327-1335.

WANG Rengang, LIN Shifeng, WANG Zili, YU Shizhou, ZHANG Jie, YU Jing, LONG Mingjin, LI Li. Identification of a novel eIF4E1 allele in tobacco and the development and application of its codominant molecular marker[J]. Acta Agriculturae Zhejiangensis, 2026, 38(7): 1327-1335.

图/表 10

表1 试验所用引物

Table 1 Primers used in the test

用途 Usage	引物名称 Primer name	引物序列(5'→3') Primer sequence(5'→3')
eIF4E1基因5'端显性分子标记	CF2	TTTGGTTTGATAATCCTATGGCT
Dominant molecular marker at the 5'-end of eIF4E1 gene	GR11	GAAGGCAAGATATTCAGGAGCT
eIF4E1基因3'端显性分子标记	3'-F	AATTCGTGCCACGTTAGGGC
Dominant molecular marker at the 3'-end of eIF4E1 gene	3'-R	TTGAGTCGTCCTGCAATTTGC
eIF4E1基因全长序列扩增	eIF4E1-F	CAAGTACCCTTTTCCTACTAAAATCTATAACTAAG
Full-length amplification of eIF4E1 gene	eIF4E1-R	GGCATTAATTCAAAACCAAACGAG
盘县大柳叶eIF4E1新等位基因共显性分子标记	A	TCTGCAAACAGTGTTTACAATAATAgAC
Codominant molecular marker for the novel eIF4E1 allele in Panxiandaliuye	B	AACTAACTTGCTTGGGTGGcTG
	P	TGTCTGCCTTAGATGCATGTTG
	Q	CGGACGCTAACTACTGCTCCAC

图1 烟草对PVY的抗性鉴定

Fig.1 Resistance identification of tobacco to PVY

图2 盘县大柳叶中eIF4E1基因的分子标记检测 A,5'端分子标记检测结果;B,3'端分子标记检测结果;M,DL 2 000 DNA marker;1,TN90;2,红花大金元;3,盘县大柳叶。

Fig.2 Molecular marker detection of eIF4E1 gene in Panxiandaliuye A, Detection results of molecular marker at 5' end; b, Detection results of molecular marker at 3' end; M, DL 2 000 DNA marker; 1, TN90; 2, Honghuadajinyuan; 3, Panxiandaliuye.

图3 盘县大柳叶中eIF4E1基因的PCR扩增产物 M1,DL 2 000 DNA marker;M2,DL 10 000 DNA marker;1,以cDNA为模板的扩增产物; 2,以基因组DNA为模板的扩增产物。

Fig.3 PCR amplification products of eIF4E1 gene in Panxiandaliuye M1, DL 2 000 DNA marker; M2, DL 10 000 DNA marker; 1, Amplification products using cDNA as template; 2, Amplification products using genomic DNA as template.

图4 盘县大柳叶和红花大金元的eIF4E1基因与基因组DNA序列比对红色方框内为elF4E1.Pan基因的提前终止密码子(TAA)。

Fig.4 Comparison of eIF4E1 gene of Panxiandaliuye and Honghuadajinyuan and genomic DNA sequence The premature termination codon (TAA) in the eIF4E1.Pan gene is marked with red box.

图5 双向等位基因特异性PCR引物设计

Fig.5 Primer design for bidirectional PCR amplification of specific alleles

图6 Bi-PASA遗传标记引物对抗感材料基因组DNA的扩增产物 A,P/B引物扩增结果;B,A/Q引物扩增结果;C,P/B/A/Q引物扩增结果;M,DL 2 000 marker;1,盘县大柳叶;2,红花大金元×盘县大柳叶;3,K326×盘县大柳叶;4,贵烟5号×盘县大柳叶;5,红花大金元;6,K326;7,贵烟5号。

Fig.6 Amplification products of genomic DNA of resistant and susceptible materials by Bi-PASA genetic marker primers A, PCR amplification with primers P and B; B, PCR amplification with primers A and Q; C, PCR amplification with primers P, B, A and Q; M, DL 2 000 marker; 1, Panxiandaliuye; 2, Honghuadajinyuan×Panxiandaliuye; 3, K326×Panxiandaliuye; 4, Guiyan 5×Panxiandaliuye; 5, Honghuadajinyuan; 6, K326; 7, Guiyan 5.

图7 Bi-PASA遗传标记在F2群体和亲本中的扩增 M,DL 2 000 marker;P1,红花大金元;P2,盘县大柳叶;1~22,F2代单株;R,抗病单株;S,感病单株。

Fig.7 Amplification of Bi-PASA genetic marker in F2 population and parents M, DL 2 000 marker; P1, Honghuadajinyuan; P2, Panxiandaliuye; 1-22, F2 individuals; R, Resistant individual; S, Susceptible individual.

图8 Bi-PASA遗传标记在回交后代部分单株中的扩增 M,DL 2 000 marker;P1,红花大金元;P2,盘县大柳叶;1~22,回交后代单株。

Fig.8 Amplification of Bi-PASA genetic markers in some individuals of backcross progenies M, DL 2 000 marker; P1, Honghuadajinyuan; P2, Panxiandaliuye; 1-22, Backcross population.

图9 转移单隐性基因的显性分子标记辅助选择育种(A)与共显性分子标记辅助选择育种(B)比较示意图

Fig.9 Schematic comparison between dominant molecular marker assisted selection breeding (A) and co-dominant molecular marker assisted selection breeding (B) for transferring single recessive genes

参考文献 21

[1]	唐俊昆, 周志成, 易图永, 等. 烟草马铃薯Y病毒病的发生与综合防治[J]. 现代农业科技, 2011(11): 195-197.
	Tang J K, Zhou Z C, Yi T Y, et al. Occurrence and integrated control of tobacco PVY disease[J]. Modern Agricultural Sciences and Technology, 2011(11): 195-197.
[2]	Koelle G. Genetische analyse einer Y-virus-(rippenbräune) resistenten mutante der tabaksorte virgin A[J]. Der Züchter, 1961, 31(2): 71-72.
[3]	Miller R D. Registration of ‘TN 86’ burley tobacco[J]. Crop Science, 1987, 27(2): cropsci1987.0011183X00270002005-9x.
[4]	Lewis R S. Transfer of resistance to potato virusY (PVY) from Nicotiana africana to Nicotiana tabacum: possible influence of tissue culture on the rate of introgression[J]. Theoretical and Applied Genetics, 2005, 110(4): 678-687.
[5]	Noguchi S, Tajima T, Yamamoto Y, et al. Deletion of a large genomic segment in tobacco varieties that are resistant to potato virus Y (PVY)[J]. Molecular and General Genetics, 1999, 262(4): 822-829.
[6]	Julio E, Denoyes-Rothan B, Verrier J L, et al. Detection of QTLs linked to leaf and smoke properties in nicotianatabacum based on a study of 114 recombinant inbred lines[J]. Molecular Breeding, 2006, 18(1): 69-91.
[7]	王贵, 刘勇, 卢秀萍, 等. 烟草PVY抗性的遗传分析与分子标记筛选[J]. 分子植物育种, 2012, 10(1): 97-103.
	Wang G, Liu Y, Lu X P, et al. Inheritance of resistance to potato virus Y in tobacco and molecular marker indentification[J]. Molecular Plant Breeding, 2012, 10(1): 97-103.
[8]	Julio E, Cotucheau J, Decorps C, et al. A eukaryotic translation initiation factor 4E (eIF4E) is responsible for the “va” tobacco recessive resistance to potyviruses[J]. Plant Molecular Biology Reporter, 2015, 33(3): 609-623.
[9]	Dluge K L, Song Z B, Wang B W, et al. Characterization of Nicotiana tabacum genotypes possessing deletion mutations that affect potyvirus resistance and the production of trichome exudates[J]. BMC Genomics, 2018, 19(1): 484.
[10]	Lin S F, Dewey R E, Wang R G, et al. Discovery of a novel eIF4E1.S allele conferring PVY resistance in Chinese tobacco (Nicotiana tabacum) landraces[J]. Plant Breeding, 2021, 140(4): 693-702.
[11]	林世锋, 王仁刚, 潘飞, 等. 双向等位基因特异性PCR技术在烟草SNP分型中的应用[J]. 中国烟草科学, 2022, 43(1): 6-13.
	Lin S F, Wang R G, Pan F, et al. Application of bi-directional PCR amplification of specific alleles in single nucleotide polymorphism genotyping of tobacco[J]. Chinese Tobacco Science, 2022, 43(1): 6-13.
[12]	Lin S F, Dewey R E, Wang R G, et al. Discovery of a naturally-occurring allele of eIF4E1.S in Nicotiana tabacum and development of a co-dominant marker[J]. Euphytica, 2021, 217(7): 140.
[13]	田波, 裴美云. 植物病毒研究方法:上册[M]. 北京: 科学出版社, 1987.
[14]	GB/T 23222—2008 烟草病虫害分级及调查方法[S].
[15]	刘勇, 宋中邦, 童治军, 等. 烟草PVY隐性抗病基因的分子标记及其适用性[J]. 中国烟草学报, 2015, 21(1): 76-81.
	Liu Y, Song Z B, Tong Z J, et al. Molecular marker from recessive gene resistant to potato virus Y of tobacco and its suitability[J]. Acta Tabacaria Sinica, 2015, 21(1): 76-81.
[16]	林世锋, 王仁刚, 任学良, 等. 烟草种质资源抗马铃薯Y病毒病基因型鉴定[J]. 中国烟草学报, 2021, 27(5): 37-44.
	Lin S F, Wang R G, Ren X L, et al. Genotypic identification of Potato virus Y resistance in tobacco germplasm resources[J]. Acta Tabacaria Sinica, 2021, 27(5): 37-44.
[17]	Liu Q, Thorland E C, Heit J A, et al. Overlapping PCR for bidirectional PCR amplification of specific alleles: a rapid one-tube method for simultaneously differentiating homozygotes and heterozygotes[J]. Genome Research, 1997, 7(4): 389-398.
[18]	Nielsen M T, Jones G A, Collins G B. Inheritance pattern for secreting and nonsecreting glandular trichomes in tobacco[J]. Crop Science, 1982, 22(5): cropsci1982.0011183X-002200050038x.
[19]	Nielsen M T, Akers C P, Järlfors U E, et al. Comparative ultrastructural features of secreting and nonsecreting glandular trichomes of two genotypes of Nicotiana tabacum L[J]. Botanical Gazette, 1991, 152(1): 13-22.
[20]	秦瑞英, 殷三, 李娟, 等. 基因组编辑技术在作物育种中的应用及监管现状[J]. 中国农学通报, 2019, 35(6): 96-100.
	Qin R Y, Yin S, Li J, et al. Application and regulatory status of genome editing technology in crop breeding[J]. Chinese Agricultural Science Bulletin, 2019, 35(6): 96-100.
[21]	杨震, 彭选明, 彭伟正. 作物诱变育种研究进展[J]. 激光生物学报, 2016, 25(4): 302-308.
	Yang Z, Peng X M, Peng W Z. Progress of study on crop mutation breeding[J]. Acta Laser Biology Sinica, 2016, 25(4): 302-308.

一个烟草eIF4E1新等位基因的鉴定及共显性分子标记的开发与应用

Identification of a novel eIF4E1 allele in tobacco and the development and application of its codominant molecular marker

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 10

参考文献 21

相关文章 3

编辑推荐

Metrics

本文评价

[1]	程亮. 青海省部分地区马铃薯Y病毒cp基因序列分析[J]. 浙江农业学报, 2019, 31(11): 1888-1895.
[2]	宋西娇, 谢礼, 宣裕吉, 王芳, 严成其, 陈剑平. 复合侵染的马铃薯花叶病病原诊断[J]. 浙江农业学报, 2018, 30(1): 99-105.
[3]	郑滔;陈炯;陈剑平. 杭州郊区菜豆花叶病病原的分子鉴定[J]. , 2002, 14(3): 0-181.