小麦<i>TaSOD-B1</i>基因克隆及KASP标记开发与验证

doi:10.7668/hbnxb.20195519

摘要/Abstract

摘要：

克隆小麦籽粒超氧化物歧化酶(SOD)基因,开发与SOD活性相关的竞争性等位基因特异性PCR(KASP)标记,对选育高SOD活性小麦品种具有重要意义。根据基因ID,设计特异性引物克隆得到TaSOD-B1基因gDNA序列;在小麦基因组遗传变异及Ensembl Plants数据库比对得到单核苷酸多态性(SNP)位点,开发与小麦SOD活性密切相关的KASP标记,并通过287份来自国内外冬小麦品种(系)SOD活性与基因型间的相关性分析进行标记的实用性验证。利用6对特异性引物扩增中国春品种TaSOD-B1基因片段,拼接得到5B染色体上的TaSOD-B1基因,基因全长为6 491 bp,包含1 650 bp的开放阅读框(ORF),共编码549个氨基酸,预测分子质量为60.80 ku,由12个外显子及11个内含子构成,内含子符合典型的GT-AG结构。基于TaSOD-B1基因第1外显子的第44碱基处经测序验证确实存在的差异位点开发KASP标记,检测结果表明,等位变异类型为TaSOD-B1a的基因型为AA,与高SOD活性相关,用荧光基因FAM(显示为蓝色)标记;等位变异类型为TaSOD-B1b的基因型为GG,与低SOD活性相关,用荧光基因HEX标记(显示为红色)。对287份国内外冬小麦品种(系)进行检测发现,不同基因型的SOD活性差异达到显著水平。基于TaSOD-B1序列成功开发出1组与SOD活性相关,并可用于SOD活性遗传改良的KASP标记。

关键词: 普通小麦, 超氧化物歧化酶, 基因克隆, KASP标记

Abstract:

Cloning the wheat grain superoxide dismutase(SOD)gene and developing competitive allele-specific PCR(KASP)markers related to SOD activity are of great significance for breeding wheat varieties with high SOD activity.According to the gene ID,specific primers were designed to clone the gDNA sequence of TaSOD-B1 gene.The single nucleotide polymorphism(SNP)loci were obtained by comparing the wheat genome genetic variation and Ensembl Plants database,and the KASP markers closely related to the SOD activity of wheat were developed.The practicability of the markers was verified by the correlation analysis between SOD activity and genotypes of 287 winter wheat varieties(lines).The TaSOD-B1 gene fragment of Chinese spring variety was amplified by six pairs of specific primers,and the TaSOD-B1 gene on chromosome 5B was obtained by splicing.The full length of the gene was 6 491 bp,including an open reading frame(ORF)of 1 650 bp,which encoded a total of 549 amino acids.The predicted molecular weight was 60.80 ku,the gene was composed of 12 exons and 11 introns.The intron conformed to the typical GT-AG structure.The KASP marker was developed based on the 44th base of the first exon of the TaSOD-B1 gene,and was verified by sequencing.The results showed that the genotype of the allelic variation type TaSOD-B1a was AA,which was associated with high SOD activity,and was labeled with the fluorescent gene FAM(shown as blue).The genotype of TaSOD-B1b was GG,which was associated with low SOD activity and was marked with the fluorescent gene HEX(shown as red).The detection of 287 winter wheat varieties(lines)at home and abroad showed that the SOD activity of different genotypes was significantly different.Based on the TaSOD-B1 sequence,a set of KASP markers related to SOD activity was successfully developed and could be used for genetic improvement of SOD activity.

Key words: Common wheat, Superoxide dismutase, Gene cloning, KASP marker

中图分类号:

S336生物技术育种方法

侯培珂, 程宇坤, 王继庆, 孙玲, 王建鹏, 耿洪伟. 小麦TaSOD-B1基因克隆及KASP标记开发与验证[J]. 华北农学报, 2025, 40(3): 1-8. doi: 10.7668/hbnxb.20195519.

HOU Peike, CHENG Yukun, WANG Jiqing, SUN Ling, WANG Jianpeng, GENG Hongwei. Cloning of Wheat TaSOD-B1 Gene and Development and Verification of KASP Marker[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 1-8. doi: 10.7668/hbnxb.20195519.

http://www.hbnxb.net/CN/Y2025/V40/I3/1

图/表 5

表1 克隆TaSOD-B1基因引物序列

Tab.1 Primer sequence for cloning TaSOD-B1

引物名称 Primer	引物序列(5'—3') Sequence of primer (5'—3')	PCR产物/bp PCR product	退火温度/℃ Tm
5B1	F: AAAGGCCGCATGTTGAGAGA	1 121	58
	R: GGGATGACAGAGTTCGGTT
5B2	F: ACGCGTCTATATACATCCGATT	1 231	61
	R: CTTTAGAGATTGTAGAAAACTT
5B3	F: GCCATTATCGTGCCTGTG	932	61
	R: TGTCCTCGCAAAATGTCG
5B4	F: AGACCCTGAAACTCGCTGA	1 001	58
	R: CGACTAATGGCAGTTGCGAA
5B5	F: AAAAAGGTTCATGCCGATAAAGTTC	2 123	58
	R: GCCATTATCGTGCCTGTG
5B6	F: CCACAGACAACACAGGACCTT	948	58
	R: CGACCTTGCATTTTTGCCCTT

图1 中国春TaSOD-B1基因扩增结果 A:M.Trans 2k DNA Marker,1.引物5B1,2.引物5B2,3.引物5B3,4.引物5B4,5.引物5B6;B:M.Trans 5k DNA Marker,1.引物5B5。

Fig.1 Amplification results of TaSOD-B1 gene in Chinese Spring A:M.Trans 2k DNA Marker,1.Primer 5B1,2.Primer 5B2,3.Primer 5B3, 4.Primer 5B4,5.Primer 5B6;B:M.Trans 5k DNA Marker,1.Primer 5B5.

图2 287份国内外小麦材料TaSOD-B1标记的开发与验证 A.TaSOD-B1标记对287份国内外小麦材料基因分型图;B.8份材料TaSOD-B1位点测序结果。

Fig.2 Development and verification of TaSOD-B1 markers in 287 domestic and foreign wheat materials A.TaSOD-B1 marker genotyping map of 287 domestic and foreign wheat materials;B.TaSOD-B1 locus sequencing results of 8 materials.

表2 TaSOD-B1a、TaSOD-B1b等位变异在287份国内外小麦品种(系)的检测结果

Tab.2 Detection results of allelic variation of TaSOD-B1a and TaSOD-B1b in 287 domestic and foreign wheat varieties(lines)

环境 Environment	等位变异 Genotype	数量/份 Number	SOD活性均值/(U/g) Mean SOD activity	标准差/(U/g) Standard deviation	变化范围/(U/g) Range
Ⅰ	TaSOD-B1a	13	1 816.56a	54.82	1 729.22~1 930.84
	TaSOD-B1b	35	1 788.87a	46.70	1 667.57~1 988.86
Ⅱ	TaSOD-B1a	15	1 826.63a	82.69	1 548.95~1 890.39
	TaSOD-B1b	5	1 786.30a	42.88	1 721.47~1 837.62
Ⅲ	TaSOD-B1a	21	1 777.94a	39.04	1 711.34~1 847.39
	TaSOD-B1b	95	1 769.88a	58.10	1 558.24~1 937.09
Ⅳ	TaSOD-B1a	4	1 854.92a	63.20	1 793.81~1 937.56
	TaSOD-B1b	34	1 749.11a	112.61	1 354.13~1 899.57
Ⅴ	TaSOD-B1a	4	1 766.94a	109.56	1 655.08~1 907.58
	TaSOD-B1b	61	1 790.28a	76.52	1 478.07~1 923.75
全部品种	TaSOD-B1a	57	1 804.19a	66.83	1 548.95~1 937.56
All varieties	TaSOD-B1b	230	1 776.96b	74.03	1 354.13~1 988.86

图3 TaSOD-B1a、TaSOD-B1b等位变异在287份国内外小麦品种(系)的分布频率 Ⅰ.北部冬麦区;Ⅱ.西南冬麦区;Ⅲ.黄淮冬麦区;Ⅳ.长江中下游冬麦区;Ⅴ.国外品种。

Fig.3 Distribution frequency of TaSOD-B1a and TaSOD-B1b alleles in 287 domestic and foreign wheat varieties(lines) Ⅰ. Northern China Winter Wheat Region;Ⅱ.Southwest China Winter Wheat Region; Ⅲ. Yellow and Huai River Valley Winter Wheat Region; Ⅳ. In the middle and lower reaches of Yangtze River Winter Wheat Region; V.Foreign varieties.

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小麦TaSOD-B1基因克隆及KASP标记开发与验证

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