ALS抑制剂类除草剂抗性水稻功能标记的开发与验证

doi:10.16819/j.1001-7216.2018.7091

摘要/Abstract

摘要：

【目的】培育具有乙酰乳酸合酶(ALS)抑制剂类除草剂抗性的品种是防治水稻直播田杂草危害最经济、有效的途径之一,而利用分子标记辅助选择有助于提高其品种选择效率。【方法】在明确除草剂抗性突变体黄华占M-1 ALS基因编码区碱基差异的基础上,利用四引物扩增受阻突变体系PCR(Tetra-primer ARMS-PCR)技术,设计引物对不同品种(品系)和淮稻5号/黄华占M-1的F₂群体进行基因型检测,并结合除草剂的田间试验对标记的准确性进行验证。【结果】ALS基因编码区序列比对分析表明,尽管籼、粳稻之间具有多处差异碱基,但只有第1642和1643碱基TG到AT的变异能导致位于高度保守域的第548位编码氨基酸由色氨酸突变为甲硫氨酸,进而使水稻产生对ALS抑制剂类除草剂的抗性。依据PCR扩增产物的电泳带型,可以准确区分出3种不同的基因型,其基因型与苗期除草剂抗性的表型完全一致。【结论】利用Tetra-primer ARMS-PCR技术,可以实现对两个连续变异碱基位点基因型的快速检测,从而加快ALS抑制剂类除草剂抗性水稻品种的选育进程。

关键词: 乙酰乳酸合酶基因, 除草剂抗性, 碱基突变, 四引物扩增受阻突变体系PCR

Abstract:

【Objective】Breedingrice varieties with resistance to ALS-inhibiting herbicide is the mosteconomical and effective method to preventweeds in direct-sowing field, and molecular marker-assisted selection canimprove its efficiency. 【Method】The dominant molecular marker, designed for genotypic detection in different varieties and an F₂ population derived fromHuaidao5/Huanghuazhan M-1(mutant with resistance to ALS-inhibiting herbicide) was developed with Tetra-primer ARMS-PCR technique, based on the clarified mutation in CDS of ALS gene. 【Result】 The sequence alignment analysis of ALS gene encoding regionshowed that only the mutation from T₁₆₄₂G₁₆₄₃ to A₁₆₄₂T₁₆₄₃ leading to the 548th amino acid substitutionfrom tryptophan to methioninein highly conserved regionwas the critical factors to produce resistance to ALS-inhibiting herbicide inHuanghuazhan M-1, despitethe existence of multiplebase differencesbetween different indica and japonica rice varieties.The results of PCR detection indicated that three different genotypescould be accurately distinguished by electrophoretic bands, and it was exactly consistent with the phenotype of herbicide resistance at the seedling stage. 【Conclusion】 The genotype associated with two continuous base mutations could be rapidly detected by Tetra-primer ARMS-PCRtechnique, and the efficiency for varieties with resistance to ALS-inhibiting herbicidecould be improved in breeding process.

Key words: acetolactate synthase, herbicide resistance, basemutation, tetra-primerARMS-PCR

中图分类号:

陈涛, 张善磊, 赵凌, 张亚东, 朱镇, 赵庆勇, 周丽慧, 姚姝, 赵春芳, 梁文化, 王才林. ALS抑制剂类除草剂抗性水稻功能标记的开发与验证[J]. 中国水稻科学, 2018, 1(1): 137-145.

Tao CHEN, Shanlei ZHANG, Ling ZHAO, Yadong ZHANG, Zhen ZHU, Qingyong ZHAO, Lihui ZHOU, Shu YAO, Chunfang ZHAO, Wenhua LIANG, Cailin WANG. Development and Verification of a Functional Marker Associated with Resistance to ALS Inhibitor Herbicide[J]. Chinese Journal OF Rice Science, 2018, 1(1): 137-145.

图/表 6

表1 四引物扩增受阻突变体系PCR技术检测水稻ALS基因碱基变异的引物序列

Table 1 Sequences of primer to detect the base variation by tetra-primer ARMS-PCR system in rice.

引物名称 Primer	引物序列(5′-3′) Sequence (5′-3′)
ALS-O-F	AGGTGAGGCAATCATCGCTAC
ALS-O-R	ATGGGTCTATTCAGGTCAAACA
ALS-I-F	AACCAACATTTGGGTATGGTTGTGCTAA
ALS-I-R	CTATTTGCCTTGTAAAACCTATCCTTCC

图1 用于检测水稻ALS基因连续双碱基变异的Tetra-primer ARMS-PCR体系的设计策略序列中黑色碱基表示外显子, 蓝色碱基表示基因的非编码区序列, 红色碱基表示引物序列, 阴影表示差异碱基, 阴影斜体表示产生ALS抑制剂类除草剂抗性的关键碱基。

Fig. 1. Strategy for Tetra-primer ARMS-PCR system to detect the two continuous base mutationsfor ALS gene in rice. The exon,non-coding regionsand primers are showed in black, blue and red bases, respectively. The differential bases in sequences are in shadow and the key bases involving in the resistance to ALS inhibitor herbicides are in italic and shadow.

图2 Tetra-primer ARMS-PCR对不同水稻品种(品系)ALS基因型的检测 M–DNA分子量标准(DL2000);1–黄华占M-1;2–黄华占;3–南粳2728;4–淮稻5号;5–镇稻99。

Fig. 2. Electrophoresis detection of ALS genotypes by Tetra-primer ARMS-PCR in different rice varieties or lines. M, DNA marker (DL2000); 1, Mutant of Huanghuazhan M-1; 2, Huanghuazhan; 3, Nanjing 2728; 4, Huaidao5; 5, Zhendao 99.

图3 不同品种(品系)对ALS抑制剂类除草剂咪唑乙烟酸的抗性测试 A―黄华占M-1;B―南粳2728。

Fig.3. Resistance test of different varieties or lines to ALS inhibitor herbicideimazethapyr. A, Huanghuazhan M-1; B, Nanjing 2728.

图4 Tetra-primer ARMS-PCR对淮稻5号/黄华占M-1 F2群体部分单株ALS基因型的电泳检测 M–DNA分子量标准(DL2000);1―黄华占M-1;2―淮稻5号;3―淮稻5号/黄华占M-1 F1;4~17―部分F2分离单株。

Fig. 4. Electrophoresis detection of ALS genotype by Tetra-primer ARMS-PCR in F2 population derived from Huaidao 5/Huanghuazhan M-1. M,DNA Marker (DL2000); 1, Huanghuazhan M-1; 2, Huaidao5; 3, F1 (Huaidao5/ Huanghuazhan M-1); 4-17, Parts of plants from F2 population.

图5 淮稻5号/黄华占M-1 F2群体对ALS抑制剂类除草剂咪唑乙烟酸的抗性测试 A–不含ALS基因突变纯合基因型单株;B–含ALS基因突变纯合基因型植株;C–含ALS基因突变杂合基因型单株。

Fig.5. Resistance test of F2 population derived from Huaidao5/Huanghuazhan M-1 to ALS inhibitor herbicideimazethapyr. A, Plants without homozygous genotype containing mutant ALS gene; B, Plants with homozygous genotype containing mutant ALS gene; C, Plants with heterozygous genotype containing mutant ALS gene.

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