控制水稻光响应基因ELM1的图位克隆

doi:10.13560/j.cnki.biotech.bull.1985.2024-1246

摘要/Abstract

摘要：

目的探究水稻开花期的调控基因与图位克隆，阐明其开花遗传与分子机制，完善水稻抽穗期基因的调控网络，为水稻生产育种提供实践意义。方法以水稻长日照条件下延迟开花突变体elm1为材料，统计开花期等农艺性状，通过正反交实验构建群体，统计F₂群体表型进行遗传规律分析，与籼稻Dular构建图位克隆群体并进行基因定位，并对精细定位区间内候选基因进行测序，结合生物信息学等手段对候选基因进行预测与分析，使用AlphaFold2软件预测蛋白结构的变化。结果 elm1突变体在长日照条件下开花时间显著增加，其受一对单隐性核基因控制，图位克隆到ELM1基因，ELM1基因编码SET结构域组蛋白甲基转移酶，等位于已报道的lvp1突变体，测序结果显示在elm1突变体中LOC_Os09g13740基因的第5外显子（ATG下游3 293 bp处）发生点突变（G变为T），导致该突变位点由甘氨酸（亲水）突变为缬氨酸（疏水），AlphaFold2蛋白结构预测表明该突变导致蛋白构象发生改变，对蛋白功能可能有一定的影响。田间试验表明，在合适纬度的区域种植elm1突变体，突变体表现穗粒数明显增多且增产的表型。结论 elm1突变体的突变位点为一弱等位突变，该等位突变体在适宜的纬度区域下育种可提升水稻产量，研究证明突变体elm1是一份优异的等位变异材料。

关键词: 水稻, 开花期, elm1突变体, 图位克隆, 弱突变, 蛋白结构

Abstract:

Objective Exploring the regulatory genes of rice flowering date and conducting positional cloning research, the genetic and molecular mechanisms of rice flowering can be clarified, the regulatory network of rice heading date genes can be improved, and certain theoretical and practical significance can be provided for rice production and breeding. Method Using the late-flowering mutant elm1 under long-day conditions in rice as the material, we analyzed agronomic traits such as flowering time. Then we constructed a population through reciprocal crosses and analyzed the genetic patterns by phenotyping the F₂ population. Further we also developed a mapping population with the indica rice Dular for positional cloning and gene localization. Finally, we sequenced the candidate genes within the fine-mapped interval, predicted and analyzed the candidate genes using bioinformatics tools, and predicted the changes in protein structure using AlphaFold2 software. Result The elm1 mutant demonstrated a significantly prolonged flowering time under long-day conditions, which was controlled by a single recessive nuclear gene. The ELM1 gene through positional cloning was cloned. The ELM1 gene encoded a SET-domain histone methyltransferase and was allelic to the previously reported lvp1 mutant. Sequencing results showed that a point mutation (G to T) in the elm1 mutant occurred in the 5th exon of the LOC_Os09g13740 gene (3 293 bp downstream of the ATG start codon), resulting in a change from glycine (hydrophilic) to valine (hydrophobic) at this site. Protein structure prediction using AlphaFold2 indicated that this mutation caused a conformational change in the protein, which may affect its function. Field experiments showed that it showed a phenotype with significantly increased number of grains per panicle and higher yield when the elm1 mutant was grown in suitable latitudinal regions. Conclusion The mutation site in the elm1 mutant is a weak allele. Breeding this allelic mutant in suitable latitudinal regions may enhance rice yield. Further studies have demonstrated that the elm1 mutant is an excellent allelic variation material.

Key words: rice, heading date, elm1 mutant, map-based cloning, weak mutation, protein structure

杜量衡, 唐黄磊, 张治国. 控制水稻光响应基因ELM1的图位克隆[J]. 生物技术通报, 2025, 41(5): 82-89.

DU Liang-heng, TANG Huang-lei, ZHANG Zhi-guo. Map-based Cloning of Light-responsive Gene ELM1 in Rice[J]. Biotechnology Bulletin, 2025, 41(5): 82-89.

图/表 7

图1 野生型与突变体elm1的开花期表型分析A：野生型（WT）与突变体elm1在北京试验田的开花期植株比较（Bar=20 cm）；B：野生型（WT）与突变体elm1的在海南三亚试验田开花期植株比较（Bar=20 cm）；C：野生型（WT）与突变体elm1在北京试验田的花期比较（长日照），***表示P<0.001显著差异；D：野生型（WT）与突变体elm1在海南三亚试验田的花期比较（短日照），ns表示无差异

Fig. 1 Phenotypic of the flowering period between WT and elm1A: Comparison of wild type (WT) and elm1 mutant at the flowering stage in the experimental field in Beijing (Bar=20 cm). B: Comparison of WT and elm1 mutant at the flowering stage in Sanya, Hainan (Bar=20 cm). C: Comparison of heading date between WT and elm1 mutant in the experimental field in Beijing (long-day), *** indicates a significant difference at P<0.001. D: Comparison of heading date between WT and elm1 mutant in the experimental field in Sanya, Hainan (short-day), ns stands for not significant

表1 野生型与elm1突变体的农艺性状比较（海南）

Table 1 Comparison of agronomic traits between WT and elm1 mutant (Hainan)

性状类别

Trait category

旗叶叶长

Flag leaf length （cm）

旗叶叶宽

Flag leaf width （cm）

株高

Plant height （cm）

分蘖数

Tiller number

千粒重

Thousand-grain weight （g）

每穗粒数

Grain number per panicle

elm1

表2 F2代分离群体统计结果

Table 2 Statistical results of F2 generation segregation population

统计类别 Statistical category	日本晴× elm1 Nipponbare×elm1	elm1×日本晴 elm1×Nipponbare
植株总数 Total number of plants	396	374
正常植株数 Number of normal plants	288	276
不开花植株数 Number of non-flowering plants	108	98
分离比 Segregation ratio	3∶1	1∶3
χ²	0.066 7	0.051 2

图2 突变体elm1基因连锁分析部分电泳图A：Indel-3标记在F2代单株的连锁分析（1‒32：32个F2代突变体表型单株的电泳条带结果）；B：Indel-5标记在F2代单株的连锁分析（1‒37：37个F2代突变体表型单株的电泳条带结果）；Nip：日本晴；Du：Dular

Fig. 2 Gel electrophoresis of gene linkage analysis for the mutant elm1A: Linkage analysis of the Indel-3 marker in individual F2 plants (1‒32: Electrophoresis banding results of 32 individual plants). B: Linkage analysis of the Indel-5 marker in individual F2 plants (1‒37: Electrophoresis banding results of 37 individual plants). Nip: Nipponbare. Du: Dular

图3 基因ELM1图位克隆A：突变体elm1的基因初定位，每个标记之间的距离以重组频率（以cM为单位）表示；B：突变体elm1的基因精细定位；C：候选区域；D：突变体elm1突变发生LOC_Os09g13740基因的第5外显子，其中lvp1-1突变发生在第5外显子79 bp碱基的缺失，第8外显子与第9外显子之间插入115 bp碱基的突变

Fig. 3 Map-based cloning of gene ELM1A: Preliminary gene localization of elm1 mutant. The distance between each marker is represented by the recombination frequency, measured in centimorgans (cM). B: The fine mapping of the gene in the mutant elm1. C: Candidate region. D: The mutation in mutant elm1 occurs in the fifth exon of the LOC_Os09g13740, where the lvp1-1 mutation was a deletion of 79 base pairs in the fifth exon, and a mutation involving the insertion of 115 base pairs between the eighth and ninth exons

图4 野生型与突变体蛋白ELM1的结构预测A：野生型的ELM1蛋白结构预测；B：突变后ELM1的蛋白结构预测

Fig. 4 Prediction of the ELM1 protein structure in WT and mutantA: Prediction of the ELM1 protein structure in the wild type. B: Prediction of the ELM1 protein structure after mutation

图5 野生型与突变体elm1的农艺性状比较A：野生型与elm1突变体的穗型比较（Bar=5 cm）；B：野生型与elm1突变体的每穗粒数比较，**表示P<0.001显著差异；C：野生型与elm1突变体的株高比较；D：野生型与elm1突变体的千粒重比较；E：野生型与elm1突变体的结实率比较；F：野生型与elm1突变体的分蘖数比较

Fig. 5 Agronomic traits comparison between wild type and elm1 mutantA: Panicle comparison between WT and elm1 mutant (Bar=5 cm). B: Comparison of grain number per panicle between WT and elm1 mutant. ** indicates a significant difference at P<0.001. C: Comparison of plant height between WT and elm1 mutant. D: Comparison of thousand-grain weight between WT and elm1 mutant. E: Comparison of seed setting rate between WT and elm1 mutant. F: Comparison of tiller number between WT and elm1 mutant

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