水稻分蘖调控基因HTD3的克隆与功能研究

Cloning and Functional Analysis of Rice Tillering Regulatory Gene HTD3

【目的】克隆水稻分蘖相关基因,为构建理想株型水稻,提高粮食产量提供理论基础及有利基因资源。【方法】在常规大田种植条件下分别比较突变体htd3(high-tillering dwarf 3)与野生型在幼苗期、抽穗期和成熟期表型及主要农艺性状差异,利用图位克隆方法克隆候选基因,利用荧光定量PCR方法分析HTD3及独脚金内酯和脱落酸相关基因的表达水平,测序比对分析HTD3在147份种质资源中的自然变异情况。【结果】与野生型相比,突变体htd3的分蘖芽生长较快,分蘖数和有效穗数显著增多,株高、一次枝梗数和每穗粒数显著降低,结实率和千粒重没有显著变化。遗传分析表明,htd3多分蘖的性状受一对隐性核基因控制。图位克隆将HTD3基因定位在第12染色体CM8和CM10之间约63.5 kb的物理区间内,互补转基因实验证明该区间内LOCOs12g21710为控制突变体多分蘖表型的基因。HTD3在野生型和突变体中呈组成型表达,该基因突变会引起部分独脚金内酯和脱落酸相关基因的表达水平上调。水稻品种中HTD3编码区G2674A自然变异使得分蘖数显著增多。【结论】HTD3是最近报道的T20/MIT1基因的一个新的等位基因,HTD3突变导致水稻出现分蘖适度增加,株高略矮的表型,在培育理想株型水稻和高产育种上具有较大的应用潜力。

【Objective】Cloning of rice tiller-related genes lays a theoretical basis and provides beneficial genetic resources for constructing ideal plant-type rice and increasing grain yield.【Method】We compared phenotypes and main agronomic traits between the mutant htd3(high-tillering dwarf 3) and its wild type under conventional field planting conditions,used map-based cloning method to clone candidate genes, analyzed the expression levels of HTD3, SL and ABA-related genes by fluorescence quantitative real-time PCR. Sequencing comparison was performed to analyze the natural variation of HTD3 in 147 germplasm resources.【Result】Compared with the wild type, the axillay bud of the mutant htd3 grow faster, the number of tillers and effective panicles were significantly increased, the plant height, the number of primary rachis branches and grains per panicle were significantly decreased, and the seed setting rate and 1000-grain weight were not significantly changed. Genetic analysis indicated that the high-tillering trait of htd3 was controlled by single recessive nuclear gene, which was mapped to a 63.5 kb region between the marker CM8 and CM10 on chromosome 12, and the complementary transgenic experiment proved that LOCOs12 g21710 was the gene controlling the high-tillering phenotype. HTD3 is constitutively expressed in wild-type and mutant, and the mutation of this gene could up-regulate the expression level of some stratolactones and ABA-related genes. The natural variation of G2674 A in the HTD3 coding region in rice varieties significantly increase the number of tillers.【Conclusion】HTD3 is a new allele of the recently reported T20/MIT1 gene. The HTD3 mutation leads to the phenotype of moderate increase in tillers and slightly shorter plant height in rice, which has great application potential in cultivating ideal plant type rice and high-yield breeding.