水稻叶片融合基因LF1的图位克隆及功能分析

Map-base cloning and functional analysis of leaf fusion gene LF1 in rice

[目的]克隆并解析水稻叶片融合基因LF1(Leaf Fusion 1)的功能,为阐明水稻叶片发育的分子机制奠定基础。[方法]lf1为水稻品种‘02428’组织培养获得的叶片融合突变体。利用水稻叶片融合突变体lf1与籼稻品种‘N22’构建遗传分离群体,精细定位lf1;结合突变体重测序分析确定候选基因,经基因敲除克隆LF1;分析该基因的表达模式和蛋白的亚细胞定位。[结果]从lf1/N22的F_(2)群体中,挑选10株突变个体和10株正常个体,将lf1定位在第6染色体上InDel6-6和RM30标记之间;进一步利用256株极端个体,经加密标记,最终将lf1精细定位在标记InDel6-6和RM6818之间约3.2 Mb区间内;通过对野生型‘02428’和lf1进行重测序分析,发现‘02428’与lf1在定位区间的9个基因存在差异,其中6个为转座子基因,1个为表达蛋白基因,1个为假定蛋白基因,1个为NAC转录因子基因Os06g0344900。与野生型‘02428’相比,lf1中Os06g0344900第1外显子存在1个33 bp的缺失,导致11个氨基酸的缺失,据此将该基因确定为LF1的候选基因。利用CRISPR/Cas9技术,获得4个不同类型的敲除株系,均表现出突变体的表型。上述结果表明,Os06g0344900即为目的基因LF1。qRT-PCR分析表明,LF1呈现组成型表达,且在幼穗中表达量最高。亚细胞定位显示LF1定位于细胞核中。[结论]水稻突变体lf1的叶片融合性状由6号染色体的Os06g0344900基因突变所致。该基因的克隆及初步功能分析为解析水稻叶片的发育调控机制奠定了基础。

[Objectives]Cloning and analyzing the function of rice leaf fusion gene LF1(Leaf Fusion 1)were intended to lay the foundation for elucidating the molecular mechanism of rice leaf development.[Methods]lf1 was a leaf fusion mutant obtained by tissue culture of rice variety‘02428’.The rice leaf fusion mutant lf1 and indica rice variety‘N22’were used to construct a genetic segregation population,and finely map lf1.The candidate genes of LF1 were determined by resequencing analysis and verified by knocking out experiment,and the expression pattern of LF1 and the protein subcellular location were further analyzed.[Results]lf1 was primarily located in the region between molecular markers InDel6-6 and RM30 on chromosome 6 using 10 mutant individuals and 10 normal individuals selected from lf1/N22 F_(2) population.Further,lf1 was finely mapped in the range of about 3.2 Mb between the molecular markers InDel6-6 and RM6818 by 256 extreme individuals and increasing the density of molecular markers.Compared with wild type‘02428’,nine genes showed sequence differences in lf1 at the fine mapping interval.Among of them,six genes were encoding transposons,one was annotated as expressing protein gene,and another one was a NAC transcription factor gene Os06g0344900.A 33 bp deletion in the first exon of Os06g0344900 resulted in a deletion of 11 amino acids in lf1 compared with the wild type‘02428’.Then,Os06g0344900 was identified as the candidate gene for LF1.Using CRISPR/Cas9 technology,four different type knockout lines of Os06g0344900 were obtained.Similarly with lf1 mutant,all knockout lines displayed leaf fused phenotype.These results indicated that Os06g0344900 was the target gene of LF1.Further,qRT-PCR analysis showed that LF1 were constitutively expressed in various tissues,and relatively higher in the young spikes.The subcellular location showed that LF1 was located in the nucleus.[Conclusions]The leaf fusion phenotype of rice mutant lf1 is caused by the mutation of Os06g0344900 on chromosome 6.The cloning and preliminary functional analysis of this gene lay the foundation for elucidating the molecular regulation mechanism of rice leaf development.