水稻晚开花突变体lft1的鉴定与基因克隆

Identification and map-based gene cloning of a late flowering mutant lft1 in rice( Oryza sativa L.)

[目的]通过对水稻晚开花突变体lft1(late flowering time)的鉴定及基因定位,解析水稻响应光周期调控开花的分子机制。[方法]从‘日本晴’T-DNA插入突变体库中筛选得到一个在长日照下晚开花的突变体lft1。调查了lft1/日本晴F_2分离群体的开花期表型,分析晚开花表型与T-DNA插入之间的相关性。比较日本晴/9311 F_2群体和lft1/9311 F_2群体的开花期分布,选取群体中具有突变效应的极端晚开花单株进行基因定位。比较突变体lft1和野生型‘日本晴’中已知开花期基因的表达水平,分析基因在调控网络中的作用。[结果]相比于野生型‘日本晴’,突变体lft1在长日照条件下晚开花20 d,粒长和千粒质量均显著增加。对lft1/日本晴F_2群体调查发现,晚开花性状由1对隐性基因控制,且与T-DNA插入呈现非共分离。相比于对照日本晴/9311 F_2群体,在lft1/9311 F_2群体中出现了极端晚开花的个体,从中选取了58株极端个体用于基因的初定位。利用‘日本晴’和‘9311’之间的多态性分子标记对极端晚开花个体进行了图位克隆,将基因限定在第9染色体短臂上,分子标记RM219与RM3912之间,物理距离为2.94 Mb的范围内。在开发标记的过程中,发现分子标记SJ9-32能够在‘日本晴’和‘9311’中实现扩增,但是在lft1及其后代的极端个体中均不能有效扩增。通过进一步设计标记,我们最终验证了在lft1中存在DNA片段的缺失,该缺失区段覆盖了SDG724基因。等位性测验进一步证实lft1的晚开花表型是由于SDG724基因的缺失而引起的。q RT-PCR结果表明,SDG724可以调节Os MADS51和RFT1的表达水平。[结论]突变体lft1中由于SDG724基因的缺失,导致Os MADS51和RFT1的表达水平下调,从而引起晚开花性状。通过调控SDG724基因的表达水平来调节水稻开花期,这在生产上将具有重要意义。

[ Objectives ] Identification and genetic mapping of a rice late flowering mutant/ill （late flowering time）helps uncovering the underlying mechanism of photoperiodic regulation of rice flowering. [ Methods] A＇ Nipponbare＇ T-DNA insertion mutant with late flowering time under long-day condition was selected. The relevance between late flowering and T-DNA insertion event was further established by analyzing /fil/Nipponbare F2 population. Two F2 populations, Nipponbare/9311 and /fil/9311 were investigated simultaneously and compared. Individuals with extremely late flowering time in lfil/9311 F2 population were performed on gene mapping. The expression level of flowering time related genes was compared between the wild type Nipponbare and/ill to clarify the gene role in network. [ Results] Compared to wild type Nipponbare,/fil delayed flowering about 20 days under long-day condition, accompanied by increase of grain length and 1 000-grain weight. After investigation in/fil/Nipponbare F2 population, noco-segrega- tion with T-DNA insertion was observed and the late flowering trait was controlled by a pair of recessive gene. Compared with control F2 population,extremely late flowering individuals were found, from which 58 were chosen for initial gene mapping. Genotyping of these individuals was performed by polymorphism markers developed between Nipponbare and 9311. The gene region was restricted on short arm of chromosome 9,flanked by molecular marker RM219 and RM3912, a length of 2.94 Mb region. In the process of further development of markers,we found that one molecular marker SJ9-32 can be detected by Nipponbare and 9311, but not by the /ill and 58 extreme late flowering individuals. A large missing DNA segment covering SDG724 gene did exist. Anallelism test further confirmed that deletion in SDG724 was a causative factor of the late heading in/ill. The qRT-PCR results showed that SDG724 could regulate the expression of OsMADS51 and RFT1. [ Conclusions ] Due to the lack of SDG724 gene, corresponding expression levels of OsMADS51 and RFT1 were down regulated in/filunder long-day condition, therefore a late flowering trait was presented. It is feasible to adjust rice flowering by modulating the expression level of SDG724 gene, and this will work for rice breeding.