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在60 Co-γ射线辐射诱变籼稻中恢8015的突变体库内发现了一个无花粉型雄性不育突变体gamyb5。gamyb5的株型、株高、分蘖数等农艺性状与野生型中恢8015无差异,而其花药细长且呈白色半透明状,花药中无花粉粒。花药半薄切片观察结果表明,gamyb5的小孢子母细胞减数分裂异常,没有形成正常的四分体和小孢子,并且绒毡层异常伸长,细胞程序性死亡延迟。对以gamyb5为母本,与野生型中恢8015和广亲和粳稻品种02428分别配制的杂交组合遗传分析表明,gamyb5突变性状受一个隐性核基因控制。利用gamyb5和02428杂交的F2定位群体,最终将突变基因精细定位于第1染色体长臂的ZF-29和ZF-31两个标记之间,物理距离约16.9kb。对该区域内2个完整的开放阅读框测序分析发现,编码受赤霉素诱导的MYB转录因子基因LOC_Os01g0812000的第2外显子存在8个碱基的缺失,导致翻译提前终止。qRT-PCR检测到影响花药发育的调控因子UDT1、TDR、CYP703A3和CYP704B2的表达量在突变体中比野生型中极显著降低。进一步证明GAMYB在花药减数分裂和绒毡层细胞程序性死亡过程中起关键作用。
A non-pollen-type male sterility mutant, gamyb5, was found in a mutant pool of 8015 in indica rice 60 Co-γ-ray radiation. The agronomic traits of gamyb5, such as plant type, plant height and tiller number, were similar to those of the wild type, but the anthers were slender and white translucent with no pollen grains. Semi-thin sections of anthers showed that the microspore mother cells of gamyb5 were abnormally meiotic, did not form normal tetrad and microspores, and the tapetal layer was abnormally elongated and programmed cell death was delayed. Genetic analysis of a cross between gamyb5 and wild type Zhonghui 8015 and a wide compatibility japonica variety 02428 showed that the gamyb5 mutation was controlled by a recessive nuclear gene. Using the F2 locus of hybrids between gamyb5 and 02428, the mutant gene was finally located between the two markers of ZF-29 and ZF-31 on the long arm of chromosome 1 with a distance of about 16.9 kb. Two complete open reading frames (ORFs) in this region were sequenced and found that there was an 8-bp deletion in exon 2 of MY_3 transcription factor gene LOC_Os01g0812000 induced by gibberellin, leading to early termination of translation. The results of qRT-PCR showed that the expression levels of UDT1, TDR, CYP703A3 and CYP704B2, which regulate the development of anther, were significantly lower in mutant than wild type. It further proves that GAMYB plays a key role in the process of anther meiosis and tapetal cell apoptosis.