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【目的】水稻花药角质层和蜡质层是花粉囊发育重要的结构支撑和安全屏障。对花粉囊发育相关基因进行表型分析和遗传定位,为进一步克隆相关基因和分子机制研究提供基因资源和理论基础。【方法】从籼稻中恢8015辐射诱变(~60Co-γ)突变体库中分离鉴定出了一个无花粉型雄性不育突变体whf41。对野生型和突变体不同发育时期的花药进行半薄切片观察;并对其成熟期的花药进行扫描电镜观察。将突变体分别与中恢8015和02428杂交,构建BC_1F_1、F_1株系和BC_1F_2、F_2群体,对该表型进行遗传分析,采用图位克隆的方法精细定位目的基因。【结果】表型分析结果显示,whf41突变体的花药瘦小且呈透明乳白色,花药中不包含花粉粒细胞;半薄切片结果显示,突变体的小孢子无法形成正常的花粉外壁,绒毡层细胞异常膨大而不进行程序性死亡,最终膨胀的绒毡层和花粉细胞碎片逐渐融合并充满药室;扫描电镜结果进一步发现突变体花药内外壁均呈平滑状而缺乏脂类物质,花粉细胞逐渐破碎并降解。遗传分析表明,whf41突变体的无花粉型雄性不育性状受一对隐性核基因控制,我们将该基因精细定位于第3染色体短臂XD-5和XD-11两个标记之间,物理距离45.6 kb,其中包含9个开放阅读框。序列分析显示该区间内细胞色素P450基因LOC_Os03g07250的第4个外显子处存在1个单碱基替换和3个碱基的缺失,导致其翻译序列发生一个氨基酸的替换(天冬氨酸→甲硫氨酸)和一个氨基酸(缬氨酸)的缺失,致使其功能改变进而出现该表型。q RT-PCR检测结果表明,whf41突变体中CYP704B2和一系列花药脂质合成与转运相关基因的表达水平均发生了显著下调。【结论】根据本研究结果可推断,Os WHF41是CYP704B2的新等位基因,相关结果进一步明确CYP704B2在水稻花药脂质合成与花粉壁形成过程中的重要作用。
【Objective】 The stratum corneum and waxy layer of rice anther are important structural supports and safety barriers for the development of pollen sacs. The phenotypic analysis and genetic mapping of the genes involved in the development of the pollen sacs provided genetic resources and theoretical basis for the further cloning of related genes and molecular mechanisms. 【Method】 A non-pollen-type male sterility mutant, whf41, was isolated and identified from a mutant library of indica rice variety Zhonghui 8015 (~ 60Co-γ). The anthers of wild-type and mutant at different developmental stages were observed by semi-thin sections. The anthers of the mature anthers were observed by scanning electron microscopy. The mutants were crossed with Zhonghui 8015 and 02428 respectively to construct BC_1F_1 and F_1 lines and BC_1F_2 and F_2 populations. The phenotypes were analyzed by genetic analysis, and the target genes were finely mapped by the map-based cloning method. 【Result】 The results of phenotypic analysis showed that the anther of whf41 mutant was small and transparent and milky white, and the anther contained no pollen granulocytes. The results of semi-thin section showed that the microspores of mutants could not form normal pollen exine wall and tapetum cells Abnormal expansion without programmed death, the final expansion of the tapetum and pollen cell fragments gradually fusion and filled the chamber; scanning electron microscopy results showed that the mutant anther inner and outer walls were smooth and lack of lipid material, pollen cells gradually broken And degradation. Genetic analysis showed that the pollen-type male sterility of whf41 mutant was controlled by a pair of recessive nuclear genes. We mapped this gene between the two markers of XD-5 and XD-11 on chromosome 3, 45.6 kb, contains 9 open reading frames. Sequence analysis showed that there was a single base substitution and a 3 base deletion in the fourth exon of the cytochrome P450 gene LOC_Os03g07250 in this interval, resulting in an amino acid substitution in its translated sequence (aspartate → A Alanine), and one amino acid (valine), resulting in a change in function that leads to the phenotype. q RT-PCR results showed that both the expression levels of CYP704B2 and a series of anther lipid synthesis and transport related genes in whf41 mutant were significantly down-regulated. 【Conclusion】 According to the results of this study, Os WHF41 is a new allele of CYP704B2. The results further clarify the important role of CYP704B2 in lipid synthesis and pollen wall formation in rice.