水稻无花粉型核雄性不育突变体whf41的鉴定与基因定位

摘要

[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BC1F1、F1株系和BC1F2、F2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.qRT-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,OsWHF41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 anther cuticle and wax layer act as structural support and protective barriers for the development of pollen sac in rice. Phenotypic analysis and genetic mapping of the related genes can provide genetic resources and lay theoretical basis for further cloning and molecular mechanism analysis. [Method]We identified a no-pollen male sterile mutant whf41 from the mutant progeny of 60Co-γ-treated indica cultivar Zhonghui 8015. Anthers of the wild-type and whf41 mutant at different development stages were fixed for transverse section analysis and mature anthers of which were randomly selected for scanning electron microscopy analysis. BC1F1, F1 lines and BC1F2, F2 populations derived from whf41/ Zhonghui 8015 and whf41/ 02428 were used for genetic analysis and fine-mapping of the mutation site. [Result] Comparing with wild type,the whf41 mutant exhibited thin and transparent milky anther without mature pollen grains. Transverse section analysis showed that the whf41 mutant displayed aborted pollen grains,none detectable exine and swollen tapetal layer without expected PCD (programmed cell death), finally resulting in fragments of tapetal layer and pollen grains in the chamber. Scanning electron microscopy analysis further revealed the mutant had smoother anther wall, fewer lipids both on the inner and outer sides and fragments of degragated pollens. Genetic analysis revealed that whf41 was controlled by a single recessive genic gene, which was fine-mapped to a 45.6-kb region that harbored nine Open Reading Frames(ORFs) on the short arm of chromosome 3 between markers XD-5 and XD-11. Sequence analysis revealed that the whf41 mutant carried a nucleotide substitution and three nucleotide deletion, which led a substitution of one amino acid (D to M)and a deletion of one amino acid (V) in the fourth exon of one Cytochrome P450 family gene, LOC_Os03g0725, which is named as OsWHF41 and probably responsible for the no-pollen male sterility phenotype. Furthermore, Qpcr analysis showed that the expression level of CYP704B2 and seven anther lipid synthesis and transportation related regulators changed significantly in whf41. [Conclusion]Based on all these results, we deduced that the OsWHF41 gene was a new allelic to CYP704B2 gene and this further confirmed the important role of CYP704B2 in anther lipid synthesis and pollen exine formation .