水稻无花粉型核雄性不育突变体whf41的鉴定与基因定位

发布时间：2018-04-12 13:04

本文选题：水稻 + whf　；参考：《中国水稻科学》2017年03期

【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。
[Abstract]:[objective] Rice anther cuticle and waxy layer are important structural support and safety barrier for pollen sac development.Phenotypic analysis and genetic localization of genes related to pollen sac development were carried out.[methods] A non-pollen male sterile mutant whf41was isolated and identified from the mutant library of Zhonghui 8015 by radiation mutagenesis in indica rice Zhonghui 8015 and its molecular mechanism.The anthers of wild type and mutants at different developmental stages were observed by semi-thin sections, and the anthers at mature stage were observed by scanning electron microscope (SEM).The mutants were hybridized with Zhonghui 8015 and 02428, respectively, to construct the BCCs-1FS-1 and BC1FS-1 and BC1FSP-2F2 populations, and to carry out genetic analysis of the phenotype.[results] phenotypic analysis showed that the anthers of whf41 mutant were small and clear and milky and did not contain pollen granulocytes in anthers.The microspore of the mutant could not form the normal pollen outer wall and the tapetum cells expanded abnormally without programmed death. Finally the expanded tapetum and pollen cell fragments gradually fused and filled with medicine chamber.The results of scanning electron microscope further showed that the inner and outer walls of the anther of the mutant were smooth and the pollen cells were gradually broken and degraded.Genetic analysis showed that the non-pollen male sterility traits of the whf41 mutant were controlled by a pair of recessive nuclear genes. We mapped the gene between the two markers of short arm XD-5 and XD-11 on chromosome 3.Physical distance 45.6 kb, which contains 9 open reading boxes.Sequence analysis showed that there was a single base substitution and three base deletion at exon 4 of cytochrome P450 gene LOC_Os03g07250 in this region.Resulting in the deletion of one amino acid replacement (aspartic acid methionine) and one amino acid (valine) in its translation sequence.The results showed that the expression of CYP704B2 and a series of genes related to anther lipid synthesis and transport were significantly down-regulated. [conclusion] according to the results of this study, the expression level of CYP704B2 and a series of genes related to anther lipid synthesis and transport were significantly down-regulated.It can be inferred that WHF41 is a new allele of CYP704B2.The results showed that CYP704B2 plays an important role in anther lipid synthesis and pollen wall formation in rice.
【作者单位】：中国水稻研究所/水稻生物学国家重点实验室/浙江省超级稻研究重点实验室;
【基金】：国家公益性农业科技研究专项(20140302,20150308) 国家转基因重大专项(2014ZX08001-002) 中国农业科学院创新工程超级稻育种创新团队、水稻杂种优势研究机理研究创新团队资助项目(CAAS-ASTIP-2013-CNRRI)
【分类号】：S511

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