【作者】 李文强；

【导师】 张改生；

【作者基本信息】 西北农林科技大学 ， 遗传学， 2007， 硕士

【摘要】 细胞质雄性不育(CMS)是一种母性遗传性状,是作物杂种优势利用的重要基础。小麦是世界栽培面积最广的作物,对其雄性不育机理的揭示,特别是杂种优势的利用一直是本领域设法攻克的科学难关。然而,其雄性不育机理研究所积累的资料十分有限。因此,有必要将更多的关注投入到小麦雄性不育机理的研究方面,这对于加强小麦杂种优势研究与利用具有重要的理论和实践意义。对其它物种CMS的广泛研究表明,由于线粒体DNA(mtDNA)的变异所引起线粒体功能的异常,进而可导致花粉在发育中败育而产生雄性不育。本研究以小麦近缘属的四种山羊草、对应山羊草细胞质的小麦异源细胞质雄性不育系以及不育系与恢复系所组配的杂种F1为试材,在同一细胞质起源背景下利用RAPD标记对山羊草、不育系及其杂种F1的mtDNA的变异性进行了系统深入的研究,获得如下重要结果:(1)山羊草与相对应细胞质雄性不育系在mtDNA上存在明显差异。其中,引物OPY-01的RAPD扩增图谱在粘果山羊草Ae.kotschyi和不育系ms(Ae.kotschyi)-90-110间存在稳定和可重复的差异;引物S32的RAPD扩增图谱在易变山羊草Ae.variabilis和不育系ms(Ae.variabilis)-90-110间存在稳定和可重复的差异;引物S22的RAPD扩增图谱在偏凸山羊草Ae.ventricosa和不育系ms(Ae.ventricosa)-90-110间存在稳定和可重复的差异;引物S202、OPB-05、OPA-04、S153、S21均在二角山羊草Ae.bicornis和不育系ms(Ae.bicornis)-90-110的mtDNA中扩增出稳定和可重复的差异。表明在异源细胞质雄性不育系的选育过程中线粒体基因组受到质核互作的影响而发生变化;不育系与对应细胞质的山羊草相比,其mtDNA存在明显的变异性;而且不同类型的不育系mtDNA变异性不同,因此其对应的不育性也不同。这种变异性很可能会引起线粒体基因结构的变化,进而导致线粒体功能障碍。因此,以上标记可作为鉴别雄性不育相关基因的分子标记。(2)不育系与相应的可育杂种F1在mtDNA上存在明显差异。其中,引物OPY-01对不育系ms(Ae.kotschyi)-90-110和杂种F1代ms(Ae.kotschyi)-90-110×5253的mtDNA扩增产物存在稳定和可重复的差异;引物OPD-05对不育系ms(Ae.variabilis)-90-110和杂种F1代ms(Ae.variabilis)-90-110×5253的mtDNA扩增产物存在稳定和可重复的差异;引物S21对不育系ms(Ae.ventricosa)-90-110和杂种F1代ms(Ae.ventricosa)-90-110×5253的mtDNA扩增产物存在稳定和可重复的差异;引物OPP-02对不育系ms(Ae.bicornis)-90-110和杂种F1代ms(Ae.bicornis)-90-110×5253的mtDNA扩增产物存在稳定和可重复的差异。结果表明,核育性恢复基因对不育系进行育性恢复的过程,线粒体DNA再次发生变异;对于不同不育类型其线粒体DNA的变异性不同,因此各不育系的恢复性也不同。这亦表明,线粒体基因组的变异不是随机的,而是在受核基因组主导下发生各种变化,不同核背景对线粒体基因组产生的效应不同。核育性恢复基因的导入导致杂种F1代的mtDNA所发生的变异可能涉及到雄性不育相关基因区域,因此,不育系和对应的杂种F1的特异扩增片段可作为鉴别雄性不育相关基因的分子标记。(3)以小麦黄化苗为材料建立了一种小麦mtDNA的高效提取方法。通过简单差速离心、DNaseⅠ处理得到无核DNA杂质的线粒体,用SDS和蛋白酶K裂解线粒体,经酚/氯仿抽提除去蛋白,并用RNase A消化而得到单纯mtDNA。对所提取的mtDNA进行紫外吸收光度分析,A260/A280平均为1.92,A260/A230平均为2.09,平均每克黄化苗可提取mtDNA 26.85 ug;并对mtDNA进行琼脂糖凝胶电泳和RAPD扩增,均得到清晰的电泳图谱。表明此提取方法得到的mtDNA,不但产率高、结构完整,而且能有效去除核DNA、RNA和蛋白质等杂质,获得高质量的mtDNA用于PCR反应和各种遗传学分析。研究还发现,通过调整线粒体裂解温度(先50℃裂解1 h,再37℃裂解1 h),亦可大幅度提高mtDNA的产率。更多还原

【Abstract】 Cytoplasmic male sterility (CMS) is a maternally inherited trait characterized by the absence of functional pollen. Crop heterosis is often attained through the use of CMS. Wheat is one of the most important field crops in the world today. In spite of the progress made in well-studied species, the mechanism of CMS in wheat remains puzzling. So we should pay more attention to the mechanism of CMS in wheat. In several well-studied species, molecular studies of CMS have correlated the trait with mitochondrial DNA (mtDNA) mutations that disturbing mitochondrial function at a critical stage of anther development.In this thesis, mitochondrial DNAs (mtDNAs) of four Aegilops species (Ae.kotschyi, Ae.variabilis, Ae.ventricosa and Ae.bicornis), their corresponding cytoplasmic male sterile (CMS) wheat with the Aegilops cytoplasms and the fertility-restored F1 hybrids were studied by RAPD (Random amplified polymorphic DNA) analysis. RAPD fingerprint patterns were compared under a single cytoplasm background to analyse mitochondrial DNA (mtDNA) variation. The main results are as follows.(1) Reproducible differences in the mtDNAs were obtained from the Aegilops species and the corresponding CMS lines. With primer OPY-01 different fingerprint patterns were obtained from the mtDNAs of Ae.kotschyi and the male sterile line ms(Ae.kotschyi)-90-110. With primer S32 different fingerprint patterns were obtained from the mtDNAs of Ae.variabilis and the male sterile line ms(Ae.variabilis)-90-110. With primers S22 different fingerprint patterns were obtained from the mtDNAs of Ae.ventricosa and ms(Ae.ventricosa)-90-110. With primers S202, OPB-05, OPA-04, S153 and S21, different fingerprint patterns were obtained from the mtDNAs of Ae.bicornis and male sterile line ms(Ae.bicornis)-90-110. These results indicate that the genetic interactions between the common wheat 90-110 nucleus and alien cytoplasm of Aegilops species have affect the structure of the mitochondrial genome. The differences of fingerprint patterns between Aegilops species and the corresponding CMS wheat revealed that some structural alterations of the mitochondrial genomes occurred during the recurrent backcrossing program. These differences of mtDNAs can be used for molecular markers to locate CMS-associated gene regions.(2) Reproducible differences in the mtDNAs were obtained from male sterile lines and the corresponding fertility-restored F1 hybrids. With primer OPY-01 different fingerprint patterns were obtained from the mtDNAs of male sterile line ms(Ae.kotschyi)-90-110 and F1 hybrid ms(Ae.kotschyi)-90-110×5253. With primer OPD-05 different fingerprint patterns were obtained from the mtDNAs of male sterile line ms(Ae.variabilis)-90-110 and F1 hybrid ms(Ae.variabilis)-90-110×5253. With primer S21 different fingerprint patterns were obtained from the mtDNAs of male sterile line ms(Ae.ventricosa)-90-110 and F1 hybrid ms(Ae.ventricosa)-90-110×5253. With primer OPP-02 different fingerprint patterns were obtained from the mtDNAs of male sterile line ms(Ae.bicornis)-90-110 and F1 hybrid ms(Ae.bicornis)-90-110×5253. These results indicate that the mitochondrial genomes were changed in fertility restoration by the introduction of fertility restorer nucleus and fertility restoration involves a strong influence of nuclear restorer genes on mtDNA organization. And also, the differences of mtDNAs in Aegilops species, their corresponding CMS lines and the fertility-restored F1 hybrids indicate that mitochondrial genomes variation is controlled by different nuclear genomes. The differences of mtDNAs between male sterile lines ant the the corresponding fertility-restored F1 hybrids can be used for molecular markers to locate CMS-associated gene regions.(3) An efficient method for isolation of mtDNA from etiolated tissues of wheat was developed. The protocol consists of mitochondria isolation with differential centrifugation, DNase I treatment, lysis with SDS and proteinase K, removing protein by TE-saturated phenol/chloroform extraction and a final RNase A treatment for obtaining mtDNA. The mtDNA samples were tested using spectrophotometry and agarose gel electrophoresis. It was proved that the mtDNA isolated by this method not only have the high yield but also structural complete, and contains no impurities, such as nuclear DNA, RNA and protein. The result showed that this high quality mtDNA can be successfully used in PCR and other genetic studies. In addition, it was found that adjusting the lysis temperature has a noticeable effect on the mtDNA yield.更多还原