【作者】 张龙雨；

【导师】 张改生；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2013， 博士

【摘要】 化学杂交剂诱导的生理型不育(PHYMS)是小麦雄性不育系的重要来源之一。SQ-1是一种新型化学杀雄剂,它能诱导小麦产生95%-100%的雄性不育。在生产上,应用SQ-1已经组配出一批超高产、优质、多抗的杂交小麦新品种,但是对于PHYMS的机理尚不清楚,尤其是对PHYMS花粉败育的内部代谢知之甚少。为了阐明PHYMS花药发育能量代谢过程的作用机制,本研究首先通过细胞学的方法对PHYMS系的花药绒毡层发育,小孢子的细胞分裂及淀粉积累等情况进行了观察,同时测定了丙酮酸含量,初步分析了能量供应对小麦花粉发育的影响；并以[aPDK作为研究的主体,采用普通克隆技术,TAIL-PCR以及原核表达对该基因的分子特征进行了分析；其次采用半定量RT-PCR技术检测了TaPDK及其底物蛋白基因TaPDC-E1α在小麦花药中的表达；最后,利用酵母双杂交技术筛选了TaPDK的互作蛋白,并结合实时定量PCR技术分析了这些蛋白基因的表达模式,进一步探讨了这些基因与能量代谢以及花粉发育的关系。获得的主要结果及结论如下：1.细胞学观察结果表明,与正常可育系相比,绒毡层在PHYMS系的小孢子发育早期提前解体；小孢子细胞分裂出现异常,大部分小孢子细胞停止在单核和二核期不再分裂。此外,PHYMS系的成熟花粉中几乎没有淀粉颗粒的累积。丙酮酸含量的测定显示,小麦PHYMS系中的丙酮酸含量一直处于较低的运行状态。因此,致使小孢子这种异常的发育与能量供应不足密切相关。2.小麦TaPDK基因编码364个氨基酸残基,蛋白分子量为41KD,与原核表达出的融合蛋白分子量基本一致。保守结构的分析发现TaPDK含有线粒体支链α-酮酸脱氢酶激酶家族和类组氨酸ATP酶家族；同时,进一步对其启动子序列分析表明TaPDK含有多种顺式作用元件。因此,TaPDK分子序列的功能多样性,暗示了PaPDK参与了多通路的代谢调节。3.半定量RT-PCR结果表明,TaPDC-E1α在PHYMS系中表达量最低,说明经杀雄剂SQ-1诱导形成的生理型不育系在败育过程中其能量代谢途径更容易受到影响。此外,TaPDK基因在PHYMS系中表达下调,而在遗传型不育系中表达上调,表明对TaPDK基因进行调节的上游信号机制在小麦PHYMS系与遗传型不育系中不一致。4.通过酵母双杂交技术筛选TaPDK的互作蛋白,共获得24个阳性克隆,这些基因共编码8种不同的蛋白质,分别是多聚泛素,CBS结构域包含蛋白,非特异性脂质转移蛋白前体,增殖细胞核抗原,甘油醛-3-磷酸脱氢酶,肌动蛋白,鸟嘌呤核苷酸结合蛋白p亚基,查尔酮合成酶基因及功能未知蛋白。这些候选蛋白参与了蛋白降解,能量供应,细胞分裂及花粉的形成,这表明TaPDK除参与调节丙酮酸脱氢酶复合体的活性外,该蛋白在花粉发育的过程中还起着其他多样化的作用。5.对TaPDK互作蛋白基因的定量表达分析表明,TaPUbi仅在PHYMS系中表达上调,在同遗传背景的遗传型不育系和相应的近等基因系中表达没有变化。因此,说明SQ-1的喷施诱导了该基因的表达,多聚泛素介导的泛素蛋白酶体途径在PHYMS系中参与了对TCA途径的能量调节。TaPCNA在不育系和可育系中的表达一直呈现出依次下降的表达趋势,但是无论是在PHYMS系还是同背景的遗传型不育系中,TaPCNA的表达均高于可育系。TaCBS在不育系及可育系中无明显差异,但在三核期,该基因在不育系中的表达高于可育系。这可能是不育系中花粉发育后期能量严重不足,CBS结构域蛋白作为细胞能量的传感器,在SQ-1诱导花粉败育的能量应急中参与能量水平的调节。TanLTP在不育系中的高表达,应该与SQ-1喷施后花粉形成过程的防御反应有关。更多还原

【Abstract】 Currently, the physiological male sterility (PHYMS) induced by chemical hybridizing agent (CHA) is an important source of male sterile line in wheat (Triticum aestivum L.). SQ-1, a new chemical hybridizing agent, can induce95%-100%spikelet sterility in wheat. In practice, a series of new hybrid wheat cultivars with high yield, elite quality, and multiple resistance were developed by making use of SQ-1. However, very little is known about the molecular mechanism of PHYMS. The challenge remains to understand how intermediary metabolism can be regulated during pollen abortion. To elucidate energy metabolism mechanism of the abnormal pollen development in PHYMS, the cytological observation of tapetum development, cell division, starch accumulation in PHYMS were performed, and the content of pyruvic acid was detected. Furthermore, the molecular characteristics of TaPDK, as the research starting point, were analyzed via common clone, hiTAIL-PCR, and prokaryotic expression. Then the expression pattern of TaPDK and its substrate protein gene TaPDC-E1α were detected via semiquantitative RT-PCR analyses. In addition, the proteins interacting with TaPDK were screened via yeast two-hybrid technique. Subsequently, a few candidate proteins in nucleotide expression levels were detected by real-time quantitative PCR, and further discussed the relationship between these genes expression and pollen development.The main results and conclusions were as follows:1. Cytological observations showed that tapetum was premature degeneration at early microspore stage in PHYMS, and the majority of microspores failed to divide and remained bicellular status. Additionally, there was no starch accumulation in the mature pollen of PHYMS. Subsequently, the results from the pyruvic acid detection showed that the content of pyruvic acid was dramatically down-regulated in the PHYMS lines than that of the fertile lines. Collectively, the abnormal microspore development was associated with energy shortage.2. The TaPDK encoded364amino acid residues, and protein molecular weight was41KD. The protein molecular weight of TaPDK from prokaryotic expression was consistent with software prediction. Conservative structure analysis found that TaPDK contains BCDHK-Adom3superfamily and HATPase-c superfamily. Subsequently, the upstream regulation sequences of TaPDK contained multiple cis-elements. Therefore, the structure diversity of TaPDK molecular sequence suggested that the TaPDK may be involved in the metabolic regulation of multiple pathway.3. According to semiquantitative RT-PCR analyses, the expression levels of TaPDC-E1α was lowest in PHYMS, demonstrating that the energy metabolism pathway was more susceptible in the PHYMS. TaPDK was obviously down-regulated expression in PHYMS, and up-regulated expression in genetic male sterile lines, which indicated that the upstream signal mechanism of mediating TaPDK was inconsistent between the PHYMS and the genetic male-sterile lines.4. We obtained twenty-four colonies coding for eight different proteins via yeast two-hybrid screen, namely non specific lipid transfer protein precursor, polyubiquitin, glyceraldehyde-3-phosphate dehydrogenase, proliferating cell nuclear antigen, CBS domain containing protein, actin, guanine nucleotide-binding protein beta subunit, chalcone synthase, and unknown function protein. These proteins involved in protein degradation, energy supply, cell division, and pollen formation, which further suggested that TaPDK plays multiple roles in pollen development, besides participating in regulating pyruvate dehydrogenase complex activity.5. The quantitative RT-PCR analysis indicated that the expression level of TaPUbi was only obviously up-regulated in PHYMS lines, but no significant difference in TaPUbi transcripts was observed in the corresponding genetic male sterility and fertility. It suggested that the SQ-1spraying induced TaPUbi expression, and ubiquitin proteasome pathway may participate in regulating the energy status of TCA pathway in PHYMS. The TaPCNA expression in sterile and fertile lines gradually exhibited down-regulated pattern with the pollen developmemt, but its expression was still higher in PHYMS lines and genetic male sterile lines than in fertile lines. Whereas the expression of TaCBS maintained stable, no significant differences were detected in leaf and anther at the uninucleate and binucleate stage between PHYMS and fertile lines. Intriguingly, TaCBS expression was much more increased at the tricellular stage in PHYMS lines than in fertile lines. It was speculated that the energy supply was serious shortage in later pollen development of PHYMS lines, and TaCBS, as cell energy sensor, adjusted energy levels to deal with energy crisis during pollen abortion in PHYMS lines. The expression of TanLTP was higher in PHYMS lines than in fertile lines. This indicated that the highest expression of TanLTP might protect pollen against potential disruption after SQ-1treatment.更多还原