【作者】 张磊；

【导师】 金危危；

【作者基本信息】 中国农业大学 ， 作物遗传育种， 2016， 博士

【摘要】 脂质代谢相关基因在高等植物花药孢粉素和角质素合成过程中具有重要作用,这些基因的突变常常导致花粉外壁和花药角质层发育障碍,并最终导致雄配子败育和雄性不育的发生。尽管目前在玉米中发现了几十个雄性不育突变体,然而仅有少数基因被克隆。玉米经典雄性不育突变体ms33被发现于1995年,其不育表型稳定,具有很强的商业应用潜力。然而,导致该突变的基因却尚未被克隆。在本研究中,我们通过图位克隆的策略获得了玉米MS33基因,并采用分子遗传学,细胞生物学,发育生物学以及分析化学手段对相关突变体进行了全面研究。作为在禾本科植物中被克隆的第一个GPAT蛋白编码基因,SA1/MS33在控制花药相关聚酯合成中具有关键作用,参与指导花药角质层和花粉外壁形成。获得的主要结果如下：1.在国家玉米改良中心构建的玉米MuDR突变体库中,发现了一个隐性雄性核不育突变体shrinking anther 1 (sa1),该突变体营养生长阶段正常,花粉完全败育,不育性状不受环境影响。通过细胞学分析表明,sal突变体花药绒毡层在小孢子发育早期发生降解,花粉外壁异常,花药表皮角质层不能正常形成。2.等位测验表明,sa1突变体与一个经典的玉米雄性不育突变体male sterile 33(ms33)等位。我们用sal突变体分别与郑58,B73白交系构建了F2定位群体,将突变位点定位在玉米2号染色体190 Kb的区间内,随后对区间内全部基因进行测序分析,获得了候选基因GRMZM2G070304。测序发现,在sal突变体中,该基因第二外显子存在247 bp片段插入；在ms33-6019等位系中,基因第一外显子缺失479 bp片段；在ms33-6024等位系中,基因编码区第507碱基位置插入2个碱基,发生移码导致翻译提前终止。SA1/MS33基因编码一个甘油醛-3-磷酸酰基转移酶(GPAT),该基因是在禾本科植物中克隆的第一个GPAT基因。3. qPCR和RNA原位杂交分析表明,SA1/MS33主要在根系以及花药绒毡层中表达。通过气相色谱-质谱分析,发现sa1突变体花药聚酯单体含量的显著降低,然而根系中的聚酯单体成分并未发生显著变异。因此,我们推测基因突变导致花药绒毡层内聚酯合成障碍可能是导致花药外壁和花药表皮角质异常的主要原因。综上所述,本研究证明玉米SA1/MS33基因在花药绒毡层中参与花药相关聚酯合成,这些物质进一步参与花药表皮角质和花粉外壁的形成。这一过程的异常导致小孢子发育早期败育,并最终表现雄性不育表型。研究结果对阐释玉米花药发育和脂质代谢过程具有重要意义,并为雄性核不育基因在育种上的应用提供了新的遗传资源。更多还原

【Abstract】 Lipid metabolism related genes play an important part in biosynthesis of pollen sporopollenin and anther cutin in high plants. Mutant in these genes often result in damage in pollen exine and anther cuticle development, and lead to microspore abortion and male sterile finally. Although a large number of male sterile mutants exist in maize, very few of them have been functionally characterized. A classical maize male sterile mutant ms33 was reported in 1995. This mutant showed stable sterile phenotype and has potential commercial value. However the related gene has not been identified before.In this study, we identified the maize MS33 gene via map-based strategy. And comprehensive analysis was conducted using various methods, including molecular genetics, cytobiology, developmental biology and analytic chemistry. As the first isolated GPAT coding gene, SA1/ MS33 showed an irreplaceable role in biosynthesis of maize anther related polyesters, directing the formation of anther cuticle and pollen extine. The main results are listed below:1. A male sterile mutant shrinking anther 1 (sa1) was identified by screening a MuDR library which was create in National Maize Improvemental Centre of China. This mutant exhibited normal vegetative development and no pollen grains were produced in anther. Analysis showed sal displayed defective pollen exine and anther cuticle development, as well as the consequent premature microspore degradation.2. Genetic test showed sal mutant was allelic to the classical maize mutant male sterile 33 (ms33). Through map-based cloning and sequencing, the related gene SA1/MS33 was isolated. SA1/MS33 encodes a putative glycerol-3-phosphate acyltransferase (GPAT), and this gene was the first reported GPAT gene in grasses.3. Data from qPCR and RNA-in situ indicated that SA1/ MS33 mainly expresses in roots and tapetal layers in developing anther. Gas chromatography-mass spectrometry (GC-MS) revealed dramatic reduction in lipidic polyester monomers in the anthers of the ms33 mutant. However, no obvious difference was detected in root polyester monomers content between wild type and ms33 mutant. It is suggested that MS33 might mediate pollen exine and anther cuticle formation by regulating biosynthesis of lipidic polyester in maize.In conclusion, this study demonstrated SA1/MS33 gene participates in anther related polyester biosynthesis which involves in the formation of anther cuticle and pollen extine. Disruption in this process might result in microspore abortion in early stage and lead to male sterile. These results have important significance in illustration of anther development and anther related lipid metabolism in maize, and provide new genetic resource for molecular breeding based on the utilization of male sterile related gene.更多还原