【作者】 金海燕；

【导师】 王君晖；

【作者基本信息】 浙江大学 ， 遗传学， 2011， 硕士

【摘要】 沉默调节蛋白Sirtuin是一组重要的蛋白质,它们在衰老、胁迫和代谢的过程中起着十分重要的调节功能。哺乳动物中共有7个同源蛋白,而在拟南芥中只有二个,分别为AtSIRTl(At5g09230)和AtSIRT2(At5g55760)。本文主要研究表达在线粒体中AtSIRT1的基因功能。AtSIRT1具有多个转录本,可以产生4种成熟的mRNA,本文中分别命名为S1.3,S1.4,S1.5,S1.7。半定量RT-PCR分析表明,它们在不同组织中表达行为有一定的差别。启动子分析表明AtSIRT1旺盛表达于分生组织,且受光照调节。利用Gateway重组克隆体系构建了N端绿色荧光蛋白的融合蛋白表达株系,发现除了S1.4定位于细胞核,其余均定位于线粒体。用FLAG蛋白标签过量表达这四种转录本,分别命名为S1.3-FLAG, S1.4-FLAG, S1.5-FLAG和S1.7-FLAG,本文重点研究S1.3-FLAG和S1.7-FLAG的功能。另外,本文还鉴定了AtSIRT1的两个T-DNA插入突变体株系131994C和N433426。对S1.3-FLAG, S1.7-FLAG,131994C和N433426四个株系进行表型观察,发现S1.7-FLAG株系的种子萌发迟缓,萌发率低,幼苗个体偏小,在高糖培养基中,有停止生长、甚至致死的表型；而S1.3-FLAG,131994C和N433426株系的种子萌发速度快,幼苗个体大,在高糖培养基中生长旺盛。植物体内的葡萄糖含量测定表明,在正常B5和高糖培养基中,131994C和N433426的葡萄糖含量稍低于野生型；S1.3-FLAG在正常B5培养基中的葡萄糖含量与T-DNA突变体齐平,而在高糖培养基中却明显低于野生型和T-DNA突变体；S1.7-FLAG株系的葡萄糖含量在两种培养基上均明显低于野生型。在测定谷氨酸脱氢酶(GDH)同工酶活性中发现,S1.7-FLAG株系无论是在B5还是高糖培养基中,其活性远远高于野生型；T-DNA突变体在高糖培养中GDH活性与野生型相同,而在B5培养基中则略弱于野生型；S1.3-FLAG的GDH活性无论在什么条件下,均弱于野生型。初步的机理探讨认为,S1.7-FLAG过量表达可以提高GDH酶活性,可能改变了进入TCA循环的碳骨架供应,进而出现致死现象。S1.3可以看做缺少活性的S1.7蛋白,所以它的效应与S1.7相反。更多还原

【Abstract】 Sirtuins have emerged as important proteins in aging, stress resistance and metabolic regulation. Mammals contain seven sirtuins (SIRT1-7), but Arabidopsis thaliana contain 2 homologous proteins, named AtSIRT1 (At5g09230) and AtSIRT2 (At5g55760). In this study, we pay attention to AtSIRTl, a locus encoding mitochondrial protein.AtSIRT1 has 7 transcripts, and processes 4 mature mRNA, named S1.3, S1.4, S1.5 and S1.7. Semi-quantitative RT-PCR analysis revealed that, the expression levels of these transcripts in different tissues differ. Promoter analysis showed that AtSIRT1 strongly expressed in meristem tissues, and the expression was under light regulation. Using Gateway recombinant cloning system, Arabidopsis plant expressing N-termined GFP fusion proteins were generated. We found that S1.4-GFP was located to the nucleus, and the others were located to the mitochondria. Four transcripts were overexpressed harbouring the FLAG tag. We choose S1.3-FLAG and S1.7-FLAG for further study here. In addition, we characterized two T-DNA insertional mutants of AtSIRTl.Transgenic plants overexpressing the S1.7-FLAG displayed, low seed germination rate and delayed seedling development. When they grow on B5 medium supplemented with high levels of sucrose (e.g 150 mM), they fail to develop green expanded cotyledons and true leaves, and even die. Transgenic plants overexpressing S1.3-FLAG and the T-DNA mutants exhibited vigorous growth both on B5 and high levels of sucrose media. The glucose contents in these seedlings were determinated. The T-DNA insertional mutants had slightly lower glucose contents in comparison with wild-type. While the S1.7-FLAG line had significantly lower contents of glucose in comparison with wild-type.Determination of the isoenzyme activity of glutamate dehydrogenase (GDH) revealed that the S1.7-FLAG seedlings had significantly higher GDH activity than wild-type when they grow on B5 and high sucrose media. The GDH activity in the T-DNA mutants was slightly lower than wild-type when they grow on B5 medium, and displayed no difference with wild-type when they grow on the high sucrose medium. The activity of GDH in S1.3-FLAG seedlings was much lower than wild-type.We suggested that the phenotype of S1.7-FLAG may resulted from increased GDH activity and alternated carbon skeleton supply. The phenotype of S1.3-FLAG may resulted from defect of the function of S1.7.更多还原