【作者】 王琴；

【导师】 刘选明； 林辰涛；

【作者基本信息】 湖南大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 以GUS-CCT2 (CRY2的C端)为诱饵,筛选拟南芥cDNA文库,筛选到了与之相互作用的蛋白RAT1(Related to acetyltransferase 1),通过同源分析,找到与RAT1高同源性的蛋白(89.7%),称为RAT2。RAT1和RAT2与γ-CA1、γ-CA2、γ-CA3(Y碳酸酐酶)是同源的,同时也与细菌的丝氨酸乙酰转移酶同源。它们都与线粒体复合体Ⅰ有关。RAT1和RAT2蛋白定位在线粒体中,RAT1还定位在细胞核内。rat1和rat2单突变体没有特殊表型。在rat1单突变体与rat2单突变体的杂交后代中得不到rat1rat2双突变体。通过对R1r1r2r2和r1r1R2r2的花粉及胚胎观察,发现rat1rat2双突变体会胚胎致死,且通过转入35S-RAT2到R1r1r2r2植株中,这种致死表型能够恢复。由于得不到rat1rat2双突变植株,构建了rat2/RAT1-RNAi植株。发现rat2/RAT1-RNAi植株能够正常生长发育,只是生长发育延迟,在长日照条件下开花晚。实时定量PCR研究结果表明,在rat2/RAT1-RNAi植株体内,FT的表达降低了,其它开花基因的表达水平与野生型相当。所以rat2/RAT1-RNAi植株开花晚是由于FT的减少引起的。在对RAT1和RAT2的生化分析中发现,在体外,RAT1没有碳酸酐酶活性,但有明显的乙酰转移酶活性。我们推测由于RAT1与RAT2定位于线粒体中,且能与复合体1作用,RAT1和RAT2就很可能通过影响能量代谢来起作用。在突变体中,由于能量供应不足,而导致了上述的一系列表型。另,rat1、rat2单突变体没有表型,而rat1rat2双突变体胚胎致死,说明RAT1与RAT2在植物体内的功能是冗余的。RAT1进核是否与CRY2的功能有关还需要继续研究。更多还原

【Abstract】 RAT1 was found to interact with GUS-CCT2 (C-terminal of CRY2) in the screening of Arabidopsis cDNA library, using GUS-CCT2 as the bait. RAT2 is 89.7% identical to RAT1. They are 61-64% identical to Arabidopsis gamma carbonic anhydrases 1,2, and 3 (γCA1-3), and are about 33% identical to bacterial serine acetyltransferases. They were reported to associate with mitochondria complexⅠ. RAT2 was showed to localize in mitochondria, and RAT1 could be detected both in mitochondria and nucleus. The rat1 and rat2 monogenic mutants showed no abnormal phenotype, but we can not find the ratlrat2 double mutant plants in the cross progenies. After analysis of R1r1r2r2 and r1r1R2r2 plants’ pollens and embryos, we found that ratlrat2 double mutant is embryo lethal. And the embryo lethal phenotype can be rescued by transgenic expression of RAT2 in R1r1r2r2 (35S-RAT2/R1r1r2r2). The rat2 knock-out and RAT1-knock-down (RNAi) lines (r2rli) showed retarded growth and delayed development. Under long day conditions, the r2r1i lines are late-flowering, correlating with reduced FT expression. In vitro biochemical experiments indicated that the RAT1 protein showed no carbonic anhydrase activity but a protein acetyltransferase activity. We postulate RAT1 and RAT2 protein tightly associated with mitochondrial energy metabolism, for they were localized in mitochondrial and can interact with complexⅠ. So in mutant plants, the abnormal phenotypes may were all caused by insufficient supply of energy. On the other hand, we can see that RAT1 and RAT2 act redundantly in vivo. The function of RAT1 in the nucleus is unclear.更多还原