【作者】 沈静；

【导师】 徐国华；

【作者基本信息】 南京农业大学 ， 植物营养学， 2009， 硕士

【摘要】 植物细胞膜上的质子泵(H+-pump)或H+-ATPase(EC 3.6.1.35)是一类通过水解ATP产生能量,将细胞质中的H+逆浓度泵出细胞的运输蛋白。其主要功能是在细胞膜两侧产生H+浓度梯度和膜电位,以质子驱动力的形式推动各种离子和小分子代谢产物进行跨膜运输。细胞膜质子泵是由一个多基因家族编码的,可将其分为五个亚家族。研究表明,细胞膜质子泵基因的表达具有一定的组织特异性,反映了不同组织的生理需求。由于质膜H+-ATPase是由多基因编码的蛋白,因此其转录水平上的变化在一定程度上决定了其活性的变化。水稻是我国重要的粮食作物,而且也是一种典型的模式作物。因此本论文通过RT-PCR方法分析了全铵,全硝,缺氮(包括恢复供氮),缺磷(包括恢复供磷)以及pH、模拟干旱或盐胁迫下质膜质子泵基因的表达特征。结果发现：(1)在水稻根和叶中大量表达的质膜H+-ATPase基因均属于亚家族Ⅰ的OsPMA1,2,3和属于亚家族Ⅱ的OsPMA7,其余的未见表达。在缺磷后,根系中的OsPMA1、2、3、7的表达都降低,但是恢复供磷后这些基因的表达又开始逐渐上调,但是叶片中这些基因的表达则没有发生明显的变化。缺氮时根系中仅OsPMA7的表达有所降低,但是氮素形态(供铵或供硝)并没有造成其基因表达上的差异,并且与对照之间也无明显的差异。叶片中的OsPMA基因的表达均不受缺氮或供氮形态(供铵或供硝)的影响。(2)随着根际pH的降低,OsPMA7的表达量升高,但是OsPMA1、2、3的表达随着胁迫时间的延长出现下调的趋势,并且当胁迫时间达到96小时后,OsPMA7的表达量也开始降低。而叶片中的质子泵表达没有受到根际pH的影响。盐胁迫下,根系中OsPMA1、2、3的表达未见明显变化,但是OsPMA7的表达受到抑制。在叶片中,上述质子泵基因的表达都有所上调。模拟干旱胁迫(PEG处理)下,无论根系还是叶片中的质子泵基因的表达均未受到影响。(3)对14-3-3蛋白家族在转录水平的检测发现,它们的表达强度在总体上未受到缺磷、缺氮或是不同氮形态供应的影响,仅Os14-3-3 10基因在缺磷时表达上调。本研究通过对上述基因表达模式的分析,为逆境胁迫下水稻细胞膜质子泵的在转录水平上的调控提供了初步的信息。更多还原

【Abstract】 Plasma membrane H+-ATPase is a universal electrogenic H+ pump, which uses ATP as energy source to pump H+ across plasma membranes into the apoplast. The key function of this enzyme is to generate an H+ electrochemical gradient, thereby providing the driving force for the active influx and efflux of ions and metabolites across the plasma membrane Plasma membrane H+-ATPase is encoded by multiple genes, which have been grouped into five sub-families. The familiesⅠandⅡrepresent most expressed genes in plant species. In addition, the expression of plasma membrane H+ ATPase genes were reported in specific tissues or organs, which reflected some special physiological requiment of these tissues. Because of the involvement of plasma membrane H+-ATPase in plant physiological and biochemcal process, the transcriptional level of H+-ATPase is a major factor in regulation of the H+ ATPase activity.Rice is an important cereal food crop and has been taken as a model for plant biology reseach. In this study, we analyzed expression patterns of 10 isoforms of plasma membrane H+-ATPase of rice cultivar (Nipponbare) under different nutrition supply status and environmental stress including sole ammonium nutrition, sole nitrate nutrition, N starvation, P starvation, pH stress, dryness stress and salt stress with the semi-quantity RT-PCR in cultured Nipponbare rice. The results indicate that:(1) Most of the abundant expressed genes are belonging to the subfamiliesⅠ&Ⅱ, which including OsPMA1,2,3, and 7. P deficiency depressed the expression of OsPMA1、2、3 and 7 in the roots. But their expression was up-regulated after the restore of P supply. However, P deficiency had no apparent effect on the expression of these genes in the leaves. N deficiency only decreased the expression of OsPMA7 in the roots. However, N forms (ammonium or nitrate) had no strong effect on the expression of these genes. The expression patterns of OsPMAs in leaves were not affected either by N deficiency or by N forms. (2) The expression of OsPMA7 increased with the decrease of pH in the culture solution, but the expression of OsPMA1、2 and 3 was decreased with the extending of the culture time. After 96 h, the expression of OsPMA7 was also decreased. However, change of the pH in the culture solution had not affect on the expression of these genes in the leaves. Under salt stress, the expression of OsPMA7 in the roots was down-regulated, while the expression of OsPMA1、2、3 genes had not been affected in the roots. These genes in leaves were all up-regulated in the transcript levels by salt stress. Osmotic stress mimicked by PEG had not affect on the expression of OsPMAs both in the roots and the leaves.(3) Similar expression patterns of 14-3-3 protein iso-genes were observed under P deficiency, N deficiency and treatments of different N forms (NO3- and NH4+), however, one iso-gene,14-3-3 protein 10, was highly up-regulated by P deficiency.The study provided the insight of the regulation of plasma membrane H+-ATPase at the transcriptional level and post-transcriptional level under environmental stress.更多还原