【作者】 郭永豪；

【导师】 马向东；

【作者基本信息】 河南农业大学 ， 微生物学， 2005， 硕士

【摘要】 在自然界中,酵母生长于一个随时都有可能发生剧烈变化的环境中,面临着高渗、高温、PH变化、营养缺乏等一系列的胁迫压力。长期以来,酵母进化形成了一套完备的抗逆机制,能够在一定的胁迫条件下正常生长。作为酵母重要的抗逆因子——海藻糖具有重要的抗逆作用,生物合成海藻糖的关键酶是海藻糖-6-磷酸合成酶(TPS1),因此研究胁迫条件下tps1基因mRNA的表达情况具有重要的意义。 本文以三株生长生理生化特性各异的酵母为研究材料,通过基因克隆,得到tps1基因,经过测序以后,分析tps1基因序列突变情况和TPS1蛋白氨基酸序列的突变情况。发现与序列X68496相比,WFB的tps1基因序列存在8个差异,FHS存在9个差异;YY存在13个差异。 按照DNA序列,利用生物学软件推测出来的氨基酸序列,WFB和FHS的TPSI没有引起氨基酸序列的改变,而YY的TPS1氨基酸序列发生了3处改变,分别为67位氨基酸(Gly/Ala),69位氨基酸(Glu/Gln),388位氨基酸(Val/Leu)。通过GeneBank分析保守序列发现,这三处的突变均处在保守序列内部,并且与假丝酵母、黑曲霉、裂殖酵母等的海藻糖-6-磷酸合成酶氨基酸保守序列相比,YY TPS1氨基酸序列的67位与388位氨基酸突变较为明显。 三个菌株经过不同的温度热激以后,做Dot bloting。发现tps1的mRNA的胞内积累量不尽相同。三个菌株在经受37℃的热激处理以后,与对照相比,WFB和FHS的mRNA积累量略有升高,YY则稍微下降:在经受42℃处理以后,三个菌株tps1的胞内mRNA水平均有所升高,其中WFB和FHS达到了最高的水平;当再用48℃处理时,WFB和FHS的tps1 mRNA量有所下降,而YY则继续升高。可以说在生长温度低于40℃时,YY耐高温的生长特性与tps1 mRNA的积累量无关,只有当生长温度升高到40℃以上时,YY才会开始大量积累tps1 mRNA。 当经过高渗处理以后,tps1基因mRNA的表达量并不像热激的情况下那样,具有明显的变动。虽然在高浓度的NaCL处理下,tps1基因mRNA的表达量略有升高,但是与对照相比,差异不明显。且YY一直在升高,而FHS和WFB在高浓度的NaCL处理下,tps1基因mRNA的表达量有所下降。更多还原

【Abstract】 Under the condition of nature, yeasts are exposed to a highly variable environment with respect to the availability and quality of nutrients, temperature, pH, radiation, access to oxygen, and water activity. But yeast has one of the precise system to adapt to the variable environment after long evolution. Trehalose, as an important stress-resistance factor, play a key role in the yeast to resistant to the various stress. As a key process, UDP-glucose is converted first to trehalose-6-phosphate by trehalose-6-phosphate synthase (TPS1) and then to trehalose. As a result, it is remarkable to research the expression of gene tpsl.In this paper, three yeast stock, which have different growth properties, are used as material. After the cloning of gene tpsl, sequencing revealed that there are eight mutation site of WFB tpsl, and accordingly, FHS is nine and YY is thirteen.On the other hand, the protein sequence of TPS1 of FHS and WFB haven’t any difference compare to one protein sequence of TPS1 reported by GeneBank. But the TPS1 sequence of YY has three mutations sites. The locus of three mutation site are 67, 69 and 388. Based on the analysis of conserve sequence of protein, finding reveals that the mutation is remarkable.After heat shock, the results of Dot Bloting reveal that tps1 mRNA of three yeast have different expression level. Under the condition of 37°C , tps1 mRNA of FHS and WFB have a high abundance, but tpsl mRNA of YY is low. When subject to 42°C, the expression of tps1 mRNA of FHS and WFB reach their maximum. Correspondingly, YY reach its maximum at 48 °C.When subject to the solution of NaCL, the abundance of tpsl mRNA of three yeast have no remarkable change. This result prove trehalose have less relative to theresistance of yeast to the stress of high external osmolarity更多还原