【作者】 王翠；

【导师】 刘箭；

【作者基本信息】 山东师范大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 研究植物耐热分子机制的方法很多,其中筛选高温敏感突变体是一种有效的方法。至今,大量的热敏感突变体的研究表明植物热激反应是一个复杂的网络系统,涉及热激蛋白及热激转录因子、抗氧化系统、激素和钙等多个方面。拟南芥AtFes1基因家族包括:At3g09350 (AtFes1A)、At3g53800 (AtFes1B)和At5g02150 (AtFes1C)。芯片数据显示At3g09350是含量最丰富的热激响应基因,该基因编码一种受高温诱导的armadillo/beta-catenin repeat蛋白,与动物HspBP-1和酵母Fes1p同源,但其生物学功能迄今不详。为了研究拟南芥AtFes1A的功能,我们从ABRC订购了该基因被敲除的T-DNA插入突变体Salk-021784和Salk-072075。利用基因组DNA PCR和Western-blotting方法,我们对突变体进行了筛选,获得AtFes1A基因被敲除的纯合突变体。回交实验证明突变体中T-DNA为单拷贝;通过表型分析发现,两个独立突变体5天幼苗的获得耐热性均明显下降;互补实验使得突变体耐热性恢复;这些结果充分说明AtFes1A缺失会导致拟南芥耐热性下降。为进一步探究AtFes1A在拟南芥中的分子机制,我们利用Northern和Western方法对突变体中相关热激响应基因的变化进行了分析,结果发现高温条件下atfes1a突变体中Hsp70等基因热激响应较野生型更为强烈,但Hsp70蛋白水平却明显低于野生型,因此我们推测敲除AtFes1A导致Hsp70在高温胁迫下发生了降解。基于Hsp70对于植物耐热的重要性,我们认为atfes1a突变体的热敏感表型就是由于Hsp70降解引起的。免疫共沉淀实验已经证实AtFes1A与Hsp70能够相互作用,为进一步证实这种互作作用我们利用酵母双杂交系统进行了分析。结果发现,Hsp70在酵母体内可以正常表达,但是AtFes1A在酵母中表达异常,而转双基因酵母也不能在四缺陷培养基上生长。此外,我们构建了AtFes1A过量表达载体并转化野生型拟南芥。研究发现,过量表达AtFes1A基因对Hsp70及Hsp101的表达均没有产生明显影响。更多还原

【Abstract】 Screening high temperature sensitive mutants is an effective way to explor the molecular mechanisms of plant thermotolerance. An increasing number of mutants have extended our understanding that plant heat shock response is a complex network, involving heat shock proteins, heat shock transcription factors, antioxidant system, hormones and calcium.Arabidopsis AtFes1 consists of three gene family: At3g09350 (AtFes1A), At3g53800 (AtFes1B) and At5g02150 (AtFes1C). Bioinformatics data indicate that At3g09350 gene encodes an armadillo / beta-catenin repeat protein which is induced by high-temperature, but its biological function is unknown yet.We obtained mutant lines Salk-021784 and Salk-072075 from the ABRC. Homozygous mutants were confirmed by using PCR and Western-blotting. Backcross experiment result indicates that Salk-021784 has only one locus of T-DNA insertion .We found that the mutants present a defection of acquired-thermotolerance in the 5d seedling. The phenotypic defection of Salk-021784 mutant were recovered by complementary experiment. These results indicate that absence of AtFes1A will lead to the defection of thermotolerance in Arabidopsis thaliana. To find the possible cause for defective acquired thermotolerance in AtFes1A, we determined the transcript levels and the expression profiles of some Hsps and Hsfs at different temperatures by Northern and Western. We found that the expression of Hsp70 in atfes1a mutants was more stronger, but its protein level was significantly weaker than the wild type. This indicate that Hsp70 was degradated under high temperature in the mutans, which may lead to the defection of thermotolerance in Arabidopsis.The interraction beteen AtFes1A and Hsp70 had been demonstrated. In this experiment we used a yeast two-hybrid system to further analysis the interraction between AtFes1A and Hsp70 . We found that the expression of Hsp70 in yeast was normal, but AtFes1A expressed with diversity immuno-hybridied bands, and the genetically modified yeast can not grow in the four defects medium.In addition, we constructed AtFes1A overexpression vector and transformed it into the wild type. We find that overexpression of AtFes1A have no effects on the accumulation of Hsp70 and Hsp101 transcripts and their corresponding proteins in Arabidopsis.更多还原