【作者】 赵琼；

【导师】 郭新红；

【作者基本信息】 湖南大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 GHMP超级激酶家族是真核生物体内的一大类重要的蛋白激酶,主要作用是将ATP的磷酸基转移到底物特定的氨基酸残基上,催化蛋白质的磷酸化作用,参与多种信号转导过程。拟南芥中已确定7个基因属于GHMP激酶家族,但对其生理功能的研究尚不多见,因此对该基因家族成员进行生理功能研究具有重要的意义。本研究主要对拟南芥GHMP家族中的GHMP1基因的表达模式及缺失突变体的功能进行了分析,取得了以下主要研究结果:首先,对GHMP1基因的表达模式进行了分析。实时定量RT-PCR结果表明,GHMP1基因在拟南芥根和花中的表达量较高,而在叶、叶柄及茎中的表达量较低,表明该基因具有组织表达特异性。该基因表达受ABA和NaCl的诱导,用10 uM ABA处理1 h时和100 mM NaCl处理2 h时,GHMP1基因表达量最大,推测该基因可能介导了ABA和盐胁迫诱导的相关信号途径。用RT-PCR方法比较分析了用10 uM ABA处理1 h的突变体ghmp1-1和野生型中一系列胁迫应答基因(abi1,abi2,KIN1/2,SOS2/3,OST1,RD29A/B,RAB18,RD22,DREB1/2A)的表达情况,发现GHMP1基因功能缺失使这些胁迫相关基因的转录水平出现不同程度的变化。表明GHMP1基因在ABA信号转导过程中起作用。其次,通过“双引物法”和RT-PCR方法筛选、鉴定得到拟南芥GHMP1基因的T-DNA插入纯合体突变体ghmp1-1和ghmp1-2。第三,利用反向遗传学方法,对拟南芥GHMP1基因的缺失突变体功能进行了研究。对突变体ghmp1-1、ghmp1-2和野生型的种子萌发率、根长、气孔开度及幼苗失水率等表型进行了比较分析,结果表明,ghmp1-1和ghmp1-2突变体对不同浓度的外源ABA胁迫比野生型敏感性低,进一步证明GHMP1基因作用于拟南芥胁迫应答信号途径,激活胁迫相关转录因子,参与ABA正调控。GHMP1基因的研究结果为GHMP基因家族其他成员的生理功能分析提供了有价值的参考资料。更多还原

【Abstract】 GHMP super kinase family is important protein kinases in eukaryotes. The main role is to catalyze protein phosphorylation by transfering the phosphate of ATP to amino acid residues of the specific substrate, which was involved in a variety of signal transduction. So far, the seven genes have been identified in Arabidopsis belonging to GHMP super kinase family, but little was known of its physiological functions, so the study of functions of these genes is significant. In the present study, the expression patterns of GHMP1 gene and functional study of T-DNA mutant in Arabidopsis were analysed. The main results of this paper can be summarized as follows.Firstly, the GHMP1 gene expression patterns were studied. The real time PCR results showed that there were a high transcription level in roots and flowers, but low level in leaf, petiole, and stem, indicating GHMP1 belonged to tissue-specific gene. The expression of GHMP1 gene was induced by ABA and NaCl. When the young seedlings for 14 days old were treated with 10 uM ABA for an hour or with 100 mM NaCl for two hours, the transcription level of GHMP1 gene was up to its peak, suggesting that GHMP1 gene may mediate the related to ABA and salt stress-induced signaling pathways. The transcription levels of a series of stress response genes (abi1, abi2, KIN1/2, SOS2/3, OST1, RD29A/B, RAB18, RD22, DREB1/2A) related to ABA and salt responses were compared by real-time PCR between the ghmp1-1 mutant and wild type with 10 uM ABA treatment for an hour, the distinct differences in transcription levels in these genes were displayed as the deletion of GHMP1 gene. Therefore, we can presume the GHMP1 gene play an important role in ABA signal pathway. Secondly, two GHMP1 gene T-DNA homozygous mutant ghmp1-1 and ghmp1-2 were screened through the“double-primer”method and were identified by the RT-PCR method. Thirdly, the functions of T-DNA insertion mutants of the GHMP1 gene in Arabidopsis were studied by the reverse genetics research methods. The changes in phenotype such as seed germination rate, root length, stomatal aperture, rate of water loss between the mutant and wild-type were observed. The results showed that mutants are less sensitive to exogenous ABA and NaCl in seed germination and root elongation compared with wild-type. The further evidence showed that the role of GHMP1 gene response to salt and ABA signaling pathways in Arabidopsis, which proved that its activation role as a stress-related transcription factor. This work provided some invaluable references for the physiological functions of the other GHMP genes.更多还原