【作者】 刘浩；

【导师】 宋纯鹏；

【作者基本信息】 河南大学 ， 植物学， 2011， 博士

【摘要】 种子萌发受ABA和GA的调控。ABA抑制种子萌发,诱导休眠;GA促进种子萌发,解除休眠。最近研究表明ABA与GA之间在体内含量之间的动态平衡是调控种子萌发的关键因素。然而由于GA和ABA信号通路的复杂性,目前我们对其调控机制仍然了解有限。本研究中我们利用T-DNA库,从中筛选出了一种诱导相关基因超表达后对ABA不敏感的突变体,通过遗传学分析发现突变体的表型能够稳定遗传,因此我们把它命名为gai4 （gain function of ABA insensitive 4, gai4）。利用TAIL-PCR技术我们发现突变体中T-DNA插入到GAI4的启动子区,在拟南芥中GAI4编码一个依赖于2OG-Fe（II）的氧化还原酶。在不加雌二醇诱导超表达的时候,该基因的表达量相比野生型来说得到了削弱;而在雌二醇诱导超表达后,它的表达量升高了有50倍之多。通过生物信息学分析发现它可能参与了GA的生物合成。接下来对其生理功能进行了分析,结果发现永久超表达植株对NaCl、H₂O₂在萌发上也有一定的抗性,并且超表达植株显示出不需要冷驯化就可以萌发。在远红光和红光交替处理时,超表达植株在萌发以及下胚轴伸长上都要优于野生型和相对应的突变体,这些结果都暗示了该基因确实参与了GA的生物合成。通过生物化学方法,进一步发现它可能编码一个GA20ox双加氧酶,并且能够利用GA₁₂作为底物产生一种新的GA种类。为了进一步观察它的表达部位,我们利用RT-PCR技术对其根、茎、叶、花中的表达量进行了分析,发现它在幼根中表达最强。此外,原生质体瞬时表达的结果显示GAI4定位于细胞质中。GAI4调控了跟DELLA蛋白代谢以及ABA相关基因的表达,而且它本身的表达受冷和红光的诱导。综上所述,我们认为GAI4参与了GA的生物合成并且能够产生一种新的GA种类,从而特异性的调控了种子萌发过程,对其进行功能分析将有助于我们更好的了解GA与ABA信号转导之间的互作。更多还原

【Abstract】 Seed germination is regulated by ABA and GA. ABA can inhibit germination and induce dormancy; GA stimulate seed germination and release dormancy. Recent studies have shown that the balance of abscisic acid （ABA） : gibberellin （GA） levels was the major factor. However, because GA and ABA signaling transduction are complicated, so we still know little about its regulation mechanism.In this study, using the T-DNA library we isolated one ABA mutant. Through genetic analysis of the mutatant we found its phenotype could be stable geneticed, so we named the mutant gai4 （gain function of ABA insensitive 4, gai4）. Using TAIL-PCR technology we found that T-DNA was inserted into the promoter region of GAI4. In Arabidopsis, GAI4 encode a 2OG-Fe（II） oxidoreductase. Under normal conditions, the expression levels of GAI4 has been weakened. After the ultra-induced expression of estradiol, the expression levels of GAI4 was increased about 50 times. Through bioinformatics and biochemistry analysis we found that GAI4 maybe involved in the GA biosynthesis. We further analyzed its physiological function. Results showed that over-expression lines were insensitive to NaCl, H₂O₂ on germination and they did not need cold acclimation to promote germinate. Under red and far-red light, the over-expression lines were growing better than WT and gai4 on germination and hypotocyl length. These results gived us a clue that GAI4 was involved of GA biosynthesis. Using biochemistry analysis, we found that GAI4 coded one member of GA20oxs and it could use GA₁₂ as substrate to produce one new type of GA. Then we found GAI4 had higher expression in root by RT-PCR. Fluorescence co-localization result of GAI4::GFP fusion proteins using transient expression in protoplast of mesophyll cells showed GAI4 enzyme protein localized in cytoplasm. GAI4 its expression could induced by cold and red light. In gene expression aspects it could regulate the gene related to DELLA and ABA metabolic.In summary, we believe that GAI4 is involved in GA biosynthesis and can produce one new type of GA, then specially regulate the seed germination. Through the functional analysis of GAI4 can help us better understand the interactions between GA and ABA signal transduction.更多还原