【作者】 周莹；

【导师】 张启发； 练兴明；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 水稻不仅是我国主要的粮食作物,还养活着世界一半的人口。在水稻氮代谢途径中,有一些重要的酶(例如GS、GOGAT、GDH、AAT和AS),调控着水稻氮素的代谢。有研究发现,尤其是编码天冬氨酸转氨酶的AAT基因,与控制蛋白质和氨基酸含量的QTL有对应的关系。本研究在水稻品种中花11(来自与中国农业科学院作物科学研究所的商业品种)中超量表达了AAT基因(包括来源于水稻的3个AAT基因:OsAAT1、OsAAT2和OsAAT3;来源于大肠杆菌的1个AAT基因:EcAAT),同时抑制表达AAT这个基因。得到转基因植株后,分析了转基因植株的生长和代谢水平与对照植株的差异,研究了这两个基因的表达情况和作用机理。综合分析2004年和2007年数据,得出以下几个主要结果:1、得到了超量表达AAT的转基因植株(如上所述,共试图超量表达4个片段)。2、t测验(t-test)表明,超量表达AAT基因的转基因植株,不管在正常生长条件下,还是各种胁迫生长条件下(包括低氮胁迫,NaCl胁迫,ABA胁迫),其生长(包括株高和干重)都与对照没有显著差异。3、t测验(t-test)表明,超量表达AAT基因(包括OsAAT1,OsAAT2和EcAAT基因)的转基因植株的米粉中,氨基酸含量和蛋白质含量明显高于野生型对照。4、超量表达OsAAT1基因的阳性植株的种子中的氨基酸总含量比其对应的阴性植株的含量提高了16.1%,蛋白质含量提高了22.2%;超量表达OsAAT2基因的阳性植株的种子中的氨基酸总含量比其对应的阴性植株的含量提高了12.0%,蛋白质含量提高了21.1%;超量表达EcAAT基因的阳性植株的种子中的氨基酸总含量比其对应的阴性植株的含量提高了5.4%,蛋白质含量提高了11.1%。t测验(t-test)表明,超量表达AAT基因的转基因阳性植株和其对应的阴性植株的米粉中,氨基酸含量和蛋白质含量在阳性植株中显著(P≤0.05)高于其对应的阴性植株。说明氨基酸含量和蛋白质含量的升高是由转基因作用引起的。5、验证AAT基因家族的表达情况,发现AAT基因家族的3个成员的表达受各种胁迫的影响。同时这3个成员在植株的不同部位和不同的生长时期,表达也存在着差异。6、超量表达EcAAT基因,能使植株体内OsAAT1和OsAAT2基因的表达上升。但是水稻体内的3个OsAAT基因的表达并不能相互调控,即超量表达任何一个OsAAT基因,并不能引起其它两个OsAAT基因表达的改变。更多还原

【Abstract】 Aspartate aminotransferase(AAT) is a key enzyme in the synthesis of amino acids.It plays an important role in regulation of carbon and nitrogen metabolism in almost all organisms.In this study,we over-expressed in rice separately all three AAT genes from rice(OsAAT1～3) and one AAT gene from Escherichia coli(EcAAT). Over-expression was driven by the CaMV 35S promoter and transformed into rice by Agrobacterium tumefaciens-mediated transformation.Compared with control plants, the genetically engineered transformant plants showed significantly increased leaf AAT activity and seed amino acid and protein contents despite the lack of observed phenotypical changes.The total leaf AAT activities in OsAAT1,OsAAT2 and EcAAT over-expressed plants were 26.6,23.6 and 19.6 A/mg/min,which were significantly higher than that in the wild type control(17.7 A/mg/min).The amino acid content in seeds of transgenic plants over-expressing OsAAT1,OsAAT2,and EcAAT was 119.36 mg/g,102.84 mg/g and 113.72 mg/g,respectively,which were 16.1%,12.0%and 5.4%higher,respectively,than that in the control plants.The transgenic plants over-expressing OsAAT1,OsAAT2,and EcAAT had significantly higher protein contents(increased 22.2%,21.1%,and 11.1%,respectively) than wild type plants.No significant changes were found in leaf AAT activity,seed amino acid content or protein content in OsAAT3 over-expressed plants.The expression patterns of the three OsAAT genes and their different functions are also discussed.We do some RT-PCR and real-time PCR to check the expression of AAT genes. As result shows that OsAAT1 and OsAAT2’s expression level are higher than OsAAT3, and when over-expressing EcAAT gene in transfer rice,it would result in higher expression of OsAAT1 and OsAAT2,but OsAAT3 expression level is lower.Except OsAAT1 gene has higher expression level in OsAAT2 transgenic rice,over-expressing one of OsAAT genes,it couldn’t induce other OsAAT genes changed their expression.更多还原