【作者】 戚金亮；

【导师】 韩振海；

【作者基本信息】 中国农业大学 ， 果树学， 2003， 博士

【摘要】 本实验以中国农业大学园艺植物研究所筛选到的一个苹果铁高效基因型——小金海棠（Malus xiao jinensis Cheng et Jiang）为试材，分别克隆了小金海棠的抗缺铁相关基因MxNramp1基因的752bp基因组DNA片段和Fe（Ⅱ）-转运蛋白基因（MxIrt1）的cDNA全长，为深入探讨小金海棠抗缺铁的分子机理奠定了基础。 （1）以异源的小麦Nramp基因片段和玉米Fe（Ⅱ）-转运蛋白基因（ZmIrt）片段为探针进行了杂交分析。Southern杂交结果表明：小金海棠基因组中存在该两家族基因。Northern杂交结果表明：在根中和叶中，Nramp基因均能得以转录且受缺铁胁迫的诱导而加强。Fe（Ⅱ）-转运蛋白基因在根中的转录也受到缺铁胁迫的诱导，并随缺铁胁迫处理天数的增加而加强。 （2）根据植物Nramp基因家族的功能保守区序列设计引物，通过常规PCR法从小金海棠基因组DNA中扩增出了MxNramp1基因的752bp的特异性产物；序列分析表明，该片段所推导的氨基酸序列与小麦Nramp基因片段（杂交用的探针片段）及水稻OsNramp3基因分别具有98％和83％的同源性。该片段的克隆为进一步克隆MxNramp1基因的全长提供了条件。 （3）根据植物Irt基因家族的功能保守区序列设计引物，首先通过常规PCR法从缺铁胁迫处理的小金海棠根系cDNA文库中克隆了Fe（Ⅱ）-转运蛋白基因MxIrt1的490bp的片段，然后根据测序结果设计引物，通过RACE法从该cDNA文库中克隆了MxIrt1基因的cDNA全长。序列分析表明，该基因的cDNA全长为1398bp，开放阅读框为1095bp，编码一个364个氨基酸的多肽，与番茄和豌豆中的该家族基因分别具有71％和69％的氨基酸同源性，是一种膜蛋白，具有1个信号肽序列，7个跨膜螺旋，3个N—糖基化位点和1个O—糖基化位点，分子量大约为39.174kD，等电点为8.07。 （4）成功地构建了小金海棠Fe（Ⅱ）-转运蛋白基因MxIrt1的酵母表达载体pDB^1eu—MxIrt1，并正在进行该基因的确切功能和亚细胞定位研究。 分析认为该两基因可能在小金海棠抗缺铁机理中起到重要的作用，为深入研究其功能奠定了基础。更多还原

【Abstract】 705bp DNA fragment of MxNrampl gene and full cDNA of Mxlrtl gene which were related to resist iron stress were cloned by using Malus xiaojinensis Cheng et Jiang-the first Iron-efficient genotype in the genus Malus in the world as material.(1) Using fragment of Nramp gene from wheat and Fe(ll)-transporter gene fragment of maize (Zmlrt~) as probes, we analysed these genes by blotting hybridization technique in Malus xiaojinensisCheng et Jiang. Southern blot suggested that the genome of Malus xiaojinensis Cheng et Jiang has these genes. Northern blot suggested that the transcription of Nramp gene is induced by iron stress in both roots and leaves and strengthened by iron stress in leaves; the transcription of Irt gene can be induced and strengthened by iron stress in roots.(2) We have cloned 752bp fragment of MxNrampl gene from the genomic DNA of Malus xiaojinensis Cheng et Jiang by using common PCR method with primers designed according to the conserved domain sequences of plant Nramp gene families. Sequence analysis suggested that the predicted amino acid sequence of this fragment has 98% and 83% identities to the Nramp gene fragment of wheat and OsNrampS of rice respectively.(3) 490bp cDNA fragment of Fe(II)-transporter gene Mxlrtl was cloned by using common PCR method from iron-stressed root cDNA library of Malus xiaojinensis Cheng et Jiang with primers designed according to the conserved domain sequences of plant Irt gene families. Then we cloned the full cDNA of Mxlrtl gene by RACE method from this library with primers designed according to the sequence of 490bp cDNA fragment. Sequence analysis showed that the full length of this cDNA which encodes 364 amino acids is 1398bp. It has 71% and 69% identities to Lycopersicon esculentum and Pisum sativum respectively in amino acid level. This gene is a membrane protein which has one signal peptide, seven transmembrane helices, three N-glycosylation sites and one O- glycosylation site. The mass of it is predicted to be 39.2kD and its pI is 8.07.(4) We constructed the yeast expression vector pDBleu-Mxlrtl successfully. Now we are studying the role and subcellular location of Mxlrtl gene.更多还原