Biotechnology Research Of Iron-efficient Genotype In The Genus Malus--Cloning Of MxNrampl And MxIrtl

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.