Cloning And Expression Of MAPK Gene In Malus L. And Signal Transduction Under Water Stress

Drought stress effects on many changes of morphology and physiology , biochemistry, as well as gene expression in plant. Generally, the response of physiological and biochemical activity of different species under drought stress are the basis of measuring plant drought-tolerance .The expression and regulation of gene related to drought-tolerance and signal transduction are still little known. The experimental materials are Malus hupehensis (Pamp) Rehd. that is a sensitive type to drought; M. sieversii (Ldb) Roem is a drought-tolerance type ; M. micromalus Makine is a middle type. A MAPK (mitogen-activated protein kinase) homologous gene, MaMAPK were cloned from M. micromalus under water stress. The expression of MaMAPK and the changes of MAPK activity and signal transduction related to MAPK were studied in order to explore the molecular mechanisms of drought-tolerance in Malus, and to provide the basis for further screening and utilizing drought-tolerance materials. The results were as follows:1. A full length cDNA sequence enciding MAPK was cloned from Malus micromalus by RT-PCR with primers designed according to the conservation domain of MAPK gene in higher plant for the first time. The cDNA sequence was named MaMAPK GenBank accession number AF435805. It was found that there were higher homology between MaMAPK and MMK4(a. MAPK gene from Medicago sativa, GenBank accession number X82270),the identity of nucleotide and amino acid were 81% and 82%, respectively. The cDNA sequence was about 1540bp long and encoded 417 amino acid . There were eleven conserved subdomains and phosphorylation site "TEY" , which was the typical characteristic of MAPK gene family, in the cDNA sequence . Results of Southern blotting suggested that there were all a small gene family of MaMAPK, and MaMAPKgene may be small copies in genomic DNA of three species of Malus. This is the first time to study the MPKhomology gene induced by water stress in Rosaceae.2. The expression and regulation of MaMAPKgene were reported in this study, the mechanism of drought-tolerance of three species in Malus were clarified in molecular level. Northern blotting showed that MaMAPK gene can be expressed in roots and leaves of three species after seedlings being treated with 20% PEG for different time . The expression of MaMAPK gene were gradually increased after treatment under water stress . The peak of expression emerged when the seedlings were treated for 1. 5h, then the expression were decreased gradually. The liquid kinase assays indicated that the dynamic changes of kinase activity was very similar to that of the mRNA relative expression of MaMAPK .The difference between two change is that the peak of kinase activity was emerged later than that of the mRNA relative expression. Although the increased amount of kinase activity was decreased after the peak , the increased amount was still higher than that of control until the end of treatment. 1mmol/L tyrosine phosphatase inhibitor PAO inhibited partly the MAPK activity ,the results suggested that the dephosphorylation of tyrosine phosphatase might regulate the phosphorylation of MAPK. The results indicated that MaMAPK gene were regulated not only by water stress in transcription level but also by phosphorylation and dephosphorylation in protein level.Although there were difference on the mRNA relative expression of MaMAPK gene and the activity of kinase in three species , the dynamic changes of kinase activity was similar to that of the mRNA relative expression. There were the highest MAPK activity and the highest mRNA relative expression in M. sieverii, followed by in M. micromalus and M. hupehensis. The results indicated that the mRNA relative expression and the activity of MAPK correlated with the ability of drought-tolerance in three species. Therefore, the differences on the mRNA expression of MaMAPK gene and the activity of MAPK of three species in Malus are one of molecular mechanism of different drought-tolerance .3. The relation between protein kinase phosphorylation and ABA synthesis...