Selection And Isolation Of GsPK & GsLRPK Gene Early Responding To Osmotic Stress In Glycine Soja

Low temperature,drought and high salt are the major environmental factors that place limits on plant growth,development,geographical distribution and adversely affect crop production in the world.Enhancing the crops' resistant ability to osmotic stress is very important for increasing the crop production and solving the famine problem.Therefore,improving stress tolerance in crops has been an important aspect of modem agricultural research.With the rapid development of plants' molecule reaction and signal transduction mechanism to osmotic stress,using the bioengineering strategies to transfer or improve a key controlling gene(eg: kinase gene,responding and transducing the stress signal) is a more efficient way found to slove the problem.If the key kinase gene regulating signal transduction pathways in the plant resistance to stress could be gained,it would provide important gene resource for molecular breeding and theoretics support for the study of plant molecular mechanism resistant to osmotic stress.Wild soybean(Glycine soja) is characteristic of better stress-resistance and adaptive capacity. It is an important gene resource in molecular breeding by means of transgenic technology.In this study,we predict the expression patterns of all wild soybean ESTs downloaded from dbEST database by gene expression profiles of wild soybean under drought,salinity and cold stress,which had been established in our laboratory before.At the same time,Two ESTs are selected reponding to NaCl and 4℃treatment,separately.And then,the full-length gene corresponding to these ESTs were obtained by In silico cloning and SMART-RACE technique.Protein products were analyzed by kinds of bio-informatics tools,so that the character,structure and function of the proteins were predicted.This research will provide key gene for plant genetic engineering of osmotic stress and some information for stress tolerance researches.The main results were summarized as follows:1.Selection of kinase ESTs early responding to osmotic stresses from GsojaESTs of Glycine soja downloaded from dbEST database were aligned with more than 60 000 probe-sets of the Affymetrix soybean chip,and the expression patterns of ESTs were infered using gene expression profiles of wild soybean under drought,salinity and cold stress,which had been established in our laboratory before.The initial result showed that there were 18 kinase ESTs up-regulated under the 4℃treatment,20 kinase ESTs up-regulated under the NaCI treatment and 27 kinase ESTs up-regulated under the PEG treatment.According to the annotations of BlastN and BlastX,we choose the ESTs of GsPK and GsLRPK for the full-length genes isolation.2.Isolation of protein kinase genes early responding to osmotic stresses from GsojaGsPK gene was isolated by in silico cloning method and the full-length cDNA is 1020bp, encoding a 399 amino acid protein.Using the modified SMART-RACE technique,the complete sequence of GsLRPK was obtained.The full-length cDNA of GsLRPK gene is 2145bp,encoding a 714 amino acid protein.3.Bioinformatics analysis of the protein productsAnalysis of physical features,conserved domains,secondary structures,subcellular positions of the two gene products were carried out with softwares,such as DNAman,Antheprot and so on, as well as databases and tools published in the interact.The bioinformatical analysis revealed as follows:The molecular mass of predicted GsPK protein is 38483.8D,and its pI is 6.84.GsPK protein contains the catalytic domain of protein kinase family in its C terminal,but without signal peptide in its N terminal.GsLRPK protein's molecular mass is 77689.5D,and its pl is 5.60.The GsLRPK protein contains the feature of LRR-RLK family,containing LRRs motifs,transmembrane region and protein kinase catalytic domain,and have signal peptide which can introduce the protein to the secretory vacuole.What's more,these two proteins have potential phosphor-sites,secretory vacuole site and myristyl-sites,which suggests these proteins might be regulated or activated by post-translation modifications.