Construction Of Mutant Population For Soybean And Cloning Of Single-Strand-Specific Nuclease Genes

Soybean（Glycine max（L.） Merri.） is an important oil crop.Recent increase of the sequence datas for soybean genome has resulted in an explosion of information on genes. The challenge for the post-sequencing era is to identify the biological functions for these genes.Of all the approaches used for discovering the gene function,the more useful one is based on a saturated mutagenesis population,which is a straight and efficient way to understand the role of all the genes.In all the methods for constructing the mutant liberary, the physical and chemical mutangenesis might be the main methods,which were simple and effective.They can be used to produce a large number of mutants with high mutagenesis frequence in a short period and to establish a mutant population.Targeting induced local lesions in genomes（TILLING） is a novel reverse genetics technology that provides rapid and effective characterization and targeting screen for mutations.The purpose of the present experiment is to creat the mutant library by treating the seeds of "Nannong94-16" with NaN₃-⁶⁰Coγrays or EMS.Firstly,we obtained 54 mutants of leaves,stalk,and flower,seed and cotyledon by treatment of NaN₃-⁶⁰Coγrays,and 66 mutants by EMS separately.Comparing with EMS mutagenesis,more mutants were screened in NaN₃-⁶⁰Coγray population.Mutants including high protein,high oleic acid, low protein and conglycinin subunit mutants were found in present experiment.We gained 14 Water-logging tolerance mutants,all of which displayed polymorphism with the control by SSR.A yellow green leaf character mutation was isolated,which displayed yellow green from cotyledon to muture stage all along.Genetic analysis showed that the yellow green leaf character was controlled by one pair of recessive nuclear genes.The mutant library created for "Nannong94-16" will be helpful for breeding in rapeseed and gene functional analysis of soybean.CELI is a key enzyme for TILLING research,which is the first eukaryotic nuclease known that cleaves DNA with high specificity at sites of base-substitution mismatch and DNA distortion.In this study,two CEL I-like genes from soybean,designated as GmEN1 and GmEN2,were cloned and characterized by bio-informatics.GmEN1 has no intron and contains a complete ORF of 927 bp which encodes a protein with 308 amino acids. Similarly,GmEN2 has one intron and contains a complete ORF of 894bp which encodes a polypeptide of 297 amino acids.The sequence analysis of GmEN1 and GmEN2 protein in NCBI Blast revealed that both the two genes contained the S1-P1_nuclease domain conserved in the most single-strand-specific nucleases.GmEN1 and GmEN2 shared 73%、46%identy with CEL I separately,based on phylogenetic analysis,GmEN1 and GmEN2 proteins were classed into two subgroups with different potential functions.Southern blotting analyses suggested that GmEN1 had one copy while GmEN2 had two copies. RT-PCR analysis indicated that the espression of GmEN1 was higher in seed than in other tissues,while GmEN2 was constitutively expressed in soybean.Furthermore,real-time PCR analysis revealed that GmEN1 and GmEN2 were induced during both hormone stress and naturally occurring leaf,stem senescence.To our knoeledge,both GmEN1 and GmEN2 are the genes associated with senescence.