Cloning And Expression Analysis Of RNA-Dependent RNA Polymerase Genes GmRDR6a, GmRDR6b And GmRDR1 In Soybean(Glycine Max)

RNA-dependent RNA polymerases (RDRs) were known as a big family, they can participate in a variety of biological processes such as plant growth and development regulation, biotic and abiotic stresses respond and epigenetic modifications through gene silencing pathways. RNA-dependent RNA polymerase 6 gene (RDR6) and RNA-dependent RNA polymerase 1 gene (RDR1) were important components of RDRs gene family, which had been identified with special functions. Two novel soybean RDR6 genes and one RDR1 gene were isolated using homologous cloning method, and which were named GmRDR6a, GmRDR6b and GmRDR1 respectively. Bioinformatics analysis and characterization of gene expression were done for further study of these novel soybean genes.Firstly, Two novel RDR6 genes were identified in soybean cultivar’Kefeng No.1’by homologous cloning method, named as GmRDR6a and GmRDR6b. Sequences of the two genes were analyzed by bioinformatics softwares, Genes expression in different tissues and their reponse to biotic and abiotic stresses were performed using real-time PCR, subcellular localization analysis and construction of plant overexpression vectors. The results showed that GmRDR6a was located on chromosome 6 of soybean genome, and the length of it was 4389 bp, inclulded an 744 bp intron. The ORF of GmRDR6a was 3615 bp and encoded 1204 amino acids (297.5103) with an isoelectric point of 4.86. GmRDR6b gene was located on chromosome 4 of soybean genome and the length of it was 4002 bp, contained an intron of 387 bp. The ORF of GmRDR6b was 3615 bp and encoded 1204 amino acids (296.89103) with an isoelectric point of 4.86. The consereved motifs’DLDGD’of RDRs family were identified in GmRDR6a and GmRDR6b. The expression of the two novel genes were identified in checked tissues of soybean, and showed the higest expression level in flower. Real-time PCR analysis revealed that the mRNA levels of these two genes in the resistant soybean cultivar ’Kefeng No.1’ were significant higher than the susuceptible soybean cultivar "Nannong 1138-2’after soybean mosaic virus (SMV) treatment. For abiotic stress. GmRDR6a showed high expression level in roots, stems and leaves under the treatments of salinity and drought, and showed the highest expression level in roots under salt stress. There was an early response after ABA treatment. There was no significant change of expression level of GmRDR6a gene under chilling stress. GmRDR6b showed higher expression level under salt stress and ABA treatment and there was an early response under chilling stress. There was no significant difference of expression level of GmRDR6b gene under drought stress. Therefore, a preliminary judgment was that these two genes were involved in the resistant response in soybean, and their resistance mechanism needs further study. The fusion expression vectors were introduced into onion epiderma cells by Agrobacterium-mediated method into onion epidermal cell revealed that the two genes were located in the nucleus. Overexpression vectors of pMDC83-GmRDR6a and pMDC’83-GmRDR6b were constructed to verify their functions of disease resistance.In addition, a novel soybean gene named GmRDR1 was identified in soybean cultivar ’Kefeng No.1’ using homologous cloning method. Sequence of this gene was analyzed by bioinformatics softwares. Expression pattern of the gene in different tissues and its reponse to biotic and abiotic stresses were performed using real-time PCR. The transformation system of onion epidermal cells mediated Agrobacterium was established for subcelluar localization. The results show that GmRDR1 gene was located on chromosome 2 in soybean and the length was 3956bp. The ORF of GmRDR1 was 3 378 bp and encoded 1125 amino acids (279.72 103) with an isoelectric point of 4.63. The consereved motif "DLDGD" of RDRs family was identified in GmRDRl. The expression pattern of this gene was identified in checked tissues of soybean, and showed the higest expression level in leaves of soybean. Real-time PCR analysis revealed that mRNA levels of this gene in the resistant soybean cultivar’Kefeng No.1’ were much higher than the susceptible soybean cultivar ’Nannong 1138-2’ after soybean mosaic virus (SMV) treatment. For abiotic stress, GmRDRl showed high expression levels in stems and leaves under salt stress, and showed the highest expression level after 24 h treatment. The expression level of GmRDR1 in roots had a brief rise after 6 h under salt stress and then had a downward trend. GmRDR1 showed high expression levels in roots, stems and leaves after drought treatment within 48 h, and showed the highest expression level after respectively 6,48 and 24 h, the expression level of this gene had a continued rising in stem. There was an early response after 6 h ABA treatment, the expression level of GmRDR1 gene showed a suddenly up regulation after 24h under chilling stress. therefore, a preliminary judgment was that GmRDR1 gene had the function of salt and drought tolerance, and this gene was involved in the disease resistant response in soybean. Subcellular localization result showed that GmRDR1 gene encoding the protein was located in the nucleus. Overexpression vector of pMDC83-GmRDR1 was constructed for further research this gene by transgenic method.