大豆RNA依赖的RNA聚合酶基因GmRDR1的克隆与表达特性分析

Cloning and Expression Pattern Analysis of a RNA-dependant RNA Polymerase Gene GmRDR1 in Soybean

为了寻找大豆抗病新基因,培育大豆新型抗性品种,利用同源克隆的方法从大豆品种科丰1号中分离出1个新的GmRDR1基因,并对其进行序列分析,组织表达、抗逆境胁迫表达分析及该基因的亚细胞定位研究。结果表明:GmRDR1基因位于大豆基因组的第2号染色体,基因全长为3 956 bp,其中ORF为3 378 bp,编码1 125个氨基酸,相对分子量和等电点分别为279.72×103和4.63;GmRDR1含有RDRs家族的保守序列"DLDGD";该基因在所有被检测组织中均表达,并且在叶中的表达量最高;荧光定量结果发现:在大豆花叶病毒(Soybean mosaic virus,SMV)处理下,GmRDR1在抗病材料科丰1号中的表达量显著高于感病材料南农1138-2。盐及干旱胁迫下,48 h之内,该基因的表达量明显升高,SA诱导条件下该基因在6 h出现了早期响应,冷害处理下24 h表达量出现了突然的升高。GmRDR1基因的亚细胞定位结果表明:该基因所编码的蛋白定位在细胞核里。根据以上结果判定GmRDR1基因参与了大豆对SMV的抗性反应,并且能够强烈响应盐和干旱的胁迫,因此该基因在大豆抗逆分子育种中具有较好的应用价值。

In order to find new resistant gene and cultivate new resistant varieties of soybean,a novel soybean gene named GmRDR1 was identified using homologous cloning method. Sequence of the gene was analyzed by bioinformatics software,expression pattern of the gene in different tissues and its response 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 showed that GmRDR1 gene was located on chromosome 2 in soybean and the length was 3 956 bp. The ORF of GmRDR1 was 3 378 bp and encoded 1 125 amino acids（ 279. 72 k Da） with an isoelectric point of 4. 63. The consereved motif‘DLDGD＇of RDRs gene family was identified in GmRDR1. The expression pattern of the gene was identified in all tissues of soybean,and showed the highest expression level in leaves of soybean; Real-time PCR analysis revealed that mRNA levels of the gene in the resistant material Kefeng 1 were much higher than the susceptible material Nannong 1138-2 after soybean mosaic virus（ SMV） treatment. For abiotic stress,GmRDR1 showed high expression levels under salt stress and drought treatment.There was an early response after 6 hours ABA treatment,the expression level of GmRDR1 gene showed a suddenly rise after24 hours under chilling stress. The result of subcellular localization revealed that GmRDR1 gene encoding the protein was located in the nucleus. Therefore,a preliminary judgment was that GmRDR1 gene had the function of salt and drought tolerance,and it was involved in the resistant response in soybean. The new gene showed the value for breeding resistant soybean cultivar by molecular breeding method.