| Soybean is an important resource of high-quality protein and edible oil. Cultivated soybean demands more water during the whole growth cycle, under-developed root system,water deficit are bottleneck problems for improving the soybean yield and quality. In China, losses caused by drought each, year are about60million tons. Therefore, the research about physiological and biochemical reactions, and the molecular mechanisms of response under drought stress in soybean will provide the theoretical guidance for the breeding of fine soybean variety and genetic engineering in the future.Transcription factors have played an important role on cellular and molecular level in plants affected by drought, high salt, low temperature and other abiotic stress,. C2H2zinc finger protein transcription factors are involved in the growth and development of plants and the resistance to environmental stresses.By searching the soybean EST database in GenBank (http://www.ncbi.nlm.nih.gov/genbank/), C2H2zinc finger protein transcription factor domain characteristics and EST sequences were analyzed, electronic EST stitching was conducted, C2H2transcription factor family characteristics and expression were determined:(1)cDNA sequences of six C2H2zinc finger protein genes were cloned based using RT-PCR, which were named as GmZFPl, GmZFP2, GmZFP3, GmZFP4, GmZFP5and GmZFP6;(2) Their sequences were aligned using Clustalx PAUP and MEGA software, which showed that all encoding protein sequences have C2H2zinc finger protein highly conserved domains. And the protein sequences of the six genes belong to the Cl class family proteins. Where Q2-1; K2-1protein for GmZFP1GmZFP5; Q2-2: Q2-3:K2-2protein for GmZFP2GmZFP4; Q2-2:Q2-3protein for GmZFP3, GmZFP6. The phylogenetic tree analysis showed that soybean and Lotus Japonicuswere clustered into one group, in consistent with the fact that both were legumes with relatively close genetic relationship.At seedling stage, two soybean varieties were treated by PEG, the physiological indexes including content of malondialdehyde (MDA) and proline in leaves36h after treatment were analyzed. The results showed that:the MDA content of ’Jinda74’ was far less than that of ’Jinda53’,while the content of free proline is higher. So we concluded that ’Jinda74’ was a PEG stress resistance variety,’Jinda53’ was a PEG stress sensitive variety.Based on the real-time quantitative PCR (real-time PCR), we analyzed the expression level of GmZFP1GmZFP2, GmZFP5, GmZFP4, GmZFP5, and GmZFP6in soybean tissues. The results showed that the four genes except for GmZFP1and GmZFP6, the expression level in flowers was higher than that in leaves, the highest expression level was140times, and the lowest was15times. GmZFP1expression level in soybean pods was140times higher than that in leaves and the level in roots was23times as high as that in leaves. So GmZFP1was expressed specifically in pods and GmZFP2was expressed specifically in roots; the other four genes were expressed specifically in flowers.Quantitative expression analysis of six genes in soybean leaves under PEG stress, salt stress, cold stress and induction by ABA and salicylic acid was conducted using real-time PCR. The results showed:for ’Jinda74’, under PEG stress for18h, the expression levels of all six genes in its leaves were increased significantly, the highest expression level was570times higher than that in the leaves, the lowest was6times; under salt stress for0.5h, the expression levels of GmZFP6in leaves was2.4times higher than that of the control, while levels of the other five genes declined dramatically and lower than that of the control; under cold stress for24h, GmZFP1expression level in leaves was2.6times as high as that of the control; for3h, the expression level of GmZFP5was2.4times higher; for6h, GmZFP6expression level in leaves was7times higher but that of the other three genes declined sharply and lower than the control level; ABA and SA signaling molecules could activate the four genes. Therefore, we concluded that the four genes above in leaves of ’Jinda74’ were regulated by ABA or SA signaling molecules. GmZFP6was just regulated by ABA signal transduction pathways, but no response to SA signaling molecules. Interestingly, GmZFP5was not be induced by ABA or SA signaling molecules but responded to salt stress. For Jinda53, a water-sensitive variety, compared with Jinda74, the responses of the six genes were lower under PEG stress, ABA and SA treatments, which reflected that six genes of GmZFP had strong correlation with stress adaptability in soybean. |
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