Construction And Characterization Of SSH Library Induced By Low Phosphorus Stress In Soybean (Glycine Max)

Soybean is an important food and economic crop, and plays a very important role in the national economy. It is also a kind of crops which need a large amount of phosphorus, and its production is significantly limited by low phosphorus availability in the soil. Using the transgenic technology to breed high-efficient phosphorus variety is an effective method to improve the nutritional status of phosphorus in soybean. But the limited quantity of related genes is a major bottleneck. To mine and clone more phosphorus-metabolism related genes, in present study, a high quality soybean root cDNA library of low phosphorus-tolerance variety Zhong-Huang 15 was constructed under the low phosphorus stress by using suppression subtractive hybridization (SSH) technique. The results were summarized as follows:1. The results showed that the titer of the SSH cDNA library was 4.3×105 cfu.mL-1, and transformation efficiency was 75%. There were 1500 positive clones stored in this library, and the size of insert fragments was ranged from 200 bp to 1000 bp with a average size 500 bp, which was determined by positive clones randomly.2. Two hundred positive clones selected randomly were sequenced in the constructed SSH library and 150 unique ESTs were obtained. Then the ESTs similarity was analyzed via BLASTx and BLASTn software and there were 79 ESTs of homology with the known function protein sequences. The results of BLASTn confirmed that there were 70 ESTs homologous with the known ESTs sequences.3. Analysis results of the obtained soybean genes in the library induced by the low phosphorus stress showed that the known functions of the homologous genes were involved in many metabolism and response processes such as metabolism, signal transduction, disease/defence, protein synthesis, processing, and cell growth / division etc. The results also showed that the disease / defense category gene products shared a maximum of 29.1%, which was the largest type, and the signal transduction pathway genes fell into another large type, which accounted for13.9% of the total ESTs.4. Analyzed the Blast results of the sequenced ESTs, we found that some ESTs were homologous to the related genes of plant tolerance to low phosphorus stress. These homologous ESTs were as follows: EST 180 ( homologous to the vacuolar ATPase subunit E in Phaseolus acutifolius), EST 61 ( homologous to the Aldehyde dehydrogenase in Medicago truncatula), EST 114 ( homologous to MAPK in Glycine max), EST I24 ( homologous to Tyrosine kinase in Glycine max), EST F5 ( homologous to Similar to thioredoxin-related isoformⅠin Vitis vinifera), EST 113 ( homologous to bZIP transcription factor bZIP41 in Glycine max), EST K3 ( homologous to transcription factor EIL2 in Vigna radiata), EST 38 ( homologous to peroxidase in Dimocarpus longan), EST K1 ( homologous to Cationic peroxidaseⅡin Arachis hypogaea) and EST 134 ( homologous to peroxisomal betaine-aldehyde dehydrogenase in Glycine max).